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A Field Guide to Wheat Phenotyping
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1. 5 some hair to 10 very hairy 4 k 3 p F j Figure 19 6 Pubescence on the glumes of the spike score of 8 110 Physiological Breeding II A Field Guide to Wheat Phenotyping Leaf rolling Leaf rolling is most apparent on the flag leaves but can occur on lower leaves in the canopy It can be either a mechanism to reduce canopy light interception and or a response to plant water stress In general leaves start rolling from the tip of the leaf Make observations during grain filling for adaptation to terminal drought and or heat stress Figure 19 7 Scoring e Make two observations of leaf rolling as trait expression is sensitive to environmental conditions e Make observations at two times during the day early morning before 10 00h and afternoon between 13 00h and 16 00h depending on severity of stress differences between genotypes will appear on morning or afternoon scores e Observe the most recent fully expanded flag leaf recommended or all green leaves i Rate the proportion of the leaves within the plot which are affected by rolling using a scale from 0 0 to 10 100 in increments of 10 ii Rate the leaf rolling using a scale from 0 to 3 Table 19 1 The extent of leaf rolling of the most recent fully expanded leaf can be estimated by of leaf rolled 1 rolled leaf width unrolled leaf width x 100 Equation 19 1 Figure 19 7 A tightly rolled flag leaf score of
2. Berry PM Sterling M Baker CJ Spink J and Sparks D 2003 A calibrated model of wheat lodging compared with field measurements Agricultural and Forest Meteorology 119 167 180 Berry PM Sylvester Bradley R and Berry S 2007 Ideotype for lodging resistant wheat Euphytica 154 165 179 Kansas State University 1995 Spring freeze injury in wheat modified from the Kansas State University Cooperative Extension Service publication C 646 revised March 1995 Available at http www oznet ksu edu library crpsl2 C646 PDF accessed 14 August 2011 Once damage becomes apparent it is important to take repeated assessments to account for any worsening of crop condition either a fixed number of days or at a developmental stage e g heading Texas Agricultural Extension Service 2011 Freeze injury on wheat Available at http varietytesting tamu edu wheat docs mime 4 pdf accessed 14 August 2011 Tripathi SC Sayre KD Kaul JN and Narang RS 2003 Growth and morphology of spring wheat Triticum aestivum L culms and their association with lodging effects of genotypes N levels and ethephon Field Crops Research 84 271 290 Tripathi SC Sayre KD Kaul JN and Narang RS 2004 Lodging behavior and yield potential of spring wheat Triticum aestivum L effects of ethephon and genotypes Field Crops Research 87 207 220 Warrick BE and Travis DM 1999 Freeze injury on wheat Texas Agricultura
3. Physiological maturity GS87 This is when the grain reaches the maximum dry weight and the grain becomes viable It is most easily determined in field when 50 of the peduncles are ripe i e yellow and at this point the glumes which are frequently the last part of the plant to senesce will also be losing their color Measurement is typically by visual assessment of the whole plot by the same observer judging all treatments within a trial alternatively assessment can be made by assessing 50 or 100 culms per plot Figure 14 6 Figure 14 6 Determination of physiological maturity A a crop at physiological maturity GS87 B a comparison of peduncles at GS83 GS87 and GS92 and C a schematic of maturity showing GS83 GS87 and GS92 Direct growth analysis 75 Wheat growth stages The Zadoks scale decimal code is based on 10 major stages with each stage divided into 10 sub stages Table 14 1 Table 14 1 The Zadoks scale Zadoks et al 1974 GS Description GS Description Germination Booting 00 ODryseed 41 Flag leaf sheath extending 01 Water uptake imbibition started 43 Boot just visibly swollen 03 Imbibition complete 45 Boot swollen 05 Radicle emerged from seed 47 Flag leaf sheath opening 07 Coleoptile emerged from seed 49 First awns visible 09 Leaf just at coleoptile tip Heading Seedling development 51 First spikelet of head visible 10 First leaf emerged 53 of head emerged 11 First leaf unfolded
4. Position the sensor at the start of the plot see advice on taking measurements Press and hold the trigger whilst moving across the plot release the trigger at the end of the sample area A continuous beep sound is produced while the trigger is held Walk up and down the rows regardless of the experimental design see Figure 8 2 In case of an error during the sampling take note of the SAMPLE NO and correct during data processing Final measurements and completion 4 After measuring the whole trial Go to FILE gt SAVE assign a file name e g trial name and date Saved data can be downloaded with the software supplied with the instrument Data is typically downloaded as a comma delimited text file and imported into MS Excel A Data and calculations First it is necessary to re order the downloaded data where the sample number and plot ID are in a different order Three individual text files are created for each trial i a general file with NDVI and vegetation index VI Red NIR values for each measured point approximately 10 values per second ii a file of the AVG NDVI and VI data for each plot and iii a file with diagnostic information indicated with the suffix DIAG Typically the AVG data file is used NDVI values from a crop canopy range from 0 to 1 where 0 represents no green area and 1 represents maximum greenness Table 8 1 Table 8 1 Sample of the
5. Figure 3 1 Equipment used to take measurements of leaf water potential including the pressure chamber and compressed air cylinder i Figure 3 2 Leaf sampling and leaf water potential measurement A first wrap the selected flag leaf in a moist gauze B and then cut the leaf at the base C place the leaf sample within the rubber seal of the top of the pressure chamber D use a magnifying glass to observe the point at which water appears at the cut ends E water exuded from the vascular tissue and F the reading on the pressure dial 22 bar Canopy temperature stomatal conductance and water relation traits 19 If testing two leaves at the same time arrange the leaves in the opposite orientation with adaxial sides facing each other to facilitate observation 4 Secure the top to the pressure chamber Carefully check that it is located correctly 5 Move the CONTROL valve from off into the pressurize position 6 Gently open the GAS valve to gradually allow compressed air into the chamber at 1 2 bar per second and increase the pressure always keep one hand on this valve 7 Carefully observe the cut end of the leaf leaves using a magnifying glass whilst the chamber pressure slowly increases Figure 3 2D 8 At the point when water is observed arising at the cut end of the leaf Figure 3 2E close the GAS valve and record the pressure shown on the pressure gauge Figure 3 2F 9
6. as surface moisture will distort measurements due to changes in the reflection of light within the canopy As a general rule more frequent white reference panel measurements should be taken with increased environmental instability Time of day Take the majority of measurements as close to solar noon as possible typically from 11 00h to 14 00h Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from the start of stem elongation to late grain filling depending on the experimental objectives timing of peak stress To compare between genotypes do not take measurements during heading and anthesis where differences in phenology may confound results Typically take two measurements between mid tillering and the end of booting then two measurements during grain filling Intensity LF i 100 White Panel g AAmir Wavelength 1200 mm Number of samples per plot Take 3 6 readings per plot at fixed positions with at least 10 spectrum averages per reading to ensure that signal noise is reduced Procedure Take the following equipment to the field e Field spectrometer equipped with an appropriate foreoptic lens for the measurement of a wheat canopy e White spectral reference panel with the necessary support for maintaining a fixed and horizontal position in the field Advice on taking measurements The field of view of the sensor should be taken
7. crop growth is light limited Therefore measurement of light interception can be a proxy for the photosynthetic capacity of a plot at early developmental stages when ground cover is incomplete and towards the end of the crop cycle as the leaves of the canopy senesce However the more recent spectral indices such as the normalized difference vegetation index NDVI can provide possibly more reliable estimates of green area Lopes and Reynolds 2012 Leaf area index LAI or green area index GAI are precise ways of estimating the light capturing capacity of a canopy and although light interception tends to saturate at LAI gt 3 the distribution of leaves can effect radiation use efficiency Parry et al 2011 Early ground cover is also a valuable stress adaptive trait where for example it can reduce evaporative loss of soil moisture Mullan and Reynolds 2010 This can be measured using digital images captured by a camera allowing rapid and low cost screening of large populations for this trait Section 4 Direct growth analysis Several growth related traits can be estimated and genetic differences effectively established using some of the protocols described already for example in season biomass can be estimated with SR indices root capacity can be estimated with CT and even yield can be quite well estimated under a range of environments with both methods However only direct measurement can provide absolute values
8. or for rapid screening take three measurements observations and record either all three or the median or a general observation of the plot can be made Procedure Take the following equipment to the field e Ruler e Field form and clipboard Advice on taking measurements and observations Measurements and observations should be made on fully emerged main culms Culms should be clean dry intact green with no sign of disease or damage note that senescence causes some shrinkage of the tissue Most measurements can also be made in the laboratory on biomass samples For measurements choose culms at random by selecting from the base of the culm to avoid bias For observations a general observation can be made by standing at a 45 angle alongside the plot but a close inspection of the leaves stems and spikes of several individual culms is recommended Take two repetitions approximately one week apart As observations are subjective it is important that ratings are consistent e Ensure that the ratings of new observers are calibrated with those of an experienced observer who is familiar with making crop observations so that values are standardized e If several people within the group will be making observations it is recommended that all observers meet to calibrate their readings before starting and regularly thereafter e Ensure that only one person makes observations within a replicate Trait measurements
9. 5 Cut the core into a section of specific length e g 30 cm and place each into its respective plastic bag and then tie tightly to avoid loss of moisture Figure 17 2B 6 The soil samples should be processed immediately or kept refrigerated at 6 8 C 1 Root washing cleaning and weighing This method is laborious and takes the most time Figure 17 3 Carefully use water to separate root tissue from soil and other debris within the soil core Wash and clean each sample for the same duration and in the same manner to make samples comparative Each sample may take up to 1 hour to process and weigh An automated root washer can be used e g RWC Laboratory measurements Determination of soil moisture content see Schematic 17 1 1 Complete a soil sampling form including plot number depth e g 0 30 cm 30 60 cm etc and aluminum pot number with spaces for sample fresh and dry weights 2 Organize the field sample bags by plot number and depth 3 BEFORE opening the plastic bag break up and mix the soil sample as much as possible and reincorporate any humidity condensation UM 2 Delta T Devices Ltd Cambridge UK Add water to the soil samples whilst still in their plastic bag mix gently tie and leave overnight Transfer the soil and water mix to a tray stir gently by hand wait a few minutes and decant water through a 500 um sieve to recover the roots Remove large plant material and debri
10. European Journal of Agronomy 22 231 242 Richards RA 2006 Physiological traits used in the breeding of new cultivars for water scarce environments Agricultural Water Management 80 197 211 Spectral reflectance indices and pigment measurement Chapter 7 Spectral reflectance Julian Pietragalla Daniel Mullan and Raymundo Sereno Mendoza The reflectance of different wavelengths of light from the canopy is influenced by the optical properties of the plant and gives a unique spectral signature of the constituent components of the crop canopy e g proteins lignin cellulose sugar starch water etc Field spectrometers and spectro radiometers are used to measure spectral reflectance and have a typical spectral range of 350 1100 nm or with a more extended range of 350 2500 nm This continuous range encompasses the visual and near infrared regions of the electromagnetic spectrum covering the wavelengths used for most canopy related indices An understanding of the optical properties of plant canopies has allowed the development of an extremely useful series of measurements for physiological trait selection Measurement and analysis of the reflected spectra can be used to capture a large amount of information on the physiological status of a crop canopy including calculation of vegetation pigment and water indices Table 7 1 Figure 7 1 These values allow estimation of the green biomass photosynthetic area of
11. Figure 16 1A 3 Place into a pre labeled paper bag Or randomly select a 20 culm sub sample from the in season biomass sample as detailed in this volume Chapter 15 Analyse WSC content Grind stem sample Laboratory measurements Cut the spike from the stem at the spike collar 5 Oven dry whole culm samples at 60 75 C until they reach constant weight i e for at least 48h 6 Remove the leaf lamina and leaf sheath from the stems Figure 16 1B 7 Weigh the dry stem sample for calculation of WSC content per stem or unit area DW_20stems 8 Grind the stem sample e g using a mill with a 0 5 mm screen Ensure to clean the mill carefully between samples Figure 16 1C Place ground sample into a labeled envelope Analysis Analysis of prepared samples is typically outsourced to a specialist laboratory A by the Anthrone method cost USS 5 00 per sample or B or scanned by near infrared reflectance spectroscopy NIRS using a calibration curve cost USS 0 50 per sample NIRS is an indirect method but has the advantage of also giving N values when using a N calibration curve see Figure 16 2 Cut spikes from culms w4 20 fertile 4 culms Stem sample in bag Remove leaf lamina and leaf sheath from stems Dry to constant weight Schematic 16 1 Determination of WSC concentration of wheat stems 84 Physiological Breeding II A Field Guide to Wheat Phenotyping Anthrone
12. Figure 22 1A at 60 75 C until constant weight typically for at least 48h see Table 22 1 When drying samples e Do not mix fresh samples with dry samples e Organize sampling to optimize use of the oven and of oven space e Use anon draft oven for drying open container samples e g soil moisture samples Figure 22 1B 126 Physiological Breeding II A Field Guide to Wheat Phenotyping for specific information modes measurement data download etc further details and clarification Do not use an instrument before it has equilibrated with ambient temperature and relative humidity RH as this may affect the calibration and data Take the instrument out of its protective case and turn on at least 10 minutes before Starting use Do not leave an instrument in direct sunlight heat before use as this can affect calibration and may cause incorrect readings especially for instruments with black cases When reading air temperature and RH stand with your back to the sun so as not to expose the instrument to direct sun during measurements Do not operate an instrument outside specified temperature and RH range for the instrument as measurements taken may be incorrect check the user guide for specifications Excess heat and moisture RH may cause permanent damage note that instruments are typically not water resistant Do not discard instruments after use Remember to clean the instrument return it to its protective
13. e Select an empty container which is otherwise identical to that of the samples i e from the same box packet vai Table 22 4 Typical units of data expression with the same ventilation holes staples if any etc l l Sample Measured as Expressed as e Dry the empty container in the oven next to samples cinviei and aplot sorta for the same drying time biomass weights e Before weighing samples place this empty container Dry weight of culms and crop g per 20 culm g m or g culmi on the balance and press RE ZERO TARE components e g leaf sub sample e The balance should show zero with the empty lamina leaf sheath stem container on the balance plate or a negative value Root biomass g g soil g cm soil when the empty container is removed and the plate is empty 0 000 e B p B small precision 2 d p WwW ___ ao o ame Figure 22 2 Types of balances for physiological measurements showing A semi analytical C medium large precision 1 d p and D Industrial retail bench balances 0 d p 128 Physiological Breeding Il A Field Guide to Wheat Phenotyping 5 Suggestions on models of instruments imply endorsement by CIMMYT Prices quoted serve as a guideline and will vary according to accessories functionalities taxes and customs fees Table 22 5 provides details of suggested models of instruments Reference to specific instruments is made in most chapter
14. essential Table 2 Resources required for each phenotyping technique Laboratory Data Instrument processing processing Most unit cost Field time time per time per representative Measurement Instrument USS per plot plot plot environments Canopy temperature Infrared thermometer 150 500 None All Stomatal conductance Porometer 2 500 4 000 None Irrigated heat Leaf water potential Scholander pressure chamber 2 500 5 000 None Drought heat Osmotic Vapor pressure 5 000 10 000 Drought adjustment osmometer Leaf relative Semi analytical 2 000 5 000 Drought water content balance to 3 d p Carbon isotope Mass spectrometer Outsourced at All discrimination of gt 10 per sample leaf tissue grain Spectral Spectral radiometer 5 000 60 000 None All reflectance spectrometer Normalized difference Greenseeker NDVI meter 2 500 5 000 None All vegetation index NDVI Chlorophyll content Chlorophyll meter 200 3 000 None All Crop ground cover Digital camera 150 500 None All Light interception Ceptometer 1 500 None All Leaf area index and Leaf area meter 4 000 9 000 All green area index Gas exchange Infrared gas analyzer 20 000 50 000 None All Chlorophyll fluorescence Chlorophyll fluorometer 2 000 25 000 None All Determination of key None None None for All developmental stages microscopy ID In season biomass None None All Water soluble Sample
15. observers meet to calibrate their readings before starting and regularly thereafter e Ensure that the ratings of new observers are calibrated with those of an experienced observer who is familiar with assessing ground cover so that values are standardized i Cut or break the soil core in half in the horizontal plane to expose a lateral profile e Ensure that only one person makes observations within a replicate ii Count score 0 10 the number of roots that can be seen on each of the two sides Figure 17 4 and take the average of these two values B Figure 17 4 Exposed root content of soil cores for rapid root analysis where brown circles represent the cut view of soil core and yellow marks represent exposed roots A count the number of exposed roots e g 15 in example shown or B use a relative scale 0 10 Direct growth analysis 91 3 Root analysis using a digital scanner Root rc preparation for scanner analysis requires more time than the rapid method but yields more accurate results Figure 17 5 Use computer software e g Delta T SCAN Delta T devices Ltd Cambridge UK or WinRHIZO Regent Instruments Inc Quebec Canada to analyze scans of root samples and give data on the length width and surface area of the roots The preparation of root samples is not difficult but care needs to be taken as there are a number of steps where mistakes can be made Washing
16. partitioning of 20 culms on a large precision balance rather than a small precision balance When weighing samples e Do not weigh hot samples direct from the oven allow time for sample to cool to room temperature before weighing to avoid incorrect readings and or causing damage to the balance Table 22 2 Recommendations for the type of balance and minimum resolution required for the determination of sample weight of various sample types Typical Minimum Sample weight g Type of balance resolution g 2 m plot grain weight FW gt 1000 Industrial retail bench 5 2 m plot biomass FW gt 1000 Industrial retail bench 5 100 culm sub sample FW 500 Large precision 1 100 culm sub sample DW 200 Medium precision 1 Sub sample grain weight 50 Small precision 0 1 Soil moisture of 100 g 30 Small precision 0 1 20 culm stem biomass DW 20 Small precision 0 01 200 grain FW and DW 10 Small precision 0 01 Leaf samples for RWC lt 2 Semi analytical 0 001 Root biomass of 100g lt 2 Semi analytical 0 001 Where FW fresh weight DW dry weight RWC relative water content General recommendations 127 e Do not allow time for samples to absorb moisture e Note that individual container weights may vary after oven drying Once dried samples tend towards slightly Ensure to select a good representative ambient humidity over time this may be from hours TARE container to days depending on the RH and type of sample An alternative to usin
17. the negative grain yield response of dryland wheat to nitrogen fertilizer Biomass grain yield and water use Australian Journal of Agricultural Research 49 1067 1081 Xue GP Mcintyre CL Jenkins CLD Glassop D Van Herwaarden AF and Shorter R 2008 Molecular dissection of variation in carbohydrate metabolism related to water soluble carbohydrate accumulation in stems of wheat Plant Physiology 146 441 454 Chapter 17 Sampling soil for moisture nutrient and root content Marta Lopes J Israel Peraza Olivas and Manuel Lopez Arce Soil sampling provides information on the availability and use of resources i e water and nutrients and the interaction between the plant and the soil i e roots Data on the water and nutrient content of the soil allow estimation of the amount available to the plant and distribution in the soil profile allowing calculation of the amount taken up by the crop the uptake efficiency and an estimation of the use efficiency for biomass and yield production Root data give information on the specific characteristics of crop root systems depth rooting density and distribution These are important considerations when breeding for heat and drought and explaining data interactions with climate and environment variables Root systems are known to be an important component of drought adaptation Dreccer et al 2007 Lopes and Reynolds 2010 While there are many canopy targeted inst
18. viii PAR photosyntheticallly active radiation sensor ix tubes and connectors to the console x leaf within the sensor head and xi chamber fan and B in field use 68 Physiological Breeding II A Field Guide to Wheat Phenotyping Trial measurements 3 Open a new file in the New Measurements mode press 1 and then FT Open LogFile Enter the trial name and press ENTER Define parameters PAR photosynthetically active radiation FLOW CO TEMP RH relative humidity according to the experiment Remember to turn drierite tube to full bypass and check for the corresponding humidity readings Adjust the humidity to the desired value by adjusting the drierite screw observe the humidity changes while adjusting If working with a compressed CO cylinder then keep the CO screw in the full scrub position Match IRGAs once the humidity has stabilized Place the leaf in the sensor head and adjust it is important that the leaf covers the whole area of the chamber or cuvette If this is not possible e g small leaves drought stress etc it is necessary to measure the area of leaf enclosed in the chamber and make adjustments to calculated values Wait until the values are stable usually around 2 minutes and record the value press 1 and then F1 or activate the saturating flash press O and the F3 or F4 to obtain simultaneous chlorophyll fluorescence mea
19. 0 408 MPa Values of OA for wheat typically range from 0 1 to 1 2 MPa using the rehydration method 23 Canopy temperature stomatal conductance and water relation traits It is also possible to estimate turgor potential by the difference between water potential W and osmotic potential Y when water potential is measured in each genotype before re watering Y WU Y Equation 4 3 Table 4 An example of osmometer reading conversion from mmol kg to MPa Osmometer Osmometer Osmotic Osmotic reading mmol kg reading 1 000 mol kg potential MPa potential 10 MPa Non stressed 150 0 15 0 372 0 409 Stressed 300 0 30 0 743 0 817 The osmotic potential OP is corrected OP 10 for the dilution of symplastic sap by apoplastic water assuming 10 apoplastic water Troubleshooting Problem Solution Large error variance in data due to Grow genotypes in a statistical sub block lattice design or in an differences in soil water potential unreplicated design with a common check genotype in each pot Ensure that sample leaves are clean and dry use a paper tissue to clean and dry them Difficulty calibrating the osmometer The laboratory temperature must be stable Check the expiration date of calibration standards If this is due to contamination of the chamber or the thermocouple then run a clean test Clean the thermocouple when contamination level is gt 10 Erratic values from the osmometer Ensu
20. 15 C i e conditions associated with high vapor pressure deficit CT is sensitive to environmental fluxes sites days with low air temperature and or high RH are not suitable for measurement as the low vapor pressure deficit reduces transpiration decreasing the expression of CT 10 Physiological Breeding II A Field Guide to Wheat Phenotyping Time of day For irrigated low water stress treatments take measurements from one hour before to two hours after solar noon typically from 11 00h to 14 00h when the plant is most water stressed For severe stress treatments take measurements in the late morning from two hours before solar noon to solar noon to detect drought adapted genotypes under water scarcity drought adapted genotypes have the ability to recover plant water status during night allowing higher level of transpiration and photosynthetic activity during the morning than non adapted genotypes Plant developmental stage Take measurements at least two times from full ground cover to the end of booting pre heading and at least two times from the end of anthesis to late grain filling grain filling with 5 7 days between each measurement to give a reasonably heritable estimate of trait expression i Pre heading CT measurements can be started when the crop ground cover is sufficient to maximize canopy interception and should be stopped when the spike has become visible in 10 of the population
21. 2 bar per second Values are typically lower for irrigated than for drought trials In extreme water stress large amounts of pressure will be needed to reach the balance pressure gt 40 bar The two leaves typically give slightly different values If the values differ by more than 10 then two further leaves should be sampled Each time that LWP is measured from a drought trial it is useful to include some plots from irrigated trials as a reference non stressed comparison Preparations Connect the pressure chamber and cylinder Figure 3 1 Moisten the gauzes using the squirt water bottle keep inside the chamber to ensure a moist atmosphere and avoid dehydration of the sample during pressurization Trial measurements 1 Choose clean dry healthy leaves receiving sunlight typically the flag leaf after booting 2 Wrap each leaf in turn in the wet gauze and cut close to the leaf sheath to avoid dehydration of the sample Figures 3 2A and B Quickly take the sample to the pressure chamber operator 3 Place the sample s within the rubber seal of the top of the pressure chamber with the cut end protruding slightly Figure 3 2C If the leaf is too wide for the aperture of the rubber seal then the leaf lamina edge s can be carefully pealed back Pressure gauge Gas valve Control VELT Compressed air cylinder E a ae r j i s gt _ oO ae ig SS E Pressure a M chamber i
22. 3346 Kadioglu A and Terzi R 2007 A dehydration avoidance mechanism Leaf rolling The Botanical Review 73 290 302 Maes B Trethowan RM Reynolds MP Ginkel MV and Skovmand B 2001 The influence of glume pubescence on spikelet temperature of wheat under freezing conditions Australian Journal of Plant Physiology 28 141 148 Richards RA Rawson HM and Johnson DA 1986 Glaucousness in wheat Its development and effect on water use efficiency gas exchange and photosynthetic tissue temperatures Functional Plant Biology 13 465 473 Saint Pierre C Trethowan R and Reynolds MP 2010 Stem solidness and its relationship to water soluble carbohydrates association with wheat yield under water deficit Functional Plant Biology 37 166 174 Chapter 20 Observations of in season damage Alistair Pask and Julian Pietragalla In season damage to the crop may occur as a consequence of adverse weather environmental conditions pest and or disease effects In each case it is important to maintain a concise record of damage to the crop in order help explain potentially confounding effects on data Negative consequences on yield depend on the timing of the event and or the organ s affected with effects to the spike typically causing the largest reduction in yield For instance severe and or unusual weather events can cause injury to the plant early frosts on spring wheat may damage only the lower leaves giving
23. 7 The Hue Saturation and Color Range steps are of primary interest 24 Open a new sample image 25 In the Actions palette select HUE SATURATION this will initiate only this component of the DGC action The Hue Saturation window will appear 26 Now slightly adjust the levels of Hue Saturation and Lightness Figure 10 5 to reduce the effect of shades and enhance the greenness of leaves click OK 27 In the Navigator palette zoom in on the sample image to 300 28 In the Actions palette select COLOR RANGE The Color Range window will appear gt Hue Saturation gt Color Range Fill Inverse gt Fill Set Selection ka gt Record Measurements Figure 10 7 Components of the Actions palette 29 In the Color Range window it may be necessary to re select color pixels as previously described step 8 click OK Usually only very small changes in Saturation are required when first setting up the program When making adjustments try to maximize the differences between soil and leaf tissue In particular try to reduce the level of shine from the leaves as the same white color is often also present on shiny soil surfaces To assess the accuracy of the discrimination between soil and leaf tissue view the amount of spot selections in the soil it is often not possible to completely eliminate soil selection entir
24. 75 C lower temperatures Field measurements are required for specific analyses until constant weight at least 48h In the absence of a high capacity dryer biomass grain yield and harvest index HI can be based on field dry weights In this case harvest all samples and leave them for a few days to equilibrate their moisture content with ambient air humidity in order to reduce variation between plots due to differences in maturity date then weigh Oven dry a few sub samples Take the following equipment to the field to determine the overall moisture content Three methods for harvesting are described the choice of method depends on the availability of field time machinery and labor see Table 18 1 Figure 18 1 Sub sampling and grab sampling methodologies allow processing and weighing in laboratory with greater accuracy e Pre labeled paper or textile bags Note that in order to maintain germination potential e Quadrat to give total sample area of 21 m wheat seed must be kept below 12 moisture in a Method C only cool room Drying seed at temperatures gt 40 C and or for long periods of time reduces their viability It is important to avoid adverse treatment of seed which potentially may be used for future trials in which case dry a grain sub sample to determine grain moisture and Plot combine harvester thresher Methods A hence calculate total yield dry weight and B only e Field form and clipboard as require
25. B Average above canopy Do cee frie setor e 2 52 readin i i 1037 g Number of F umo we readings Average 19 its 33058 taken below canopy reading 0 35 1 1 4410 9211 04 S4 Tau Leafarea Beam Leaf Zenith C index fraction distribution angle parameter Figure 11 3 Taking measurements for calculation of light Figure 11 4 Example AccuPAR LP 80 display Adapted from interception of A above canopy PAR B canopy reflectance Decagon Devices 2010 ceptometer turned upside down and C total canopy light interception Table 11 1 Typical output from a ceptometer positioned at the bottom of the canopy with simultaneous above canopy photosynthetically active radiation measurement Where interceptance is calculated as F 1 transmitted incident Beam Leaf area Interceptance Time Plot Sample Transmitted Spread Incident fraction Zenith angle index F 11 30 1 1 49 9 0 44 1848 3 0 64 33 5 6 2 0 9730 11 30 1 2 42 6 1 56 17757 0 64 33 5 6 4 0 9760 11 30 1 3 81 6 1 87 1796 4 0 64 33 5 5 2 0 9546 11 32 2 1 18 5 0 90 1862 3 0 68 33 3 8 0 0 9901 11 33 2 2 25 6 1 15 1859 3 0 68 33 3 7 4 0 9862 11 33 2 3 26 8 2 62 1857 5 0 68 33 3 7 3 0 9856 56 Physiological Breeding II A Field Guide to Wheat Phenotyping The sensors in ceptometers measure PAR however for sensors which measure total solar radiation e g solarimeters PAR is often taken to be 50 of the total solar radiation Monteith 1972 as an approximate average of a dir
26. GS51 At early developmental Stages particular care should be taken to avoid soil targeting when pointing the IRT as the soil temperature is often much higher than that of the crop Additional information and or observations will assist data analysis and may help to explain any anomalies observed For instance record any plots with plants that have visible spikes e g by recording an S for this plot ii Grain filling CT measurements should be taken when plots have passed anthesis and should be stopped when plants have reached late grain filling as senesced tissue will not provide relevant data instead record an M for this plot Measurements should include the spike peduncle and leaf temperature recommended rather than the spike and leaf temperatures separately Figure 1 1 Additional information and or observations will assist data analysis and may help to explain any anomalies observed For instance record any plots with plants that do not have exposed spikes e g by recording an X for this plot Number of samples per plot Take two measurements per plot Procedure The following procedure describes taking measurements using the Sixth Sense LT300 IRT Figure 1 2 Take the following equipment to the field e Hand held IRT e Temperature and humidity pen e Field form and clipboard Advice on taking measurements Always take measurements of the part of the plot which is most exposed to
27. Ifa second leaf is being tested at the same time continue applying pressure until the same occurs on the second leaf close the GAS valve and record the pressure shown on the pressure gauge 10 Move the CONTROL valve into the release exhaust position and slowly release the pressure Troubleshooting Problem Top of the pressure chamber control valve is difficult to open after pressurization Pressure loss through the rubber seal 11 Remove the top of the pressure chamber and remove the leaf leaves 12 Once all measurements have been taken disconnect the pressure chamber from the cylinder Greenhouse measurements Take measurements on greenhouse grown plants when they start to show signs of water stress In the afternoon of the day before taking measurements cover all the sample plants including the pots with individual black plastic bags label each clearly name of the genotype etc to allow measurements to be easily taken on the following day Early morning the next day 5 00h 10 00h sample two leaves from each pot and measure as described above Data and calculations For irrigated trials pre dawn LWP values are typically between 5 bar soil water at field capacity and 10 bar plants not water limited During the day values of lt 10 bar indicate plant water stress limiting physiological processes For drought trials LWP values are 20 to more than 40 bar For samples where the balance pr
28. Observations of in season damage Alistair Pask and Julian Pietragalla General recommendations 120 Chapter 21 General recommendations for good field practice Alistair Pask and Julian Pietragalla 126 Chapter 22 General recommendations for the use of instruments Julian Pietragalla and Alistair Pask 130 Appendix Glossary and abbreviations iV Physiological Breeding II A Field Guide to Wheat Phenotyping Introduction Introduction Matthew Reynolds Alistair Pask and Julian Pietragalla This manual describes the use of diverse phenotyping techniques for applied crop research with an emphasis on the methods commonly used at the International Maize and Wheat Improvement Center CIMMYT The manual provides guidance on the accurate and reliable measurement of physiological traits throughout the wheat crop cycle and follows on from the theory outlined in Volume 1 of Physiological Breeding Interdisciplinary Approaches to Improve Crop Adaptation Section 1 Canopy temperature stomatal conductance and water relations traits These traits are linked by the need of plants to transpire water to fix carbon see Cossani et al Volume 1 Canopy temperature CT and carbon isotope discrimination CID have had widespread application in stress breeding as they readily integrate the effects of many plants within a crop canopy and hence reduce errors associated with plant to plant and leaf to leaf variation Cooler CT is positively as
29. RWC at wilting is around 60 70 Troubleshooting Problem Solution Lower than expected fresh weight values The transfer of cut leaf samples to the sample tube is too slow causing the leaves to dehydrate in the air Leaf sampling should be achieved as quickly and efficiently as possible and use the shade of the sampler s body when cutting and holding the samples Higher than expected turgid weight values Blot drying of leaf samples after soaking is not sufficient ensure to dry samples thoroughly of all surface moisture using dry absorbent tissue Do not completely fill the tubes with water as this will over estimate the turgid weight by filling the inter cellular spaces with water References Useful references Barrs HD and Weatherley PE 1962 A re examination of the Hewlett JD and Kramer PJ 1963 The measurement of water relative turgidity technique for estimating water deficit in leaves deficits in broadleaf plants Protoplasma 57 381 391 Australian Journal of Biological Sciences 15 413 428 Smart RE and Bingham GE 1974 Rapid estimates of relative water Stocker O 1929 Das Wasserdefizit von Gef sspflanzen in content Plant Physiology 53 258 260 verschiedenen Klimazonen Planta 7 382 387 Turner NC and Jones MM 1980 Turgor maintenance by osmotic adjustment a review and evaluation In Turner NC and Kramer PJ Eds Adaptation of plants to water and high temperature stress pp 87 103
30. Weatherley PE 1950 Studies in the water relations of the cotton plant The field measurement of water deficits in leaves New Phytologist 49 81 97 Canopy temperature stomatal conductance and water relation traits 27 Chapter 6 Carbon isotope discrimination Marta Lopes and Daniel Mullan Carbon isotope discrimination CID AC provides an integrative measurement of stomatal conductance Farquhar et al 1989 Wheat a C3 plant discriminates A against the heavier stable carbon isotope 1 C in favor of the lighter 7C and more abundant form 99 during photosynthetic carbon dioxide fixation due to a combination of diffusion effects through the stomata and enzymatic Rubisco preference This discrimination is positively related to carbon dioxide levels in the intercellular air spaces of the leaf and given a constant leaf to air vapor pressure difference is also positively related to water uptake WU i e availability and xylem conductivity and negatively related to transpiration efficiency TE Greater overall stomatal aperture allows increased rates of leaf gas exchange allowing the plant to favor C but with higher water losses Condon et al 1990 When CID is measured in plant dry matter it provides an integrated indication of TE for the period of growth of the measured organ CID has been used as a screening tool for identifying variations in water use efficiency WUE in wheat and the development of wheat v
31. a light curve before beginning any gas exchange measurements to determine the saturation point Wheat is usually grown under high radiation environments and shows saturating photosynthesis rate below 1500 umol m s The following procedure describes the measurements using a LICOR LI 6400 XT gas exchange photosynthesis system Figures 13 4 and 13 5 The most used gas exchange parameters are A o Leaf fan set to fast net CO assimilation rate Amax light saturated net CO assimilation rate g stomatal conductance C intercellular CO concentration and E transpiration rate o Stomata ratio Set to 1 if the stomatal ratio is unknown or determine the actual stomata ratio although this is time consuming e tis strongly recommended to use a compressed CO Take the following equipment to the field cylinder to reduce any problems associated with slight e Gas exchange photosynthesis system fluctuations in the concentration of incoming CO e Avoid condensation inside the cuvette or tubes as e Battery use a car battery for long periods of field a humidity can damage the instrument measurements e Itis strongly recommended to perform an A PAR Advice on taking measurements curve before beginning gas exchange measurements e It is important to perform all measurements with the to determine the photosynthetic active radiation same setting parameters and as close as possible PAR intensity inside the chamber in order
32. and not shaded For instruments that are NOT capable of simultaneously measuring above and below the canopy take three measurements of above canopy PAR using the probe by holding it above the canopy in the same place and orientation as the below canopy measurements Figure 11 3 position A For canopy reflectance take three measurements using the probe by holding it inverted above the canopy in the same place and orientation as the below canopy measurements Figure 11 3 position B then proceed to take the below canopy measurements Figure 11 3 position C Note that reflectance is very small or negligible when the canopy has sufficiently high LAI or when the reflectance of the ground is similar to that of the canopy Figure 11 2 Positioning the ceptometer correctly within the crop to take representative measurements A in a system of two raised beds each with two rows of plants and B flat broadcast planting with seven rows of plants Figure 11 1 Taking light interception measurements with a hand held ceptometer A measurements below the spike B the Decagon AccuPAR LP 80 ceptometer and C measurements at GS31 also showing the external sensor being held for simultaneous above canopy measurements Photosynthesis and light interception 55 Preparations When at least one or both of the above and below canopy measurements have been taken the other relevant data are displayed at the botto
33. and sampling protocols for growth analysis are outlined in this section This includes accurate determination of yield and its components as these express the net effect of many of the traits described and demonstrate trait interactions with environmental conditions Growth analysis should include estimation of water soluble carbohydrates in the stem which are the principal source of reserve or stored carbohydrates and especially important for grain filling under stress conditions Also presented are root and soil sampling approaches important for understanding plant water relations see Herrera et al Volume 1 This section also addresses the determination of key developmental stages which is a pre requisite for the correct interpretation of physiological data see Slafer Volume 1 Section 5 Crop observations Several anatomical and morphological traits have been associated with genetic gains in yield including erect leaves under high yield conditions Fischer 2007 wax and pubescence under abiotic stress Reynolds et al 2009 and long peduncles under drought Acevedo et al 1991 The advantage of all of these traits is that they can be measured rapidly or assessed by eye In fact visual scales can be used for almost any anatomical or morphological trait for example to assess the effects of lodging frost or hail damage Section 6 General recommendations This section is intended to provide selected tips to increase
34. and stress tolerance All these characteristics are highly heritable and typically demonstrate a large genetic variability with low environmental interaction Measurable traits include flag leaf length and width peduncle and awn lengths plant height and stem solidness The area of light intercepting surfaces and canopy architecture provides information with respect to light distribution within the canopy light penetration light use efficiency and photosynthetic potential Plant height and stem solidness both relate to harvest index HI and lodging risk and to the storage capacity of the plant They are therefore useful to breeders for rapid screening within large populations Individual traits are discussed in more detail below Easily observable traits include leaf and or spike pubescence hairiness leaf and or spike glaucousness waxiness leaf rolling leaf angle leaf orientation and leaf posture These adaptive traits confer advantages to plants under heat and or water stress conditions by providing photo protection and reducing transpiration from the canopy All these traits reduce thermal load on the canopy by either increasing the amount of reflectance of incident radiation pubescence and glaucousness or by reducing the area of exposed lamina leaf rolling angle orientation and posture Pubescence also traps a border layer of air around the leaf whilst leaf rolling traps air within the leaf both processes function to r
35. be held at a diagonal angle for row planting High error variance of data This may be due to variable or sub optimal environmental conditions e g overcast or hazy skies or variable canopy establishment References Decagon Devices 2010 Available at http www decagon com accessed 7 January 2012 Monteith JL 1972 Solar radiation and productivity in tropical ecosystems Journal of Applied Ecology 9 747 766 Useful references Monsi M and Saeki T 1953 Uber der lichtfator in den pflanzengesellschaften und seine bedeutung fur die stoffproduktion Japanese Journal of Botany 14 22 52 Monteith JL 1994 Validity of the correlation between intercepted radiation and biomass Agricultural and Forest Meteorology 68 213 220 Reynolds MP van Ginkel M and Ribaut JM 2000 Avenues for genetic modification of radiation use efficiency in wheat Journal of Experimental Botany 51 459 473 Photosynthesis and light interception 57 Chapter 12 Leaf area green crop area and senescence Alistair Pask and Julian Pietragalla The area of the leaf lamina or all green surfaces leaf lamina leaf sheath stem and spike of the crop relates to the light interception and photosynthetic potential the surfaces for transpiration water loss and the above ground biomass of the crop The leaf area index LAI is the area of green leaf lamina surface per unit of ground area and the green area index GAI is the are
36. be taken per plot and compared to ensure incorrect values errors and instrument malfunction are quickly spotted and the values discarded Repeat measurements as necessary e g where readings differ by gt 10 Do not forget to take a field trial map and individually label each plot to help orientate the observer scientist Ensure to complete a field form for each sampling event Do not start without being familiar with the methodology pre preparing equipment allowing sufficient in field time and organizing the laboratory in advance to facilitate uninterrupted processing e g leaf water potential observers start and end times environmental observations e g air temperature relative humidity etc any relevant observations e g wind crop condition etc Figure 21 4 Canopy Temperature Field Form mamam Pasy SSS Date 20 March200 Times Environment Irrigated Airtemperature 32 8 Phenological stage Vegetative Relative humidity Scents JP ndMR oo Observations Very dight wind IRT needs new batteries Figure 21 4 Sample field form for canopy temperature measurement Form enables effective and manageable recording of data and important crop site and environmental information is also recorded 123 General recommendations 5 Recording crop site and environmental information Data and observations on the crop site and environment throughout the experimental cycle and during measur
37. by standing ata 45 angle alongside the plot but a close inspection of several individual culms is recommended As observations are subjective it is important that ratings are consistent e Ensure that the ratings of new observers are calibrated with those of an experienced observer who is familiar with making assessments so that values are standardized Crop observations 113 e If several people within the group will be making observations it is recommended that all observers meet to calibrate their readings before starting and regularly thereafter e Ensure that only one person makes observations within a replicate It is often useful to take a photographic record of the damage for later reference and calibration purposes Trial measurements Spike tipping Spike tipping appears as a premature senescence of the spike caused by induced sterility of spikelets evident by 0 2 4 6 0 20 40 60 114 Physiological Breeding II A Field Guide to Wheat Phenotyping a desiccated yellowish tip The effect typically starts at the tip and progresses towards the base of the spike Figure 20 1 Scoring 8 80 Observations should be made around mid grain filling for stress effects or started within a few days of an exceptional event e g frost Rate the proportion of spikes within the plot which are affected by tipping using a scale from 0 0 to 10 100 in increments of 10 Rate the pr
38. calculate the percentage ground cover GC for the photograph using the equation GC Mean Grey Value 255 x 100 Equation 10 1 Default actions Digital Ground Cover Select this from the drop down menu Enter the location of the folder containing the digital ground cover images here Select Select ONLY the Suppress File Open Options Dialogs and Suppress Color Profile Warnings options Folder Choose Destination Select this from the drop down menu Enter the location of the empty folder i e C Program Files Adobe Adobe_Photoshop_CS3 Select File Naming Starting serial 1 Select Stop For Errors from the drop down menu Errors Select the Override Action Save As Commands toggle Leave this as default Document Name extension 52 Physiological Breeding II A Field Guide to Wheat Phenotyping Worked example Figure 10 9 below shows a photograph taken in the field for DGC and the corresponding processed image Mean grey value 24 9 ground cover 24 9 255 x 100 9 76 Figure 10 9 A worked example from A digital photograph of a plot jpg to B a processed image for DGC Troubleshooting Problem Solution There is variability in the scale of the pictures The use of a calibrated support of known fixed height to standardize the photograph scale The photographs are over exposed too much light Take photos at early hours during the morning or late
39. carbohydrate storage capacity and HI A taller stem can make combine harvesting easier although may also increase lodging and reduce HI whilst a shorter stem may reduce carbohydrate storage capacity and make combine harvesting difficult Heights typically range from lt 50 cm dwarf 50 70 cm short 70 120 cm semi dwarf and gt 120 cm tall Figure 19 1D Measurement e Measure the length of individual culms from the soil surface to the tip of the spike record to the nearest centimeter e Do not to include the awns in your measurement e Ensure that the ruler is flat on the soil surface avoiding any mounds or cracks in the soil Stem solidness Most wheat varieties have hollow stems no internal pith whilst some have stems which are partly or entirely filled with pith solidness the pith consists of undifferentiated parenchymatous cells This pith has been shown to be a store of soluble carbohydrates as stem reserves for grain filling see this volume Chapter 16 Stem solidness is known to confer resistance to wheat stem sawfly Cephus cinctus Norton Eckroth and McNeal 1953 The level of expression of stem solidness is highly heritable but is affected by the environment and stems tend to be more solid when plants are exposed to high temperature or drought during stem elongation Figures 19 2 and 19 3 Measurement e Take measurements at 7 14 days after anthesis e Measure at the mid point of the i
40. case and to the equipment store room It is important that each instrument is stored clean dry dust free and in the correct protective case Do not discard malfunctioning instruments when anomalies or problems have been noted during equipment use Repairs and or recalibrations to instruments may be required which may involve returning the instrument to the factory specialist This could take weeks or months Set dryer temperature and time depending on the type of sample estimated moisture content and capacity of the dryer Table 22 1 General drying temperatures and times for dry weight determination Note that drying time may differ according oven drying capacity Material Temperature C Time hours Relative leaf water content 60 75 24 Grain moisture 60 75 24 48 Biomass maturity 60 75 48 Root biomass 60 75 48 Biomass emergence to grain filling 60 75 48 72 Soil moisture gravimetric 105 48 Note that seed which potentially may be used for future trials should not be oven dried as drying seed at temperatures gt 40 C and or for long periods of time reduces their viability Notes for drying samples for nutrient and or metabolite analysis e Dry biomass samples at 60 75 C for N P K and water soluble carbohydrate determination e High drying temperatures gt 90 C for long periods may affect the nutrient content Some specific metabolites analyses e g enzymes proteins etc require freeze drying of sampl
41. conditions it is related to the capacity to extract water from deeper soil profiles and or agronomic water use efficiency WUE ii under irrigated conditions it may indicate photosynthetic capacity sink strength and or vascular Capacity depending on the genetic background environment and developmental stage and iii under heat stress conditions is related to vascular capacity cooling mechanism and heat adaptation CT is an integrative measurement i e scoring the entire canopy of many plants within a plot and so has advantages over other methods used for stress detection such as stomatal conductance and water potential because it integrates a larger area of plant crop measurement is non destructive does not interfere with stomata which are sensitive and is faster and not laborious However trait expression shows interaction with both developmental phase and time of day e g pre heading and or morning readings are usually lower due to lower incident solar radiation and air temperature which can be used to relate different canopy traits and stress tolerances Site and environmental conditions Measurements must be taken when the sky is clear and there is little or no wind It is important that the plant surfaces are dry and not wet from dew irrigation or rain Studies at CIMMYT have shown that CT is best expressed on warm sunny cloudless days with low relative humidity RH lt 60 and warm air temperature above
42. d p e Distilled water e Blotting paper e Oven Advice on taking measurements Select the top most fully expanded leaf receiving sunlight typically the flag leaf or select leaves down the canopy profile Leaf sampling should be achieved as quickly and efficiently as possible and use the shade of the sampler s body when cutting and holding the samples A field assistant is often useful All weights should be recorded to the nearest milligram 3 d p Preparations 1 Number and weigh empty sample tubes tubeW Figure 5 1A Field measurements 2 Select and cut six fully expanded flag leaves from randomly chosen plants in each plot Figure 5 1B 3 Cut off the top and bottom of all the leaves together and any dead or dying tissue Figure 5 1C Canopy temperature stomatal conductance and water relation traits 25 to leave a 5 cm mid section and immediately place 9 Take the leaf samples out of the tube and quickly into the pre weighed tubes and seal the lid so that and carefully blot dry with paper towel Figure 5 1F there is no moisture loss gain from the system 10 Weigh the leaf sample TW turgid weight 4 Immediately place the tube into a cooled insulated 11 Place the leaf samples in a labeled envelope and dry container at around 10 C 15 C but not frozen at 70 C for 24h or until constant mass Figure 5 1G 5 Take the tubes to the laboratory as soon as possible 45 Reweigh the leaf sampl
43. differ with species cultivars and even between different organs of the same plant They are also influenced by the water deficit level the rate of water deficit development and environmental conditions Future research is necessary to understand more precisely the nature and control of physiological processes associated with OA The ideal method should quantify solute accumulation in response to water deficit independently of solute concentration due to water loss This chapter details the rehydration method i e OP of plants that have been rehydrated as the fastest and most economical method with potential use for screening for performance under field conditions Babu et al 1999 Moinuddin et al 2005 Other methods for estimating OA are proposed see Babu et al 1999 e Regressions of relative water content RWC with OP e OPof stressed plants extrapolated to the rehydrated state and e Sustained RWC at a given OP close to wilting Site and environmental conditions Plants are grown in containers under controlled environment in a glasshouse Evaluation of field grown plants can be performed but is not recommended as results may be affected by genotypic differences in root depth which confound the level of stress expressed by the plants see recommended adaptations to procedure below Time of day Two samples are taken over two days the first on day 1 leaf water potential LWP is taken before daw
44. fluorometers which are able to generate saturating pulse over 4 000 umol m st Figure 13 3 A self made dark adaptation leaf clip using aluminum foil Figure 13 2 Hand held chlorophyll fluorometer Fluorpen FP 100 A showing i PAR sensor and ii sample leaf in sensor chamber B field measurements in daylight conditions Photosynthesis and light interception 65 e It is highly recommended to use an instrument which provides far red pre illumination for dark adaptation measurements for a rapid transfer of electrons to PS allowing the rapid re oxidation of PS II e Ensure that the measuring light is not actinic i e not light which stimulates photosynthesis Preparations Ensure that batteries are fully charged and there is sufficient memory to record measurements If required pre program the instrument with measurement parameters protocol and settings according to the user manual e g for light adapted protocol mode set the intensity duration frequency and gain of the measuring actinic saturating and far red lights Use the Settings sub menu to set the light color light intensity number and frequency of measurements date time and sound mode Trial measurements 1 Hold the SET key for 1 second to turn the fluorometer on then allow the instrument to equilibrate with the ambient temperature for around 10 minutes 2 Select the MEASURE menu and press the SET key once
45. from the top or spike to avoid selection bias e g for chlorophyll content or ii select areas by placing quadrats or choosing rows at random e g for in season biomass Do select samples systematically i select culms or plants by counting to a given predetermined position e g every 10 stem or ii select areas ata predetermined distance into the field or plot Do use sub sampling and grab sampling where it is not possible to measure a whole quadrat sample e g due to constraints in time or labor and to reduce space resource requirements Figure 21 3 Is least 50 cm A B Plot border a Sample area Shy selected sample stems plants throughout the sampling process The following points should be taken into consideration Do not sample from the borders of plot i e the outer row s and ends typically gt 50 cm of the plot as these will show unrepresentative growth When sampling repeatedly though the season do ensure to leave suitable buffers between samplings Figures 21 1 and 21 2 Do not sample from unrepresentative parts of the plot e g areas of poor establishment and or distinctly poor good growth These areas should be marked during early growth to aid identification during later developmental stages of the crop Do not choose culms plants or areas for sampling which are unrepresentative of the field or plot In general avoid visual selection of samples unless the sample
46. high plant population due to the plasticity of yield components and vice versa In the laboratory 1 Take a random sample of whole grains clean carefully to remove all broken and aborted grains and chaff but do not discard small grains 2 Count grain by hand or using a seed counting machine see Figure 18 1F 3 Either count 200 grains re dry and weigh DW_200 G TGW DW_200 Gx5 Equation 18 1 Or re dry weigh 10 g and count number of grains DW10g grains TGW 10 DW10g grains x 1000 Equation 18 2 In each case two samples per plot should be taken If the values differ by more than 10 then a third sample should be taken Measurements calculated from harvest data Determination of the biomass and grain moisture content for use in calculation of total dry weights Values of moisture content MC of green tissue biomass samples to mid grain filling are typically 70 80 this decreases to lt 20 at harvest Decreases in grain moisture content arise first through filling 102 Physiological Breeding II A Field Guide to Wheat Phenotyping with dry matter 70 to 45 GS73 77 grains stop accumulating dry matter when their moisture content falls below 45 after which they continue to lose water to 20 at physiological maturity Grain moisture content is typically between 5 15 at harvest depending on the environment In the field and laboratory 1 Take a field sub sample and weigh FW_SS 2
47. i e the above ground stem of the wheat plant can be partitioned into its component organs shown in Figure 23 1 Where A Awns B Spike C Peduncle D Flag leaf E Leaf sheath F Node G Internode H Stem Lower leaves J Crown K Roots Long slender extension of the lemma creating course hair like protrusions of the spike Which forms at the top of the culm contains the florets seeds within the spikelets see details of spike partitioning also called ear or head Uppermost internode of the stem between upper internode and spike collar Uppermost leaf lamina of a spike bearing culm the flattened portion of a leaf above the sheath with the upper adaxial and lower abaxial surfaces The lower part of a leaf wraps around and encloses the stem A small auricle exists at the point where the leaf sheath meets the leaf lamina A region on the stem where leaves are attached also called a joint The part of a stem between two nodes The pseudo stem of the culm also called the true stem Produced during late seedling development From where the tillers originate Consisting of seminal and nodal or crown roots The seminal roots form from the seed and typically grow to depths of up to 120 spring to 200 winter cm The nodal roots form from the lower nodes are associated with tillers in the upper lt 60 cm soil layers Appendix 131 The spi
48. in the afternoon when the solar radiation is lower Adjust the camera settings to account for the available light Some leaves within the photograph are shaded Care should be taken to avoid shadows and are difficult to select for green pixels Useful references Mullan DJ and Reynolds MP 2010 Quantifying genetic effects of ground cover on soil water evaporation using digital imaging Functional Plant Biology 37 703 712 Photosynthesis and light interception 53 C Chapter 11 Light interception Daniel Mullan and Julian Pietragalla Light solar radiation provides the energy to drive photosynthesis Of the light spectrum the range that can be used by plants for photosynthesis are wavelengths between 400 nm blue and 700 nm red and is termed photosynthetically active radiation PAR The amount of light within the crop canopy can be measured with a ceptometer a long thin probe with up to 80 PAR sensors along its length from which the amount of PAR intercepted by the crop can be estimated As light passes through the canopy it is absorbed or reflected and the remaining light is transmitted to the lower leaves Therefore at a particular moment the fraction of incident light radiation intercepted F depends on the green area index GAI i e the area of the crop green surfaces per area of ground and how the leaves are geometrically arranged in the canopy K canopy coefficient For cereal crops the
49. inserted How should 3D components e g stems and spikes be measured Leaves are rolling making it difficult to measure the area Useful references Solution Ensure that the transparent belt is clean of dirt marks and that the instrument is correctly calibrated Take the planar flat area of each component Cool and moisten samples e g place leaves between moistened sheets of paper for 3 4 hours Br da NJJ 2003 Ground based measurements of leaf area index Sylvester Bradley R Berry P Blake J Kindred D Spink J a review of methods instruments and current controversies Bingham I McVittie J and Foulkes J 2008 The Wheat Journal of Experimental Botany 54 2403 2417 Growth Guide Pp 30 Home Grown Cereals Authority 2nd Scott RK Foulkes MJ and Sylvester Bradley R 1994 Exploitation Edition HGCA London Available at http www hgca com of varieties for UK cereal production matching varieties to accessed 6 January 2011 growing conditions Chapter 3 pp 1 28 Home Grown Cereals Authority 1994 Conference on cereals R amp D HGCA London UK 62 Physiological Breeding II A Field Guide to Wheat Phenotyping Chapter 13 Gas exchange and chlorophyll fluorescence Gemma Molero and Marta Lopes With recent advances in the development of field portable instruments measurements of gas exchange and chlorophyll fluorescence have become increasingly valuable in precision pheno
50. into account when deciding its position above the crop and distance between the foreoptic and the canopy Generally the sensor foreoptic must be centered above a crop row for most of the vegetation pigment and water indices However for RUE determination the field of view of the sensor must include both rows and the gaps between rows to take into account the radiation interception for the entire canopy area Two ways to increase the measurement area are to i open the field of view angle and or ii increase the distance to the target The two main settings of the spectrometer are i integration exposure time i e the amount of time for which the sensor is open and registering radiation intensity This setting may differ depending on the hardware configuration e g foreoptic filters grating diffuser correctors etc and quality of sunlight or light source used and ii the number of readings averaged Spectral reflectance indices and pigment measurement 33 per data point i e the number of spectrums read and averaged to produce a single data point A higher number of readings per average reduces the data variation but increases the processing time to a maximum of 10 Important considerations which may influence the reflectance of electromagnetic radiation from a canopy e Canopy structure and morphology reflectance is affected by canopy architecture e g erectophile and planophile types glaucousne
51. less robust than infrared thermometry measurements are much slower 2 Physiological Breeding II A Field Guide to Wheat Phenotyping than CT being made on repeated individual leaves and direct contact is required to take measurements which may affect the extremely sensitive stomata Results do give repeatable real time measurements of stomatal performance in the field without the need for destructive or laboratory processing Leaf water potential LWP takes even longer to measure than stomatal conductance and is therefore not a high throughput technique however when measured during the day it provides a definitive measure of leaf water energy status and when measured pre dawn it estimates the soil water potential of the active root zone of a genotype Therefore LWP is a powerful and precise tool for estimating crop and soil water energy status and although laborious it can provide useful reference data on at least a few plots such as check rows Relative leaf water content is an alternative to estimating hydration status that does not require specialized instrumentation nor involve significant sampling costs but tends to be less precise probably because several weighing steps are involved Osmotic adjustment OA is not straightforward to measure because root zone water potential must be controlled for standardization purposes which largely precludes field screens where root depth would confound expression of OA The value
52. lines in favorable environments over a 30 year period reflects proportional increases in leaf conductance Types of leaf porometers available e Steady State e g Decagon SC 1 Figure 2 1 PP Systems PMR 5 an effectively open chamber is clamped to the leaf surface and water vapor released through the stomata sets up a RH gradient along the chamber The instrument monitors RH at two points along the flux path and once the flux gradient reaches a steady state it calculates and displays the leaf diffusion conductance the reciprocal of resistance A leaf with a rapidly changing gradient indicates that the stomata are relatively open e Dynamic Diffusion e g Delta T Devices AP4 measures the rate of RH increase in a chamber clamped to the leaf surface as water vapor is released through the stomata this causes the chamber RH to rise A relatively rapid rise in RH indicates that the stomata are relatively open e Viscous or Mass Flow e g Thermoline measures the time in 1 100 of a second to force a fixed volume of pressurized air through the leaf This gives a measure of resistance to mass flow which is inversely proportional and linearly related to conductance A relatively rapid drop in pressure or a fast flow rate means the resistances are relatively small e Null Balance e g LICOR LI 1600 measures the vapor flux and vapor gradient near the leaf surface by calculating the flow rate needed to keep
53. measurements at regular intervals from emergence until full cover at approximately 10 20 and 30 days after emergence depending on the environment in which the trial is sown or take measurements when the average ground cover is approximately 20 50 and 80 46 Physiological Breeding II A Field Guide to Wheat Phenotyping Number of samples per plot Take one assessment photograph for small plots e g with one raised bed lt 2 0 m long or if there is poor germination take two assessments photographs per bed throughout the trial Take two assessments photographs for large plots e g plots with two raised beds lt 3 5 m long or if there is poor germination take three assessments photographs per bed throughout the trial In each case ensure that the ground cover across the plot is accurately represented Procedure Take the following equipment to the field e Digital camera e Spare batteries e Field form and clipboard for visual assessment Advice on taking measurements Schedule measurements carefully stop taking measurements when the first plot reaches maximum ground cover even if three replications have not been taken as cultivars are being compared relative to each other Measurements taken after this time will lead to misinterpretation of results In planning measurements note that each visual assessment may take up to 10 seconds and digital photographs may be taken at a rate of 1 per 5 seconds mean
54. most susceptible to site variation Plot size each plot should contain sufficient crop material to provide the maximum degree of accuracy of data by reducing the variation due to uncontrolled variables and border effects so that it can be treated independently of its neighbors e g water fertilizer and or pesticide applications and harvesting techniques Too small plots will increase inter plot variation however the optimum plot size requires field experience and scientific judgment Analysis and interpretation data are assessed for i significant and consistent expression of the trait of interest and ii an association of the trait with performance among genotypes Interpretation of association between traits and performance may be confounded by other genetic factors such as differences in phenology plant type etc in non homozygous populations 120 Physiological Breeding II A Field Guide to Wheat Phenotyping 2 Sampling and sample selection For an unbiased and representative selection of culms plants and or areas within a crop it is important to maintain a uniform selection criterion for plant materials Do choose a sample size which provides the maximum degree of accuracy of data Consider the number of replicates variable studied variability between plots degree of accuracy desired experimental design and resources available Do select samples randomly i select culms or plants from the base and not
55. not affected by height variance check manufacturer recommendations Differences in plant canopy height between genotypes from emergence to the initiation of stem elongation are inconsequential however after heading plant canopy height may differ between genotypes and it may be necessary to adjust the height of the sensor head between plots in order to maintain a constant distance between the sensor head and the crop canopy A weighted string attached behind the sensor head helps the operator to maintain a constant distance between the sensor head and crop canopy see Figure 8 1C Walk at a steady speed typically 1 ms Most field portable NDVI sensors take a constant number of measurements per second while the trigger is held and then provide an average these data Walk up and down _ Plot border _ Sample area Area measured Figure 8 2 A field map and direction of measurement the order of sampled plot is 1 20 21 40 41 42 39 22 and B area sampled within each plot by passing the sensor above central crop rows and excluding plot borders 38 Physiological Breeding II A Field Guide to Wheat Phenotyping the rows regardless of the experimental design as it is generally easier to rearrange the data in the office than to follow the plot number in the field see Figure 8 2 It is necessary to control for phenology in populations with diverse anthesis dates as plants under different stages
56. of WSC concentration from randomly selected fertile main culms alternatively culms can be selected from the in season biomass samples taken at anthesis 7days see this volume Chapter 15 See Schematic 16 1 Take the following equipment to the field e Pre labeled paper bags e Secateurs knife Advice on taking measurements Collect the stem samples in paper bags which have adequate ventilation to allow uniform drying e g with holes punched in the bag It is important that samples are kept cool and processed and dried as quickly as possible to reduce respiratory losses of carbohydrates typically within 2 hours of cutting Direct growth analysis 83 Sampling for WSC is often combined with in season biomass sampling and partitioning see this volume Chapter 15 Ensure to plan sampling approach carefully to allow for maximal data collection economy of sampling e g data on partitioning weights can be collected on the same 20 culm sample The leaf lamina and or leaf sheath may also be analyzed for WSC separately or not removed from the stem for whole stem analysis Preparations 1 Prepare labeled paper bags for oven drying use medium sized bags with holes punch in them to increase oven drying efficiency use a hole punch and ensure you have a similar hole pattern in each bag Field measurements 2 Randomly select 20 fertile main culms from each plot ensuring that all culms have a well formed spike
57. of carbon for grain filling as grain demand frequently exceeds current assimilation potentially contributing 10 20 of the grain yield under favorable conditions In particular this trait has been shown to be adaptive for drought heat and or disease stress tolerance when the supply of carbohydrates from photosynthesis during grain filling is inhibited limited and stored WSC may contribute up to 50 of the grain yield For instance under terminal drought stress e g in Australian environments where deep soil water is not available WSC have been shown to buffer biomass production grain yield and harvest index HI associated with increased water uptake WU and water use efficiency WUE Trait based breeding for genotypes with greater stem storage and remobilization of WSC may result in improved grain filling and increased yields Accumulation of WSC is a function of genetic characteristics specifically the stem s storage capacity as well as environment which will influence the former as well as the subsequent availability of assimilates for storage The total amount of WSC may be 40 or more of the total stem dry mass when WSC levels peak in early grain filling Kiniry 1993 Reynolds et al 2009 WSC storage may show trade off with investment in other sinks such as deeper root growth Lopes and Reynolds 2010 tiller survival or developing spikes The major proportion of WSC are located in the peduncle and penultimate interno
58. of development have different architectures and present differences in the source sink relationships and these may confound the analysis This can be corrected by splitting the population into early and late lines and therefore making different populations to be screened A range of up to 10 days in anthesis date is quite reasonable Preparations Ensure that the sensor unit and palmtop computer PDA batteries are fully charged this typically requires gt 6 hours Check connections between the Greenseeker sensor head battery unit telescoping tube and PDA Check the sensor head angle in relation to the ground this should be horizontal and check the distance from sensor head to the canopy by adjusting the sensor head angle mount and the telescoping pole Adjust the shoulder straps to give a good balanced weight distribution of the instrument for comfortable working 1 After turning on the Greenseeker unit and PDA allow the instruments to equilibrate with the ambient temperature for around 10 minutes Go to START gt PROGRAMS and run NTECH CAPTURE software Then go to SENSOR gt START GREENSEEKER Select LOGGING PLOTS mode the display will show three cells i SAMPLE NO shows the number of measured plots ii NDVI shows NDVI value of the last plot iii AVG NDVI shows the average NDVI value of all previously recorded plots The sensor is now ready Trial measurements
59. of the crop to intercept solar radiation and consequently slows photosynthesis and or radiation use efficiency Decreased biomass production also decreases the amount of photosynthates as WSC available to be remobilized during grain filling Identifying genotypes which are able to maintain biomass production during stress conditions is an important means of identifying better adapted lines Site and environmental conditions Samples can be taken under most environmental conditions However it is important that the plant surfaces are not wet from dew irrigation or rain Time of day Samples can be taken at any time of the day although where possible samples should be taken in the morning to allow for same day processing Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from the start of tillering to physiological maturity depending on the experimental objectives timing of peak stress Sampling is typically performed at sequential developmental stages time intervals through crop growth The most important stages are start of stem elongation GS30 31 start of booting GS41 anthesis 7days GS61 7d mid grain filling GS75 and physiological maturity GS87 For time interval sampling use a defined number of days after emergence e g 20 40 60 DAE until the developmental stage becomes more apparent At early developmental stages up to the first node at 1 cm ab
60. of the trait is in resisting dehydration of cells and tissues as it permits water retention in spite of otherwise unfavorable water potential gradients It has been implicated in maintaining root growth under drought Morgan and Condon 1986 Section 2 Spectral reflectance indices and pigment measurement Spectral reflectance SR techniques using both visible and infrared IR wavelengths are quick and easy to apply in the field and do not require destructive sampling so plots can be smaller lowering costs and measurements can be repeated many times on the same crop area Many SR indices can be calculated for a range of crop characteristics vegetative pigment and water content which show genetic diversity see Mullan Volume 1 Those which have shown the most reliable association with crop performance are the water indices and to a lesser extent vegetative indices Babar et al 2006 Guttierrez Rodriguez et al 2010 The advantage of the latter is that a dedicated sensor exists the GreenSeeker Hand Held Sensor Unit 2002 Ntech Industries Inc Ukiah CA USA facilitating high throughput field screening this is still in development for the water indices ML Stone Oklahoma State University personal communication Nonetheless a single measurement with a radiometer can provide information on many potentially useful traits making it a useful investment The main disadvantage of radiometry measurements is tha
61. order to identify the date of the anthesis for such trials it may be necessary to open the flag leaf sheath to reveal the spike or to determine the date of anthesis retrospectively from the length of the developing grain It is more meaningful to use thermal time as temperature drives the rate of development Useful references Stapper M 2007 Crop Monitoring and Zadoks Growth Stages for Wheat CSIRO Plant Industry Canberra ACT Available at http www biologicagfood com au wheat management crop monitoring and zadoks growth stages accessed 10 January 2012 Sylvester Bradley R Berry P Blake J Kindred D Spink J Bingham l McVittie J and Foulkes J 2008 The Wheat Growth Guide Pp 30 Home Grown Cereals Authority 2nd Edition HGCA London Available at http www hgca com accessed 6 January 2011 Direct growth analysis 71 Chapter 15 In season biomass Julian Pietragalla Debra Mullan and Eugenio Perez Dorame Biomass sampling provides information on crop growth and rate of growth organ size leaf area and dry mass partitioning between canopy components for the calculation of radiation use efficiency and is also a starting point for morphology measurements and nutrients or metabolite analysis e g N P protein water soluble carbohydrates WSC etc Adverse environmental conditions such as drought and heat stress can greatly reduce biomass production which in turn reduces the ability
62. reduction Rht photoperiod Ppd or growth habit Vrn unless under study and vii low variation in factors which may confound analysis e g height Number of lines start with a broad range of genetic diversity for the trait in preliminary observations In subsequent cycles numbers can be reduced drastically to lines that encompass the full range of genetic diversity for detailed observations Number and type of plots is set by the number of genotypes treatments and replicates being tested according to the objectives of the experiment Replicated statistical designs are used for detailed phenotyping e g lattice design or unreplicated designs with repeated checks e g local checks are used for rapid screening of large populations Also include buffer plots around the trial to absorb external effects Experimental establishment it is important to have consistent establishment across a field experiment to reduce inter plot variation This includes consistent agronomy e g depth of sowing seed quality water availability pest and disease control avoiding neighbor effects e g shade from trees and buildings considering gradients e g block treatments along slopes row orientation i e typically in a N S direction to minimize inter plot shading especially when the sun angle is low and minimizing soil heterogeneity e g use the best and most consistent part of the field for stress treatments as these experiments are
63. samples in their bags to avoid biomass losses 2 m e Place the appropriate sized empty paper bag on the balance and re zero TARE 22 Record dry weights DW_SS50 and or DW_SS20_ stem DW_SS20_spike DW_SS20_leaf lamina etc Data and calculations Determination of WSC Remove leaf lamina and leaf sheath from the stem in the 20 culm sub sample Stems are weighed and ground using either a plant tissue mill or grinder and submitted for WSC analysis to give the soluble carbohydrate concentration see this volume Chapter 16 Determination of nutrient content Nutrient analysis e g for total N of the whole plant or of the individual plant organs e g all leaf lamina individual leaf layers leaf sheath stem spike etc requires a sub sample of 20 culms The plant material is dried milled to a fine powder and sealed in an air tight container to prevent moisture re absorption Only a small sub sample is tested typically lt 1 g is required This amount should be especially noted for small samples as material losses occur during sample processing Ensure to check the specific procedural requirements of the laboratory Table 15 1 Formulas and a worked example for the calculation of biomass and its components from the in season biomass sample Component Biomass DW Number of tillers Number of fertile culms Spike index Leaf lamina DW Leaf lamina N content Formula per quadrat FW_Q x DW_SS5
64. similar to Figure 10 4 Aloba Phoigihgp 3 crtlernded p gpg ey Fehn ip ems d HUPA NO UL Ford Errmig Tocs AE Figure 10 3 Preferred options for the processing workspace EET Camera held 4 m EEE tai plot 48 Physiological Breeding II A Field Guide to Wheat Phenotyping Figure 10 2 Photographing plots for digital ground cover estimation Maintain the camera at A a consistent height and B constant orientation centrally above the plot Creating recording and testing an Action Selection of the green leaf area 6 In the Navigator palette Figure 10 4 zoom in on The following instructions detail a series of actions the sample image to 300 for image manipulation which can be recorded by Photoshop and then automatically repeated for the 7 On the menu bar select SELECT gt COLOR RANGE analysis of all DGC photographs adjust values to FUZZINESS 0 and select 1 Open a file either by dragging a photograph from SELECTION to give a selection preview a file into the Photoshop Workspace or by selecting 8 Click on the Plus Eye dropper tool 4 and use this FILE gt OPEN to select green pixels in the sample image Sample as 4 Create a New Action click the CREATE NEW many of the green pixels on the leaves as possible to ACTION Hutton atthebotonerine neton gain the full color range Figure 10 6
65. stable RH inside the chamber including air and leaf temperature A leaf with low rate of gas exchange transpiration needs a relatively low dry flow rate to maintain a null balance Site and environmental conditions Measurements should be taken when the sky is clear and there is not more than a slight wind The operating environment for the porometer is 5 40 C and 10 70 RH It is important that the leaf surfaces are dry and not wet from dew irrigation or rain Only take measurements in reasonably well watered trials as porosity may be too low in drought trials to give a reliable reading Time of day Take measurements close to solar noon typically from 11 00h to 14 00h Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from mid tillering to late grain filling depending on the experimental objectives timing of peak stress To compare between genotypes do not take measurements during heading and anthesis where differences in phenology may confound results Typically take one or two measurements between mid tillering and the end of booting then one or two measurements during grain filling Number of samples per plot Take three readings on different randomly chosen leaves from each plot Canopy temperature stomatal conductance and water relation traits 15 Procedure The following procedure describes taking measurements using the Decagon SC 1 hand h
66. system under tension negative pressure to allow movement upwards from the roots to the leaves This tension is positively related to the amount of water stress as lower water availability requires water to be drawn with a great pressure Therefore when a sample is cut for analysis the water within the xylem system is rapidly pulled into the surrounding tissue and the amount of positive pressure required to return the water back into the xylem is an inverse measurement of the ability of the plant to maintain water status when water stressed day measurement and to recover when the water stress reduces night measurement Leaf water potential can be measured with the Scholander pressure chamber or pressure bomb which exerts a positive pressure on sample material e g leaf or stem held within a sealed chamber The chamber is gradually pressurized with compressed air until the distribution of water within the surrounding tissue and xylem vessels is returned to its initial pre excision state The water can be observed returning to the cut ends of the xylem system and at this point the balance pressure can be recorded Identifying genotypes which are able to maintain a lower balance pressure during stress conditions is an important means of identifying lines better adapted to water stress Although this method does not take into account the tissue osmotic potential or effects of tissue respiration for comparative ph
67. systems and thus ensure global food security and reduce poverty The center s outputs and services include improved maize and wheat varieties and cropping systems the conservation of maize and wheat genetic resources and capacity building CIMMYT belongs to and is funded by the Consultative Group on International Agricultural Research CGIAR www cgiar org and also receives support from national governments foundations development banks and other public and private agencies CIMMYT is particularly grateful for the generous unrestricted funding that has kept the center strong and effective over many years International Maize and Wheat Improvement Center CIMMYT 2011 All rights reserved The designations employed in the presentation of materials in this publication do not imply the expression of any opinion whatsoever on the part of CIMMYT or its contributory organizations concerning the legal status of any country territory city or area or of its authorities or concerning the delimitation of its frontiers or boundaries CIMMYT encourages fair use of this material Proper citation is requested Correct citation Pask AJD Pietragalla J Mullan DM and Reynolds MP Eds 2012 Physiological Breeding II A Field Guide to Wheat Phenotyping Mexico D F CIMMYT AGROVOC descriptors Wheat Physiology Drought stress Genetic resources Phenotypes Canopy Temperature Crop improvement Genetic markers Physiological adaptation Cu
68. the canopy the amount of photosynthetically active radiation PAR absorbed by the canopy its photosynthetic potential varietal characteristics e g glaucousness wax and canopy architecture and grain yield has also been estimated using spectral reflectance indices during different developmental stages of the crop Measurements can also be used to assess the effects of nutrient deficiencies and environmental stresses through estimations of chlorophyll and carotenoid concentrations photosynthetic radiation use efficiency RUE and water content Table 7 1 Commonly used spectral reflectance indices SRI for wheat canopy analysis where index types are VI vegetation index PI pigment related index WI water index Index Name Physiological process Type Calculation NDVI Normalized difference Green area VI Rooo Regol RoootResol vegetation index photosynthetic capacity N status R NDVI Red normalized difference Green area photosynthetic VI R0 Re701 Rzg0tRe70 vegetation index capacity N status G NDVI Green normalized difference Green area VI Rogo Rssol RrgotRssol vegetation index photosynthetic capacity N status SRa Simple Ratio Green biomass VI Ra Resol and Rooo Regol RARS Ratio analysis of reflectance Chlorophyll a content PI Re75 R400 spectra chlorophyll a RARS Ratio analysis of reflectance Chlorophyll b content PI Re7s ResoXRv00 spectra chlorophyll b RARS Ratio analysis of reflectance Carotenoid conten
69. the experimental objectives timing of peak stress e Early evaluation of a population take simultaneous measurements for all genotypes of gas exchange and or chlorophyll fluorescence at the 3 4 leaf stage e Maximum photosynthetic capacity in a yield potential trial take measurements of gas exchange and or chlorophyll fluorescence at anthesis 7 to 14 days 64 Physiological Breeding II A Field Guide to Wheat Phenotyping e Stress tolerance i Heat stress tolerance take measurements of gas exchange and or chlorophyll fluorescence at or shortly after peak temperature ii Drought stress tolerance take measurements of chlorophyll fluorescence only during the period of stress gas exchange measurements are not recommended due to stomatal closure Number of samples per plot For chlorophyll fluorescence take measurements from 3 5 leaves per plot For gas exchange take measurements from at least 2 4 leaves per plot Procedure General advice on taking measurements Note that these instruments are very sensitive and time should be taken to read the user manual carefully The following procedures describe taking measurements separately however many gas exchange photosynthesis systems allow simultaneous measurements of leaf gas exchange and chlorophyll fluorescence e g LI COR 6400 XT GFS 3000 CIRAS 2 and LCpro SD which is recommended to avoid spatial variation within a leaf For both measurements select the
70. the precision of physiological trait measurement while avoiding common mistakes that reduce data quality or waste resources Also included are recommendations concerning instruments and their correct use and a glossary of terms Introduction 3 Three summary tables are included in this introduction to assist the reader 1 An overview of wheat phenotyping techniques detailing the physiological trait measured the reason to measure it and the advantages and disadvantages of each approach 2 Resources required for each phenotyping technique detailing the instrument and the resources in cost and time required and the recommended experimental environment s 3 A timetable for phenotyping measurements providing a guide to the most typical visual developmental stages to take individual measurements during crop growth and those stages not recommended References Acevedo E Craufurd PQ Austin RB and P rez Marco P 1991 Traits associated with high yield in barley in low rainfall environments Journal of Agricultural Science 116 23 36 Araus JL Amaroa T Voltas J Nakkoulc H and Nachit MM 1998 Chlorophyll fluorescence as a selection criterion for grain yield in durum wheat under Mediterranean conditions Field Crops Research 55 209 223 Babar MA Reynolds MP van Ginkel M Klatt AR Raun WR and Stone ML 2006 Spectral reflectance to estimate genetic variation for in season bio
71. the soil profile Typical RWD values spring wheats range from 2000 g m in 0 30 cm 300 g m in 30 60 cm 100 g m in 60 90 cm and 30 g m in 90 120 cm RWD g m RW soil volume Equation 17 1 23 Direct growth analysis Root length density RLD This is the root length RL cm per unit of soil volume cm and distribution through the profile It is commonly used to describe root quality and soil exploration It typically decreases exponentially with depth theoretically RLD above 1 cm cm will allow extraction of all available soil moisture RLD cm cm RL soil volume Equation 17 2 Specific root length SRL describes the economy of root length production in relation to the ratio of root biomass investment Theoretically a high SRL would be advantageous in resource limited environments Typical SRL values for spring wheats range from 100 to 200 mgt SRL m gt RL RW Equation 17 3 Troubleshooting Problem Solution Field measurements moisture content Repeated measurements are necessary during the crop cycle References Dreccer MF Borgognone MG Ogbonnaya FC Trethowan RM and Winter B 2007 CIMMYT selected derived synthetic bread wheats for rainfed environments Yield evaluation in Mexico and Australia Field Crops Research 100 218 228 Irrometer 2011 Soil moisture measurement Available at http www irrometer com sensors html accessed 14 August 2011 Lopes MS and Reynolds MP 2010 P
72. the sun and ensure to avoid the shadow of the operator and or shadows from the neighboring plots Record from the same end of each plot with the sun behind you e g stand at the southern end of north south oriented plots in the northern hemisphere and vice versa Ensure to compare the two readings for each plot these should be similar i e within 1 C If the two readings from a plot differ by more than 1 C then both should be repeated However if the readings still differ by gt 1 C and you cannot see any errors in the method of measurement or the IRT then continue taking measurements Whole canopy temperature leaf peduncle and spike Figure 1 1 Readings during the grain filling phase should include the whole canopy spike peduncle and leaf temperature at the same time rather than of the spike and leaf temperatures separately During the grain filling phase there is typically less green area especially under stress so it may be necessary to move the IRT closer to the plants and check the angle of the IRT to intercept the area available It is very important to hold the IRT at the same distance and angle from the crop for all measurements The distance that you hold the IRT from the crop will determine the area of surface measured i e the closer the IRT is to the object the smaller the surface area measured see Figure 1 3 and the angle that the IRT is held will dictate the part of the crop from which th
73. to increase the comparability of data Relative growth rate RGR g DW day is the change in total crop dry weight per unit area per unit time Determination of RGR requires sequential biomass measurements through the growth cycle Logarithmic In transformed values for DW can be used to increase the fit of the curve RGR varies primarily with intercepted radiation see Monteith 1994 RGR DW2 DW1 t2 t1 Equation 15 1 Where DW the crop dry weight g m7 and t the time days at the first 1 and the second 2 sampling Direct growth analysis 81 Troubleshooting Problem The surface of the leaves is wet with dew irrigation and or rain There is a variation in phenology between plots within the trial Which culms should be included in the 50 culm sub sample When cutting and or sub sampling culms some material is lost All the biomass samples cannot be processed in the laboratory in the same day When partitioning the fresh culms it is difficult to remove the leaf sheath from the stem References Atwell BJ Kriedemann PE and Turnbull CGN 1999 Plant bi In Plants in action adaptation in nature performance in Solution Wait until the plant surfaces are dry e g from dew in the late morning as surface water will cause inaccuracy in biomass measurements due to sub sampling It may be necessary to cut biomass samples over a period of days to allow comparability between data at a defined d
74. to obtain to the actual crop environmental conditions and the the saturating photosynthetic rate Amax target environment e For A C curves it is important to consider leaks in the o Relative humidity set value to 50 80 leaf chamber within the sensor head for details on how to minimize the error generated by such leaks see Long and Bernacchi 2003 Flexas et al 2007 Rodegheiro et al 2007 o Temperature set the block temperature to equal air temperature To measure the leaf temperature do not change the leaf temperature settings as this will become constant across your measurements For A C curves 25 C is preferred for the calculation of Rubisco kinetics Preparations e Ensure that batteries are fully charged e Ensure that the chamber and sensor are free of dust pollen etc and that the seals and gaskets are well placed and not damaged o CO concentration set value to 350 400 ppm o Air flux set to 400 umol st A i P F B vi M i ir Figure 13 4 Console of a portable photosynthesis system LICOR LI 6400XT showing A i CO cartridge holder and regulator ii screen and keyboard iii fluorometer chamber connection and iv tubes and connectors to the sensor head and B v H O desiccant tube and vi CO scrub tube Photosynthesis and light interception 67 Check the connections between the chamber and the o Flow rate increase this to the maximum turn the conso
75. www delta t co uk Leaf porometer Delta T Devices AP4 Leaf htto www delta t co uk Decagon Devices SC 1 Leaf htto www decagon com Normalized difference NTech Industries GreenSeeker Hand Held Canopy htto www greenseeker com vegetation index Holland Scientific Crop Circle Handheld Canopy http www hollandscientific com NDVI Sensor Field Scout CM 1000 NDVI Canopy http www specmeters com Qubit Systems Z950 NDVI Leaf http www qubitsystems com Photosynthesis LI COR 6400 XT Leaf plant http www licor com system PP Systems CIRAS 2 Leaf plant http www ppsystems com CID Bio Science Cl 340 Leaf http www cid inc com WALZ GFS 3000 Leaf htto www walz com ADC LCpro SD Leaf htto www adc co uk Plot combine Wintersteiger Classic Plot htto www wintersteiger com Almaco PMC 20 SPC 20 Plot htto www almaco com Sample mill Grinder UDY Corporation Cyclone Grain biomass http www udyone com IKA MF 10 1 Grain biomass htto www ika net FOSS Cyclotec 1093 Grain biomass htto www foss dk Thomas Wiley Model 4 and Mini Grain biomass http www thomassci com Scholander pressure Soil moisture 3000 Series and 3005 Series Leaf htto www soilmoisture com chamber Equipment Corp Skye SKPM 1405 50 Leaf htto www skyeinstruments com PMS Instrument Company Model 600 Leaf http www pmsinstrument com Seed counter Seedburo 801 Count A Pak Grain http www seedburo com automatic Pfeuffer CONTADOR Grain http www pfeuffer com Seed co
76. youngest fully expanded leaf typically the flag leaf once emerged receiving sunlight to the upper surface The leaves must be clean dry green with no sign of disease or damage and should be selected from plants that are representative of the plot Ensure to select leaves of similar age life history position and orientation as photosynthesis parameters are sensitive to light intensity and temperature variation Handle the leaf as little as possible and avoid shading the leaves during measurement It is necessary to control for phenology in populations with diverse anthesis dates as plants under different stages of development present physiological differences in photosynthesis due to the stage of leaf development plant and leaf architecture e g leaf angle and source sink relationships which may confound the analysis This is especially important in environments where the temperature is linearly increasing e g during the grain filling phase This can be corrected by splitting the population into early and late lines and therefore making different populations to be screened A range of up to 10 days in anthesis date is quite reasonable A Chlorophyll fluorescence Recommendations for dark adaptation measurements e Dark adaptation of a wheat plant takes at least 20 minutes during daytime Alternatively pre dawn measurements can be taken before sunrise for Fo F values which can be used to calculate other dark The
77. 0 FW_SS50 FW_Q FW_SS50 50 fertile culms in FW_SS50 50 x tillers per Q DW_SS20 spike DW_SS20 DW_SS20_ leaf lamina DW_SS20 x DW_Q DW_leaf lamina x N content Calculation per quadrat Per quadrat Per m 3000 x 120 500 720g 900 g 3000 500 50 300 tillers 375 tillers m 35 50 x 300 210 fertile culms 263 fertile culms m 15 40 15 0 27 0 27 10 55 x 720 131 164 g m 131 x 3 3 93g N 4 91 g N m Where FW fresh weight DW dry weight Q quadrat SS sub sample 50 number of green culms in sub sample 20 number of fertile culms in partitioning sub sample Assumptions quadrat area 0 80 m FW_Q 3000 g FW_SS50 500 g DW_SS50 120 g DW_SS20 40 g and DW_SS20_spike 15 g DW_SS20 leaf lamina 10 g N leaf lamina 3 There are 35 fertile culms in the sub sample of green culms Data is typically expressed as per m calculated by multiplying the quadrat value by the fraction of area sampled by the quadrat e g quadrat length 0 5 m x width 1 6 m 0 80 m therefore 1 0 80 1 25 x per quadrat Typical nitrogen concentrations at anthesis for field grown wheat are leaf lamina 2 4 N leaf sheath 1 2 N stem 1 2 N and spike 1 3 N Nutrient remobilization studies require two or more points of sampling e g anthesis 7 days and maturity It is advantageous to mark culms that are uniform in morphology and phenology before the initial sampling
78. 0 This is when 50 of the seedlings have emerged emergence being the appearance of the first leaf lamina breaking through the soil surface the first leaf can be recognized by its round tip A visual estimate is usually adequate as emergence is typically uniform Daily counts of emerged plants can be made of plots until the number becomes constant and the date of 50 emergence determined retrospectively It takes approximately 105 Cd for a wheat plant to germinate and emerge from a depth of 3 cm Figure 14 1 A o r B Figure 14 2 Recognizing the start of stem growth A plant at E GS31 B developing spike with Terminal spikelet GS30 First node at 1 cm above tillering node GS31 GS30 is estimated as the date at which the final spikelet can be observed on the forming spike within the stem of the main culm typically measured on around 10 plants per genotype e g 5 plants across 2 replicates However determination of GS30 can be laborious and typically requires a microscope for accuracy Alternatively GS31 is estimated in field as the date at which the first node can be detected at approximately 1 cm above the tillering node and is more easily seen with the naked eye This is typically measured as per GS30 Most cultivars require approximately 80 100 Cd to produce each tiller or leaf on the main shoot Figure 14 2 Floret 2 terminal as Ep p t stem tissue removed
79. 3 Table 19 1 Leaf rolling scale Percentage of Score Description of rolling leaf rolled 0 None None 1 Leaf loosely rolled from the tip lt 33 2 Leaf moderately rolled 34 66 3 Leaf tightly rolled gt 67 Leaf angle and orientation The angle at which the leaves are held relative to the Scoring vertical axis rather than to the stem is most apparent on Score the flag leaf angle at heading and at early the flag leaves This can lead to the appearance of either grain filling stages an open canopy through which light penetrates to the lower leaves for erect or pendant leaves or a closed canopy where the upper leaves capture the majority of the incident light for horizontal leaves or erect leaves which flop mid way The degree of canopy closure is sometimes scored separately Figures 19 8 and 19 9 e Score by dividing the vertical plane into three sectors of approximately 60 Rate the leaf angle using a scale of either 1 erect leaves 0 60 2 intermediate or horizontal leaves 60 120 or 3 pendant leaves 120 180 see Figure 19 8 0 60 0 60 60 120 60 120 Vertical axis Vertical axis 120 180 3 120 180 A B Figure 19 8 Scoring of leaf angle should measure the angle at which the leaves are held A relative to the vertical axis B rather than to the stem axis Figure 19 9 F C 3 for pendant leaves 120 180 Crop observations 111 Trou
80. 55 of head emerged 12 2 leaves unfolded 57 of head emerged 13 3 leaves unfolded 59 Emergence of head complete 14 Aleaves unfolded 15 5leaves unfolded Flowering or anthesis 16 6leaves unfolded 61 Start of flowering 17 7 leaves unfolded 65 Flowering half complete 18 8leaves unfolded 69 Flowering complete 19 9ormore leaves unfolded Kernel and milk development Tillering 71 Kernel watery ripe clear liquid 20 Main shoot only 73 Early milk liquid off white 21 Main shoot and 1 tiller 75 Medium milk milky liquid 22 Main shoot and 2 tillers 77 Late milk more solids in milk 23 Main shoot and 3 tillers 24 Main shoot and 4 tillers Dough development 25 Main shoot and 5 tillers 81 Very early dough slides when crushed 26 Main shoot and 6 tillers 83 Early dough elastic dry and shiny 27 Main shoot and 7 tillers 85 Soft dough firm thumbnail mark not held 28 Main shoot and 8 tillers 87 Hard dough thumbnail impression held 29 Main shoot and 9 or more tillers 89 Late hard dough difficult to dent Stem elongation or jointing Ripening 30 Pseudo stem erection 91 Kernel hard difficult to divide 16 water 31 1st node detectable 92 Kernel hard not dented by thumbnail 32 2nd node detectable 93 Kernel loosening in daytime 33 3rd node detectable 94 Overripe straw dead and collapsing 34 Ath node detectable 95 Seed dormant 35 5th node detectable 96 50 of viable seed germinates 36 6th node detectable 97 Seed not dormant 37 Flag leaf j
81. AVG normalized difference vegetation index NDVI and vegetation index VI data Time ms Plot Count NDVI VI_2 173610 1 29 0 54283 0 30748 178610 2 25 0 45732 0 38388 184410 3 35 0 60763 0 25526 39 Spectral reflectance indices and pigment measurement Troubleshooting Problem Large error variance in data Variable reflectance values and or with high error variance An unintentional value was recorded No association between biomass and NDVI score Useful references Araus JL 1996 Integrative physiological criteria associated with yield potential In Reynolds MP Rajaram S and McNab A Eds Increasing yield potential in wheat breaking the barriers CIMMYT Mexico D F Gutierrez Rodriguez M Reynolds MP Escalante Estrada JA and Rodriguez Gonzalez MT 2004 Association between canopy reflectance indices and yield and physiological traits in bread wheat under drought and well irrigated conditions Australian Journal of Agricultural Research 55 11 1139 1147 N Tech Industries 2011 Greenseeker Available at http www ntechindustries com greenseeker home html accessed 13 August 2011 40 Physiological Breeding II A Field Guide to Wheat Phenotyping Solution NDVI meter is not held centrally over plot and or small plots with a large border effect Low battery causes a reduction in the light source intensity affecting the reflectance value i e the active sensor becomes a passive
82. Crops Research 41 45 54 30 Physiological Breeding II A Field Guide to Wheat Phenotyping Solution All materials in contact with samples mortar and pestle soatulas Eppendorf tubes etc must be well cleaned with alcohol and free of dust Additionally if a mill is used for grinding this should be carefully cleaned between samples with a compressed air hose vacuum Plants were not well irrigated at the time of sampling Make sure that your material is dried immediately after sampling as respiratory losses of carbohydrates which occur even after cutting may alter isotope ratios in the sample Useful references Araus JL Slafer GA Reynolds MP and Royo C 2002 Plant breeding and drought in C3 cereals what should we breed for Annals of Botany 89 925 940 Condon AG Richards RA Rebetzke GJ and Farquhar GD 2004 Breeding for high water use efficiency Journal of Experimental Botany 55 2447 2460 Khazaie H Mohammady S Monneveux P and Stoddard F 2011 The determination of direct and indirect effects of carbon isotope discrimination A stomatal characteristics and water use efficiency on grain yield in wheat using sequential path analysis Australian Journal of Crop Science 5 4 466 472 Monneveux P Reynolds MP Trethowan R Gonzalez Santoyo H Pefia RJ and Zapa F 2005 Relationship between grain yield and carbon isotope discrimination in bread wheat under four water regimes
83. Dry sub sample and weigh DW_SS MC FW_SS DW_SS FW_SS x 100 Equation 18 3 For example the calculation of harvested area biomass FW to DW DW_HA 100 MC x FW_HA Equation 18 4 Expression of yield or biomass per fertile culm Detailed physiological studies often express data on a per fertile culm basis To calculate values per culm it is important that the sub sample is randomly selected to give representative mix of fertile culm classes and that the number of fertile culms spikes it contains is carefully counted Alternatively the pre harvest fertile culm count data can be used For example the calculation of biomass DW per fertile culm DW_fertile culm g DW_SS number of fertile culms Equation 18 5 Determination of the chaff dry weight Values are around 0 5 g for a typical awnless spike awns add around 20 to the chaff dry weight Chaff dry weight is important in yield potential studies where it is related to the potential capacity of plants to set grains An alternative method is to use the spike dry weight at anthesis as an approximation of the chaff weight at harvest In the laboratory 1 Cut spikes from dry culms at the spike collar in the sub sample count and weigh DW_SS S 2 Thresh the sub sample of spikes and weigh the grain DW_SS G DW_chaff g spike DW_SS_S DW_SS G number of spikes Equation 18 6 Determination of the grain number m7 The number of grains m act
84. Flag leaf length and width The flag leaf uppermost leaf is the major photosynthetic site from mid booting until the end of the grain filling period The area of the flag leaf may constitute up to 75 of the light interception surface of the plant is maintained the longest and consequently contributes the most assimilates during grain filling It can therefore be related to the potential grain weight and total yield The length and width of the flag leaf are genetically controlled and are strongly correlated to the surface area of the leaf Typical ranges of length are 100 to 300 mm and width are 10 to 25 mm Figure 19 1A Measurement e Measure the length from the base to the tip of the flag leaf record to the nearest millimeter e Measure the width at the widest part of the flag leaf record to the nearest mm e Note that the flag leaf is fully expanded from mid booting f Aa 1 W i i Peduncle length The peduncle uppermost internode of the stem consists of a lower unexposed part covered by flag leaf sheath and an upper exposed part It may account for up to half the total shoot height and is a location for significant soluble carbohydrate and nutrient storage for mobilization to the grain The upper part of the peduncle also intercepts significant amounts of light and contributes to assimilate production during grain filling A long peduncle can make combine harvesting easier although it may also i
85. GD and Singsaas EL 2007 In Practice Fitting photosynthetic carbon dioxide response curves for C3 leaves Plant Cell and Environment 30 9 1035 1040 Maxwell K and Johnson GN 2000 Chlorophyll fluorescence a practical guide Journal of Experimental Botany 51 345 659 668 Photosynthesis and light interception 69 Troubleshooting Problem Chlorophyll fluorometer Variable F F Variable F F Infrared gas analyzer The equipment is making a beep sound Flow values are not stable Breathing into the chamber or console causes the CO to increase more than 2 ppm Values are not stable Anomalous values of photosynthesis CO is not stable PAR is lower than defined g values are not stable The ambient humidity is too low and the relative humdity needs to be set to gt 50 when the drierite is in full bypass CO of the reference and sample is too low Solution Make sure that the sample leaves are equally exposed to the light and remember to measure the part of the leaf that is exposed to the light Check that the saturating flash of light is sufficiently intense Some instruments have very soft saturating flashes which do not permit good light measurements Check that the fibre optics are working properly Check that your PAR sensor is measuring correctly If PAR readings are not correct then there is no way to ensure that light fluorescence measurements are being performe
86. KD Lu Z M Condon AG and Saavedra AL 1998 Wheat yield progress associated with higher stomatal conductance and photosynthetic rate and cooler canopies Crop Science 38 1467 1475 Rebetzke GJ Read JJ Barbour MM Condon AG and Rawson HM 2000 A hand held porometer for rapid assessment of leaf conductance in wheat Crop Science 40 277 280 Rebetzke GJ Condon AG Richards RA and Read JJ 2001 Phenotypic variation and sampling for leaf conductance in wheat Triticum aestivum L breeding populations Euphytica 121 335 341 Rebetzke GJ Condon AG Richards RA and Farquhar GD 2003 Gene action for leaf conductance in three wheat crosses Australian Journal of Agricultural Research 54 381 387 Reynolds MP Balota M Delgado MIB Amani and Fischer RA 1994 Physiological and morphological traits associated with spring wheat yield under hot irrigated conditions Australian Journal of Plant Physiology 21 717 730 Reynolds MP Calderini DF Condon AG and Rajaram S 2001 Physiological basis of yield gains in wheat associated with the LR19 translocation from A elongatum Euphytica 119 137 141 Canopy temperature stomatal conductance and water relation traits 1 Chapter 3 Leaf water potential Carolina Saint Pierre and Jos Luis Barrios Gonzalez Leaf water potential LWP is an estimate of the plant s water energy status Water in the plant is transported within the xylem
87. Mai 50 100 culms 4 fake prab sample GRAB SAMPLE within harvested e area and measure Record fresh area weight Record fresh weight Thresh all harvested area Schematic 18 2 Sub sample harvest 98 Physiological Breeding II A Field Guide to Wheat Phenotyping at randomly chosen and representative places within this area including all harvested rows and gaining a representative mix of culm classes and count the number of fertile culms continue until the total grab sample contains 50 or 100 fertile culms Put the total grab sample into a labeled paper or textile bag ensuring not to lose biomass Thresh all harvested area when dry using a large stationary thresher or small combine harvester Figures 18 1A and B remove chaff and weigh grain FW_HA_G Remember that the grain from the grab sample of biomass is separate Take a sub sample of harvested area grain and weigh approx 50 g and put in labeled paper envelope FW_HA_SS G In the laboratory 6 Record fresh w ight Take grain sub sample Dry to constant weight Dry the sub sample of harvested area grain and weigh DW_HA SS G Dry the grab sample of biomass and weigh DW_GB Thresh the grab sample of biomass remove chaff and weigh grain DW_GB G Thresh grab sample Record dry Record grain welght dry weight Record grain dry weight bi Dry to constant weight Method C Reduced threshing harve
88. Physiological Breeding II A Field Guide to Wheat Phenotyping Alistair Pask Julian Pietragalla Debra Mullan and Matthew Reynolds Eds CIMMYT International Maize and Wheat Improvement Center Physiological Breeding II A Field Guide to Wheat Phenotyping Alistair Pask Julian Pietragalla Debra Mullan and Matthew Reynolds Eds GRDC ER B M Z UN Federal Ministry A sates ras for Economic Cooperation a Grains QE and Development Research amp gt 4 still SAID Corporation FROM THE AMERICAN PEOPLE E c gt L X MasAgro Generation ee Programme CIMMYT International Maize and Wheat Improvement Center Acknowledgements The authors sincerely thank the following for their generous support to physiological breeding initiatives e The Grains Research and Development Corporation GRDC Australia e The United States Agency for International Development USAID e The Sustainable Modernization of Traditional Agriculture MasAgro Program Mexico e The Cereal Systems Initiative for South Asia CSISA e The Federal Ministry for Economic Cooperation and Development BMZ Germany e The Generation Challenge Programme GCP Mexico The International Maize and Wheat Improvement Center known by its Spanish acronym CIMMYT www cimmyt org is a not for profit research and training organization with partners in over 100 countries The center works to sustainably increase the productivity of maize and wheat
89. Press MENU to scroll down in the main menu and press the SET key to select the option For light measurements select the QY NPQ LC1 or LC2 mode according to the measurements being performed 66 Physiological Breeding II A Field Guide to Wheat Phenotyping 3 Place the leaf into the sensor head at the mid point of the leaf and ensure that the selected area of the leaf completely covers the aperture of the sensor 4 Press SET to run the light fluorescence measurement 5 Remove the leaf from the sensor head and place a dark adaptation leaf clip onto the leaf at this point 6 Repeat light fluorescence measurements for 3 5 leaves per plot Allow leaves at least 20 minutes for dark adaptation 8 After which time return to the previously measured leaves 9 Press MENU and select FT or OJIP for dark measurements 10 Carefully perform the dark fluorescence measurement ensuring to avoid illumination of the dark adapted leaf Final measurements and completion 11 After measuring the whole trial Go to RETURN press the SET key Press the MENU key to scroll down and select TURN OFF DEVICE by pressing the SET key 12 Saved data can be downloaded with the software supplied with the instrument Data is typically downloaded as a comma delimited text file and imported into MS Excel B Gas exchange measurements Oo Light Conduct
90. a of total green plant surface area per unit of ground area Crops with large canopies have the potential to intercept more light and be more productive but may do so inefficiently in relation to the water and nutrients required to produce and maintain them However more rapid canopy closure during early developmental stages i e up to booting can significantly increase the total amount of light interception during this phase and is strongly linked to increased biomass at anthesis and final grain yields in optimal conditions The senescent phase of plant development is a highly organized and well regulated process The stromal enzymes such as Rubisco are degraded early in senescence leading to a decline of photosynthetic Capacity Typically the upper leaves of the canopy senesce from mid grain filling onwards in favorable conditions However senescence in lower leaves can start before anthesis with the N being remobilized to the upper expanding leaves In wheat the oldest leaves senesce first and the three uppermost leaves in particular the flag leaf which contributes the most assimilates to grain filling remain active for the longest period The roots are the last vegetative part to senesce and remain active during grain filling Prolonged green leaf area duration through delayed leaf senescence stay green allows photosynthetic activity to continue and enables the plant continue producing assimilates Genotypes whi
91. a transparent colorless plastic bag Figure 4 1B This allows plants to fully rehydrate overnight prior to leaf sampling This rehydration is not expected to generate significant osmotic readjustment variations among cultivars Babu et al 1999 Day 2 Leaf sampling In the morning collect fully expanded leaves of rehydrated plants 1 Cut the leaf sample Figure 4 1C 2 Quickly dry the surface of the leaf using a paper towel 3 Place leaf sample rolled using forceps into an Eppendorf tube and seal the lid 4 Place sample tubes into the deep freeze lt 15 C to rupture the cells Figure 4 1D 5 Repeat procedure for each genotype in the pot If you are unable to get the samples to the deep freeze immediately then place the Eppendorf tubes into a plastic bag in a thermal vacuum flask with ice Field measurements Although not recommended the protocol can be adapted to field measurement with the following considerations 1 Take samples before dawn 2 Cut four leaf samples each leaf from a different plant within a plot 3 Place all samples in a labeled sample tube 4 Add 1 cm of distilled water to each tube for rehydration 5 Refrigerate the samples at 3 4 C for 4 hours in darkness 6 Dry the leaf surface very carefully using a paper towel 7 Place each sample in an individual sample tube 8 Place sample tubes into the deep freeze Laboratory measurements To measure OP with the
92. adings from the instrument are not absolute chlorophyll values instead each reading is a chlorophyll concentration index CCI ranging from 0 to 99 9 For this instrument up to 30 measurements can be stored in the internal memory although these are lost when the instrument is switched off note that some models of instruments are available with a downloadable memory For stay green or senescence studies where repeated measurements are to be taken on selected leaves it is highly recommended to mark each culm with colored tape around the peduncle to facilitate their re location 41 Spectral reflectance indices and pigment measurement 42 Preparations Ensure that the chamber is clean and that the rubber seal surrounding the chamber is intact and clean otherwise light may leak into the chamber causing incorrect readings 1 After turning on the chlorophyll meter allow the instrument to equilibrate with the ambient temperature for around 10 minutes Initial measurements 2 Calibration is required before taking the first measurement Figure 9 1C e Hold the pinchers closed with nothing in the chamber e Wait until you hear a beep and N 0 is displayed on the screen e The instrument is now calibrated lt Sensor location lt Pinchers ON OFF switch Controls for saving averaging and deleting data Average mode Calibration mode Physiological Breeding II A Field Guid
93. ain cell turgor and hence hydration as water deficit increases i e lowering of water potential WP Cell solute concentration is increased by the accumulation of compatible solutes e g amino acids sugars polyols quaternary amines ions and organic acids rather than a lowering of cell volume or a reduction of cell water content under water deficit note that protocols standardize for instantaneous hydration status These solutes can stabilize and protect macromolecules enzymes and membranes e g sugars and alcohols can also act as scavengers of activated oxygen species reducing cell damage allow turgor dependent processes e g growth and stomatal activity and overall can protect the photosystem complex during water deficit stress OA has therefore been identified as amechanism for maintaining physiological functions under drought stress conditions Osmotic adjustment is calculated as the difference in osmotic potential OP at full turgor between stressed and non stressed plants It has been proposed as a screening tool for selecting lines with adaptation to severe drought stress Measurement of OA requires only a small number of leaf samples and furthermore it is a relatively simple technique The use of this method is Supported by the genetic variability observed in OA for several crops such as wheat maize rice sorghum barley millet sunflower pea chickpea and turfgrasses e g Zhang et al 1999 Values of OA
94. ain soil types soil compaction when taking measurements can be a serious problem If this occurs while obtaining a sample you will need to take the sample again ua M i j i if F i E pU a i it hey al Ls oe a i 7 e J p Ue a E Z Fi i k i L Mt er 88 Physiological Breeding II A Field Guide to Wheat Phenotyping B Figure 17 1 Soil coring using A a tractor with Giddings hydraulic soil corer and B a hand held soil corer When using the hydraulic corer for the deeper samples typically gt 90 cm the pin in the hydraulic ram will need to be adjusted during sampling in order to achieve these depths Preparations Check tractor and hydraulic arm hose connections hydraulic oil and grease the guiding bar and hydraulic ram Ensure that the corer is level and drills in a vertical plane 1 Weigh the clean dry aluminum soil sub sample pots with lids to 2 d p empty pot weight 2 Prepare labeled plastic bags with the number of the plot and soil depth it is useful to abbreviate depths 0 30 cm 30 60 cm 60 90 cm and 90 120 cm to A B C and D respectively Field measurements 3 Insert the corer manually or hydraulically into the soil to a depth of 120 cm for spring wheats or up to 200 cm for winter wheats Care is needed to avoid compaction of the soil sample 4 Carefully extract the corer containing the soil core Determination of root content see Schematic 17 1
95. ally through the stomata Transpiration efficiency is the amount of water transpired per gram of carbon dioxide fixed calculated as photosynthesis transpiration i e A T it can be considered as equivalent to water use efficiency at leaf level Vapor pressure deficit is the difference between the saturated vapor and actual pressure of the air Vigor is the term used to describe the capacity of a seed plant or organ to grow Water potential is a parameter which describes the energy status of the water within a plant as the sum of several components gravitational matric osmotic and pressure potentials Water uptake is the amount of water extracted consumed by a plant crop during a defined period of time Water use efficiency is the amount of water taken up per gram of carbon fixed by the plant in terms of physiological processes or per gram of grain yield produced as an agronomic definition Yield potential is the yield of an adapted genotype grown under optimal management and in the absence of biotic stresses Parts of the plant and plant organs The plant can be partitioned between tillers i e the shoots originating from the base of the plant to identify the main culm i e the primary shoot that emerges first from the soil and from which tillers originate and the second and third culms from the remaining tillers typically between 3 10 in total depending on cultivar and environment Each culm
96. and cleaning Wash the roots as previously described steps 1 1 and 1 ii then rather than mixing with alcohol put them onto black paper so that you can see the roots moisten and keep in the refrigerator until cleaning Hand clean the roots as previously described step 1 vii and put them on clearly labeled moist paper wrap with plastic film and store in the refrigerator or freezer until dyeing Dyeing and preparation for scanning Preparation of dye solution i to make the concentrate solution weigh 1 g of powder of methyl violet TOXIC and dilute this in 100 ml of 100 ethanol Keep this in the dark glass bottle until use as methyl violet is light sensitive ii dilute the concentrate solution before use by diluting 1 ml of the concentrate solution in 9 ml of ethanol then further dilute by adding this 10 ml solution to 90 ml of distilled water to give a 0 1 methyl violet solution D p To dye the root sample the following is required dye solution of 0 1 methyl violet petri dishes pipettes colanders strainers absorbent paper tweezers x 2 bleach and labels a Put the root sample in the center of a labeled petri dish Cover the sample with the dilute dye solution and leave for at least an hour or overnight Rinse the roots with water and drain twice Put the roots onto a petri dish and separate the roots with the tweezers use a little water to make sepa
97. arieties with improved WUE and drought tolerance The organs sampled and their respective growing conditions determine how the results are interpreted for example CID can be measured either in the leaves during early canopy development in well irrigated trials in which case cultivar effects will be related mainly to TE or on the grains at maturity from drought trials in which case cultivar effects are most likely to be related to transpiration rate Values may also be influenced by many different environmental factors other than water stress including responses to pests and diseases nutrient availability and soil constraints In the early developmental stages before the plant has experienced any water or other environmental stress values indicate a measure of the plant s potential TE before effects such as rooting depth or phenology affect values This approach has been successful in improving grain yields in Australian rain fed wheat systems where selection favored low CID Rebetzke et al 2002 Values from the grain at maturity give an integrated almost historical measurement of WUE during the entire growth period and in this case increased grain yields were associated with increased CID i e low TE in Mexico Sayre et al 1995 28 Physiological Breeding II A Field Guide to Wheat Phenotyping Site and environmental conditions Samples can be taken under any environmental conditions For leaf sampling it i
98. artitioning of assimilates to deeper roots is associated with cooler canopies and increased yield under drought in wheat Functional Plant Biology 37 147 156 Useful references Prior SA Runion GB Torbert HA and Erbach DC 2004 A hydraulic coring system for soil root studies Agronomy Journal 96 1202 1205 Reynolds MP Dreccer F and Trethowan R 2007 Drought adaptive traits derived from wheat wild relatives and landraces Journal of Experimental Botany 58 177 186 Increase plot size in trials where destructive measurements are planned or perform measurements using sensors e g time domain reflectrometry TDR neutron probe frequency domain sensors capacitance probes electrical resistivity tomography ground penetrating radar among others however this is limited to only a few plots calibration for soil water content is required and deep sensing is very expensive See Irrometer 2011 Soil compression during sampling Motor oil can be used as lubrication to help soil penetration but ensure to avoid soil contamination with oil especially when soil water content is being determined If the operator feels too much resistance during soil penetration then it is better to start sampling again in a different part of the plot Unexpected readings closed double bagged If loss of moisture between sampling and weighing occurs ensure that the bags are hermetically Where condensation occurs on the inside of
99. as this reduces yield losses due to shattering caused by movement through the plots Under optimum conditions 200 500 spikes m could be expected to maximize yield potential Table 18 4 Formulas for calculating individual yield components from harvest data for the three different harvesting methods Formula DW_200 Gx5 yield g m TGW x 1000 DW_SS number of fertile culms biomass g m DW_fertile culm g Yield component Thousand grain weight TGW g Grains m GNO Fertile culm DW g Spikes m SNO Grains per spike GSP grains m spikes m Where DW dry weight SS sub sample G grain In the field 1 Randomly select four representative areas of the plot 2 Place a 0 10 m quadrat in each area and count the number of spike bearing culms within the quadrat See also Measurements calculated from harvest data below Determination of the number of spikelets per spike Both the total number of spikelets and the number of fertile spikelets i e those containing grain per spike should be counted towards the end of grain filling but before physiological maturity again to avoid losses due to movement through the plots The total number of spikelets per spike is highly heritable and varies little between environments whilst the number of fertile spikelets per spike is greatly affected with spikelets aborted from the base or tip of the spike Values are typically around 10 25 spikelets per
100. asurements use the Field Scout CM 1000 Do not take CCI measurements from dead material as the data will not be useful Avoid damaged areas of the crop Adamsen FJ Pinter PJ Barnes EM LaMorte RL Wall GW Leavitt SW and Kimball BA 1999 Measuring wheat senescence with a digital camera Crop Science 39 3 719 724 Babar MA Reynolds MP van Ginkel M Klatt AR Raun WR and Stone ML 2006 Spectral reflectance to estimate genetic variation for in season biomass leaf chlorophyll and canopy temperature in wheat Crop Science 46 1046 1057 Dwyer LM Tollenaar M and Houwing L 1991 A nondestructive method to monitor leaf greenness in corn Canadian Journal of Plant Science 71 505 509 Yadava UL 1986 A rapid and nondestructive method to determine chlorophyll in intact leaves HortScience 21 1449 1450 Spectral reflectance indices and pigment measurement 43 Ka g t gt l J A CA AN al i ry ei j W l Chapter 10 Crop ground cover Daniel Mullan and Mayra Barcelo Garcia Crop ground cover or the percentage of soil surface covered by plant foliage is an important measurement of crop establishment and early vigor characterized by either fast development of leaf area and or above ground biomass Genotypes with greater early cover are able to better intercept incident radiation thereby increasing soil shading and decreasing soil evaporation which increases w
101. ater status potential 4 Osmotic Cell solute adjustment concentration to maintain turgor hydration 5 Leaf relative Leaf hydration water content status 6 Carbon isotope Integrative discrimination measurement of stomatal aperture 7 Spectral Vegetative pigment reflectance and water indices 8 Normalized Canopy size difference vegetative greenness vegetation index 9 Chlorophyll content of green tissues 10 Crop ground Early vigor green cover area and biomass 11 Light Light interception interception by the canopy 12 Leaf area Area of index green photosynthetic area index leaf canopy and senescence Chlorophyll content Reason to measure trait Linked to many physiological factors stomatal conductance plant water status roots and yield performance under a range of environments Gas exchange capacity transpiration rate photosynthetic potential adaption to heat stress Adaptation to water stress estimate of soil water potential in active root zone Stomatal function is dependent on turgor photosystem function and protection and adaptation to water stress Adaptation to water stress Estimation of water uptake and transpiration efficiency TE Estimation of green biomass leaf area index LAI photosynthetic potential and plant water status Estimation of early cover pre anthesis biomass nitrogen content post anthesis stay green Indicates photosyn
102. ater use efficiency and may have increased competitiveness with weeds and potentially decrease soil erosion In particular a rapid ground cover trait has potential benefits in Mediterranean type environments where water is available early in the season but rapidly declines as the crop approaches grain fill or when planting has been delayed and may increase biomass and subsequent grain yields Accurate phenotyping of ground cover and early vigor has typically been achieved by destructive sampling methods but these are generally too time consuming to perform within breeding programs High throughput approaches to measuring ground cover are visual assessment digital analysis of photographs or normalized difference vegetation index NDVI see this volume Chapter 8 Visual assessment allows a rapid and low technology approach but is subjective and may not have the resolution to distinguish between genotypes whilst digital analysis of photographs enables a more quantitative and objective measurement Site and environmental conditions Measurements can be taken under most environmental conditions For photograph processing purposes it is best to take photographs when the light is diffuse i e there is continuous cloud cover and there is minimal shadow and when the plant surfaces are dry and not wet from dew irrigation or rain Time of day Measurements can be taken during any hour of the day Plant developmental stage Take
103. ative places in the biomass sample and gaining a representative mix of culm classes and count the number of fertile culms continue until the sub sample contains 50 or 100 fertile culms and weigh FW_SS Dry the sub sample of biomass and weigh DW_SS Thresh the sub sample of biomass remove chaff and weigh grain DW_SS G Record fresh weight Put in black plastic bag Take 50 100 culm sub sample Record fresh weight Dry to constant weight Direct growth analysis 99 Worked examples for yield biomass and HI calculation Assumptions culm density 300 per m2 FW per culm 5 0 g HI 0 40 biomass grain moisture content 5 0 In these examples each plot consists of two raised beds each with two rows there are border plots surrounding the trial Each plot is 5 0 m in length and 1 6 m in width After removing a 0 5 m buffer on each end the total harvested length is 4 0 m Methods A and B 4 0 x 1 6 6 4 m Method C 1 0 m Harvested area Formulas and calculations are presented in Tables 18 2 and 18 3 respectively Measuring individual yield components The individual yield components are either measured directly in field prior to harvesting e g spikelets per spike from harvest samples e g TGW or are calculated from the yield biomass and or HI data obtained from the three harvesting methods e g grains m summarized in Table 18 4 Measurements prior to
104. ature as a screening tool for heat tolerance in spring wheat Journal of Agronomy and Crop Science 176 119 129 Ayeneh A van Ginkel M Reynolds MP and Ammar K 2002 Comparison of leaf spike peduncle and canopy temperature depression in wheat under heat stress Field Crops Research 79 2 3 173 184 Balota M Payne WA Evett SR and Peters TR 2008 Morphological and physiological traits associated with canopy temperature depression in three closely related wheat lines Crop Science 48 5 1897 1910 Eyal Z and Blum A 1989 Canopy temperature as a correlative measure for assessing host response to Septoria tritici blotch of wheat Plant Disease 73 6 468 471 14 Physiological Breeding II A Field Guide to Wheat Phenotyping Fuchs M 1990 Infrared measurement of canopy temperature and detection of plant water stress Theoretical and Applied Climatology 42 4 253 261 Olivares Villegas JJ Reynolds MP and McDonald GK 2007 Drought adaptive attributes in the Seri Babax hexaploid population Functional Plant Biology 34 189 203 Rosyara UR Vromman D and Duveiller E 2008 Canopy temperature depression as an indication of correlative measure of spot blotch resistance and heat stress tolerance in spring wheat Journal of Plant Pathology 90 1 103 107 Saint Pierre C Crossa J Manes Y and Reynolds MP 2010 Gene action of canopy temperature in bread wheat under diverse environments Theo
105. ble optical meter e g Minolta SPAD 502 chlorophyll meter which measures the chlorophyll content via light transmittance absorbance of red light at 650 nm and infrared light at 940 nm and compensates for differing leaf thicknesses Measuring chlorophyll content as a proxy for the entire photosynthetic complex indicates photosynthetic potential Loss of chlorophyll content i e chlorosis is indicative of stress induced by heat drought salinity nutrient deficiency ageing etc and reflects a loss of photosynthetic potential However it should be noted that such chlorophyll meters give only point readings and it is often advantageous to integrate the whole canopy chlorophyll content on an area basis either by integrating measurements within the canopy leaf area or by using instruments which measure whole canopy reflectance e g NDVI sensors see this volume Chapter 8 Site and environmental conditions Measurements can be taken under any environmental conditions It is important that the leaf surfaces are dry and not wet from dew irrigation or rain Time of day Measurements can be taken at any time of the day Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from the start of stem elongation to mid grain filling depending on the experimental objectives timing of peak stress For peak chlorophyll content two measurements should be taken betwe
106. bleshooting Problem Large variation of morphology within a plot Solution Check seed origin i e confirm the seed is not mixed with other genotypes Ensure that the sowing and crop husbandry is uniform across each plot e g even sowing depth for all rows Large variation in data within a plot Observe a larger area of plot or take more samples per plot Separate into groups within a plot and ensure to make a comment on the field form e g values for short tall References Eckroth EG and McNeal FH 1953 Association of plant characters in spring wheat with resistance to the wheat stem sawfly Agronomy Journal 45 400 404 Useful references Duncan WG 1971 Leaf angles leaf area and canopy photosynthesis Crop Science 11 482 485 Holmes MG and Keiller DR 2002 Effects of pubescence and waxes on the reflectance of leaves in the ultraviolet and photosynthetic wavebands a comparison of a range of species Plant Cell amp Environment 25 85 93 Innes P and Blackwell RD 1983 Some effects of leaf posture on the yield and water economy of winter wheat The Journal of Agricultural Science 101 367 376 112 Physiological Breeding II A Field Guide to Wheat Phenotyping Izanloo A Condon AG Langridge P Tester M and Schnurbusch T 2008 Different mechanisms of adaptation to cyclic water stress in two South Australian bread wheat cultivars Journal of Experimental Botany 59 3327
107. calculations Soil moisture calculations Open SETUP and ensure that IMAGE BACKGROUND is WHITE and MAGNIFICATION 1 100 Open ANALYSIS and select LENGTH SIN O The software will analyze the files Click ENTER to see a results overview and press lt F6 gt to see the complete results Software analysis for Length Sin 0 calculates the length width area and volume of the roots This program can also be used for calculating the area of leaves and the size of soil particles Table 17 1 Example data for the determination of soil moisture content Depth Pot reference Empty pot Pot fresh Pot dry Fresh weight Dry weight Plot cm number weight g soil g soil g FW soil g DW soil g 1 0 30 127 27 62 139 87 124 91 112 3 97 3 I 30 60 128 27 11 1341 11 113 51 103 3 85 7 1 60 90 129 26 79 121 05 104 28 94 3 7715 1 90 120 130 27 41 131 09 171 55 103 7 84 1 Depth Water Gravimetric Volumetric Water Total water Daily water Plot cm content g water content water content content mm uptake mm uptake mm day i 0 30 15 0 15 4 20 0 60 0 53 5 3 57 l 30 60 17 6 20 5 26 7 80 1 33 4 2 23 1 60 90 16 8 21 6 28 1 84 4 29 1 1 94 1 90 120 19 5 23 2 30 2 90 6 22 9 1 53 Where Water content g FW soil DW soil Gravimetric water content GWC Volumetric water content VWC Water content mm Total water uptake mm Daily water uptake mm day Assumpti
108. ch maintain canopy green area and canopy greenness during the grain filling phase are associated with higher yield LAI and GAI can be measured A directly by destructively sampling a known area of ground usually at the same time as biomass measurements and measuring the area of all plant parts with a planimeter e g this level of detail is required for calculating the canopy coefficient K from GAI or B indirectly and non destructively using techniques based on light interception e g Sunscan LAI 2000 although this also includes dead and dying plant parts or photographs see this volume Chapter 10 or by visual assessment 58 Physiological Breeding II A Field Guide to Wheat Phenotyping e g these two methods can be used as rapid screening techniques for comparing genotypes For assessment of crop senescence regular assessment of the proportion of the canopy that is green and non green dead or dying is important and can be determined by visual assessment of green leaf area GLA remaining This non green plant tissue may intercept light affecting light interception measurements but does not contribute to crop photosynthesis and therefore must be excluded from measurements and calculations e g for radiation use efficiency RUE Site and environmental conditions Samples for destructive sampling can be taken under most environmental conditions However it is important that the plant surfaces are not w
109. cropping and or land use for the last 3 years Water availability existing at sowing inputs from precipitation and irrigation Environment Meteorology this should be taken as close as possible to the trial location for at least the duration of the crop cycle and on a daily basis i Temperature minimum maximum and mean often estimated as an average of the minimum and maximum ii Rainfall precipitation iii Sun hours solar radiation iv Relative humidity Current conditions during measurements observations record conditions which may affect crop physiology and or measurements observations of wind e g was the wind light or moderate or clouds e g some cloudiness etc VI U ROLLY Ld VESEY go yow pae fa AL WUIWAV SELLY WWLOL Juir Benne sage uoga dg pagddi aay ee oe Nas mu uet apasa se paycha aA f gip ka ios Je OS o y Ly CWO Bin jike i F i ig i x e Buwans aid Guproxa aura AA pA Ti E r Il 8 pnpud Ayoeds Es ji anpcud Aaeds a A janp id Aoi eA ji S4LON lan 300L3d UNA JOON ZUMA DISH T idli 0141 Jaiei oo munn an oe Figure 21 5 Sample form for yield trial notes General recommendations 125 Chapter 22 General recommendations for the use of instruments Julian Pietragalla and Alistair Pask 1 Correct use of instruments Instructions may vary according to the make and model of your instrument Refer the in
110. cted Problem The IRT is not giving an average reading e g it is reading constantly or reading the MAX MIN temperature The initial CT readings appear to be higher lower than the readings from the rest of the trial i e a step change due to automatic re calibration The IRT has not had sufficient time to adjust to ambient temperature before starting measurements Ensure to allow at least 10 minutes for the IRT to equilibrate with the ambient temperature The two readings differ by gt 1 C Check that the IRT is being held correctly and consistently and the correct part of the crop is being measured i e avoiding borders damaged senesced leaves bare soil etc Take a look through the plots experiment before starting measurements Decide on the most appropriate distance and angle at which to hold the IRT and maintain this orientation throughout all plot measurements The plots are irregular or the crop is small is approaching mid grain filling and is starting to senesce Measurements take over an hour for a trial The time taken to measure the trial is not important unless the conditions become unsuitable for measurements Gradients in environmental conditions e g an increase in ambient temperature through the morning will be accounted for using Statistical analysis such as lattice corrected adjusted means Useful references Amani I Fischer RA and Reynolds MP 1996 Evaluation of canopy temper
111. cteristics Production of grain Crop fertility determination of yield Observable wax rolling pubescence thickness angle orientation posture Measurable lengths peduncle leaves and awns and plant height Spike tipping lodging damage by climate disease Reason to measure trait Measurements relate to respiration rate photosynthetic potential adaption to heat stress and responses to environmental variables Determination of the status of the photosynthetic apparatus Essential for optimum timing of sampling gives rate of development For radiation use efficiency RUE calculation indicates photosynthetic efficiency partitioning of plant between organs morphology nutrient metabolite analysis Allows estimation of carbohydrate storage capacity of stem which contributes to grain filling Crop water uptake allows calculation of water use efficiency WUE for biomass and yield Association of roots with crop water and nutrient uptake Yield is ultimate expression of all physiological processes Determination of yield by numerical components source sink relations Photo protective adaptive traits to heat drought stress provides information on crop canopy architecture lodging risk Useful information to explain crop performance and assist data interpretation 6 Physiological Breeding II A Field Guide to Wheat Phenotyping Advantages of tool Chamber allows prec
112. cts that may obstruct measurements such as large stones Taking and processing field soil cores is a time consuming process especially in dry and or compacted soils Approximate times for taking individual cores by hand are from 5 irrigated soils to 15 dry soils minutes and by hydraulic corer from 2 to 5 minutes Laboratory Direct growth analysis 87 processing time is at least another 10 minutes Allocate time carefully it is important that samples are obtained from all plots within the same day or over two days in order to avoid confounding effects of environmental changes over time Hand coring is advantageous for a small number of samples to minimize disturbance to the plot e g while plants are in the early stages of development when field conditions make accessibility difficult and is considerably cheaper Hydraulic coring is advantageous for a large number of samples for deeper soil profiles and taking wider samples for root content However using a tractor within the field during the crop cycle may cause damage to the plot Ensure to incorporate these considerations into the experimental design sampling design and if possible fit high access wheels to the tractor see Figure 17 1A Avoid applying too much pressure on the corer e g do not allow it to lift the tractor This may permanently damage the drill cause soil compaction and can be very dangerous to the operator if the corer was to break In cert
113. d e Small sickle large knife e g a bread knife and or clippers e Field balance as required Table 18 1 Samples to be measured when using the three alternative harvesting methods for estimating yield biomass and yield components from experimental yield plots Method Samples to be measured Abbreviation A B C FW of harvested area biomass FW_HA y y FW of sub sample of harvested area biomass FW_SS y y FW of harvested area grain FW_HA_G y y FW of sub sample of harvested area grain FW_HA_SS_G y y DW of sub sample of harvested area grain DW_HA_SS_G y y DW of sub sample grab sample of biomass DW_SS DW_GB y y y DW of grain from sub sample grab sample of biomass DW_SS G DW_GB G y y y FW of 200 grains FW_200 G y y y DW of 200 grains DW 200 G y y y Where FW fresh weight DW dry weight HA harvested area SS sub sample GB grab sample G grain The grain from the SS GB of biomass is separate from the FW of harvested area grain 96 Physiological Breeding II A Field Guide to Wheat Phenotyping Method A Total biomass harvest This method is recommended for high data accuracy but requires more field time and labor than Methods B and C The total biomass of the harvested area is cut dried and threshed in field to independently measure the fresh weight FW of biomass and yield A sub sample of fertile culms is taken and dried to allow calculation of biomass dry weight DW and HI and to allow expression of da
114. d at the same light intensity Leaves were not completely dark adapted Leaves should be in complete darkness for at least 20 minutes If dark adapting with a self made dark adaptation leaf clip then ensure to use a blackout cloth covering the plant instrument and operator when removing the clip to measure the leaf Leaves are damaged and or were handled excessively before measurements were taken Check the batteries Air mufflers in the chemical tubes are clogged or broken Change or clean the tubes There is a leak breathe through a plastic straw near the chamber CO and H O desiccant connection tubes and console to localize the leak Check for leaks Is the IRGA warmed up and ready Wait for 20 minutes and check again The instrument may not be calibrated correctly Repeat the calibration process to zero and match the IRGAs Use a compressed CO cylinder Check the LEDs are working and that none are broken Check that the sensor is working touch it with a finger if the leaf temperature does not change then replace the sensor Add 10 mL of water to the soda lime and wait 30 minutes for the H0_S and H O _R to become stable Change the compressed CO cylinder 70 Physiological Breeding II A Field Guide to Wheat Phenotyping Direct growth analysis Chapter 14 Determining key developmental stages Alistair Pask A sound understanding of wheat plant growth and development is essential for a succes
115. de so taller lines with long peduncles tend to have a larger capacity WSC may be expressed as a concentration in dry mass either as a percentage WSC or as mg gt to demonstrate the potential stem storage capacity of the genotype or as the content per stem g stemt or per unit area g m to give an absolute measurement of the carbohydrates available to the grain Site and environmental conditions Samples can be taken under most environmental conditions However it is important that the plant surfaces are not wet from dew irrigation or rain Time of day Samples should be taken in the morning as this is coolest time of the day to reduce carbohydrate losses from respiration and allows time for same day processing Plant developmental stage Measurements can be taken at any developmental stage from the end of stem elongation and or at regular intervals from mid anthesis to physiological maturity depending on the experimental objectives timing of peak stress e For peak WSC take samples at anthesis 7 for drought to 14 days for favorable conditions Note that in severely stressed conditions the peak WSC may occur before anthesis e For measurement of changes in WSC accumulation and remobilization take sequential samples from anthesis to physiological maturity every 7 14 days Number of samples per plot Take one sample of 20 culms per plot Procedure The following procedure describes the determination
116. difference vegetation index Julian Pietragalla and Arturo Madrigal Vega The normalized difference vegetation index NDVI is widely used at ground level and from low high and satellite altitudes to measure vegetative greenness and canopy photosynthetic size The field portable NDVI sensor Figure 8 1 provides rapid ground level measurement of crops at a resolution to characterize the canopy for leaf area index LAI and green area index GAI biomass and nutrient content e g nitrogen Data can be used to estimate yield prediction biomass accumulation and growth rate ground cover and early vigor senescence pattern estimations and for biotic and abiotic stress detection NDVI technology is also used for making decisions in precision agriculture weed detection and herbicide spraying and rate and timing of nitrogenous fertilizer applications NDVI is calculated from measurements of light reflectance in the red and near infrared NIR regions of the spectrum A healthy green canopy will absorb most of the red light and reflect most of the NIR light as chlorophyll absorbs mainly blue and red light and the mesophyll reflects NIR light NDVI Ryig Rrea Reig Rea Equation 8 1 The majority of field portable NDVI sensors are active i e they produce their own source of light which allows measurements to be made under any light condition and for data to be comparable across date and time of day Site and
117. dobe Photoshop CS3 Extended software Photoshop or a later version as this includes the functionality required to perform and export the automated DGC A free trial copy is available at www adobe com which will allow DGC photograph processing prior to purchasing refer to the Adobe website and program instructions for minimum computer requirements The speed of digital photograph processing will depend on these specifications typical processing time of one photograph per second Interface set up Photoshop is multi functional and consequently the software functions should be customized for DGC The following description will allow a common software interface to be established 1 Open Photoshop 2 From the menu bar select WINDOW gt WORKSPACE gt AUTOMATION 3 Select YES to modify the menu and or keyboard shortcut sets and to apply the workspace pa r P z 1 Consistent height j 4 Select WINDOW gt LAYERS to remove the Layers palette as it is not required 5 Select WINDOW gt MEASUREMENT LOG to activate the Measurement Log palette 6 The Window menu should now be displayed as shown in Figure 10 3 take note of those functions indicated with a tick 7 To enable a clear working environment double click on the top of the Measurement Log box to minimize and move it to the bottom of the screen The screen should now look
118. e temperature is taken In particular ensure that the IRT is held at an appropriate angle so that measurements are not taken from the soil Figure 1 4 If the ground cover is low i e leaf area index of less than 3 it is best to point the IRT at a low angle to the horizontal to minimize the likelihood of measuring soil When taking measurements during grain filling it may be necessary to move the IRT closer to the plants to intercept the green area available To take measurements hold the trigger for 3 5 seconds as the IRT averages the temperature readings during this time Figure 1 2 and move the IRT out and back over Figure 1 2 The Sixth Sense LT300 IRT and main features A front view and B side view D S 1 10 S 5 10 D 50 100 Figure 1 3 The area of the measurement spot S is related to the distance D of the IRT from the crop by a ratio of 1 10 so the distance and angle that the IRT is held from the crop will dictate the surface area from which the temperature is taken Canopy temperature stomatal conductance and water relation traits 11 A Plot border _ Sample area the crop at a moderate speed remembering to avoid the plot border Figures 1 5 and 1 6 Ensure to record the average CT value for the sample see Figure 1 2 Be careful when aiming the IRT at the crop measurements taken when the trigger is held down but the IRT is not consistently pointed at the plot will be ve
119. e 20 3 Lodging of wheat crops A a fisdged crop during grain filling B stem lodging and C root lodging Photographs Pete Berry ADAS Ltd U K 115 Crop observations Figure 20 4 Vegetative damage of wheat crops by A frost of leaf lamina giving a bleached white appearance B leaf lamina and leaf sheath damage from rust and C bird damage to the spike during early grain filling 29 6 33 37 0 80 90 10 Figure 20 5 A rust scoring scale adapted from Roelfs et al 1992 A actual percentage occupied by rust uredinia and B rust severities of the modified Cobb scale after Peterson et al 1947 116 Physiological Breeding II A Field Guide to Wheat Phenotyping Troubleshooting Problem Solution The crop appears to be unaffected by a frost event It will take several days for the true effect of the frost event to become apparent Take a second observation after one week when the damaged tissue has started to die and turn brown Spike tipping is becoming progressively worse either in number of spikes affected and or severity of effect References Peterson RF Campbell AB and Hannah AE 1948 A diagrammatic scale for estimating rust intensity of leaves and stem of cereals Canadian Journal of Research Section C 496 500 Roelfs AP Singh RP and Saari EE 1992 Rust diseases of wheat concepts and methods of disease management CIMMYT Mexico D F 81 pp Useful references
120. e Click on the appropriate white reading button in the software e This reference line should be a straight line at 1 i e 100 reflectance However there is often some signal noise in this reading due to atmospheric disturbance e g if there is a high level of humidity in the air This measurement will give the maximum amount of reflectance possible from the available incident radiation Measurement of the reference panel provides a value for the spectra incident on the canopy and is used to obtain a ratio with the spectrum reflected by the canopy As the intensity of incident radiation is continuously changing with the zenith angle and other environmental variables it is important to perform regular measurements of the white reference panel One white reference measurement should be taken for every 15 30 plots with the frequency of white reference readings increasing with the distance of the sun from the zenith angle 6 Data may now be captured from the trial e The foreoptic is held 60 200 cm above the crop canopy either by hand or with the assistance of a boom The actual distance will vary with differences in trial designs and instruments but should take into account the canopy area plant row spacing and field of view of the foreoptic in use Maintain a constant vertical orientation of the foreoptic during measurements Figure 7 2C Data and calculations Depending on the instrument set up data is
121. e to Wheat Phenotyping During sampling regularly check the accuracy of the readings by taking multiple readings from the same leaf and comparing the values Calibration discs are provided with the SPAD 502 chlorophyll meter and should be used regularly Trial measurements 3 Randomly select five flag leaves or youngest fully expanded leaf from different plants within the plot avoiding the buffer and outer rows 4 Place the leaf in the sensor a third to half of the way from the base of the leaf with the adaxial surface facing upwards avoiding the midrib major veins or particularly thick parts of the leaf Use the sensor location markers on the pinchers to align the sample and ensure it is correctly located see Figure 9 1A MINGLA Number of readings taken Reading as CCI Figure 9 1 Using the Minolta SPAD 502 chlorophyll meter A the main parts of the instrument B measuring a flag leaf at the mid point ensuring that the midrib or main vein is not in line with the indent on the instrument showing the chlorophyll concentration index CCI reading and C the average mode and calibration mode 5 Hold the pinchers closed until the instrument beeps then release 6 A CCI reading will be displayed on the screen Figure 9 1C 7 Once five measurements have been taken N 5 will be displayed on the screen At this point readings can be reviewed and outliers can be removed and
122. ebght SUB SAMPLE Record fresh P oo weight i l Record Record fresh ef ae T fresh welght i Record tresh Record grain k g weight weight aed wriet HARVESTED AREA GP Take grain sub sample Threshed all biomass from harvested area Schematic 18 1 Total biomass harvest ba Dry to constant weight 97 Direct growth analysis Method B Sub sample harvest This method is recommended when field time and or labor is limited sampling is quicker than Method A typically takes one person less than 5 minutes per plot but care needs to be taken to ensure that representative grab samples are taken Several grab samples are taken from the area to be harvested until the sub sample contains a specific number of fertile culms This is then dried weighed and threshed to allow calculation of HI and to allow expression of data on a per culm or per spike basis The harvested area is then machine harvested or cut and threshed to measure the FW of grain and a sub sample of grain is taken and dried to allow calculation of the DW of grain and for TGW measurement The yield and HI are measured independently but the total biomass dry weight is calculated from yield HI See Schematic 18 2 In the field 1 Carefully measure the area to be harvested excluding border rows and ends of the plot 2 Take grab samples of biomass from the area to be harvested i e by grabbing handfuls of culms 6rab sample of
123. ecord proportion of the canopy that is green or dying e g by fungal disease or insects or the proportion of the spike that is damaged e g by birds or rodents Perhaps the most prominent diseases are rusts although this is a large topic and is discussed more comprehensively elsewhere e g Roelfs et a 1992 Site and environmental conditions Measurements can be taken under any environmental conditions Time of day Measurements can be taken at any time of the day Plant developmental stage Observations should be made as soon as possible after the damage has occurred Number of samples per plot Take one observation and or assessment of 10 plants culms aim for 30 per treatment per plot Procedure Take the following equipment to the field e Scale for spike tipping Figures 20 2 leaf lamina senescence Figure 12 2 and or disease scoring Figure 20 5 e Camera as required e Field form and clipboard Advice on taking measurements Take two assessments as damage often becomes more pronounced with time as the affected tissue dies and turns brown It is recommended to take an assessment immediately after the event and the second after 7 10 days In each case both the proportion of the plot affected and severity of damage within each plot is recorded Record the date days after anthesis DAE developmental stage of the crop and the probable cause of the damage A general observation can be made
124. ect beam of light 45 PAR and diffuse light within a canopy 60 PAR PAR Solar radiation 2 Equation 11 2 The PAR intercepted by the crop on a daily basis can be calculated from the fractional interception at sampling multiplied by the total daily PAR from weather station solarimeter Over the growing season e g GS31 to anthesis the cumulative intercepted PAR can be calculated by multiplying the total daily radiation above the crop by the fraction of incident light intercepted by Troubleshooting Problem What angle should the ceptometer probe be inserted into the crop the canopy assuming a linear rate of GAI increase with calendar time between sampling For the calculation of RUE where possible light measurements should be taken in the quadrat sample before it is destructively sampled in order to increase the accuracy of calculating the canopy coefficient K RUE is calculated for each plot by dividing the cumulative biomass by the cumulative PAR intercepted MJ m dt over the same period RUE g MJ MJe MJ DW DW Equation 11 3 Where MJ the cumulative PAR intercepted MJ m7 and DW the cumulative crop dry weight g m at the first t1 or the second t2 sampling Solution Ensure that the ceptometer is measuring a representative part of the crop with the correct proportion of plant and gap i e the space between the rows of plants It is recommended that the ceptometer should
125. educe the differences between varietal means to increase the success of statistical analysis Do follow standard procedures and comprehensively train observers operators especially for subjective measurements observations Remember to record the name of the observer operator on the field form see example Figure 21 4 Do plan for possible inaccurate readings when measuring large trials Partition a large trial into small areas i e replications blocks rows or columns to reduce errors and operator fatigue An assistant is useful and can help spot errors Do be familiar with expected values for observations measurements and readings for instruments typical for each treatment environment examples given in each chapter Remember to check the label with the plot number 4 Field form and field map Each field form should contain name of trial date of sampling environment e g either irrigated drought or heat etc and or treatment plant developmental stage names of scientists operators throughout the measurement process When using instruments also see the general recommendations for the correct use of instruments this volume Chapter 22 Do not change observer instrument operator during sampling It is important that the same person takes all measurements within a sampling event or experimental unit e g repetition or block Do not take single measurements Two or more values should
126. educe transpiration losses from the canopy Leaf angle orientation and posture have been related with optimization of radiation use in high yielding environments by affecting light penetration within the canopy However some of these canopy traits may not be desirable under favorable high yielding conditions due to the reduction in light intercepted by the photosynthetic tissues e g leaf rolling is always associated with a reduction in yield potential in favorable conditions Site and environmental conditions Measurements can be taken under any environmental conditions However it is easier to make observations when the plant surfaces are dry and not wet from dew irrigation or rain 106 Physiological Breeding II A Field Guide to Wheat Phenotyping Time of day Measurements can be taken at any time of the day Observe leaf rolling two times during the day early morning before 10 00h when the plants are least stressed and in the afternoon between 13 00h and 16 00h when plants are most stressed Plant developmental stage Measurements should be taken at early grain filling in favorable conditions Observations should be taken from mid anthesis to mid grain filling Take both measurements and observations earlier in severely stressed conditions as plants will senesce more quickly Number of samples per plot For precision phenotyping take measurements observations of 10 plants culms per plot aim for 30 per treatment
127. either processed directly by the built in software or can be downloaded and imported into MS Excel Data is usually presented in five columns representing e Wavelength nm e White reference intensity in counts e Dark reference intensity in counts Intensity counts e Sample spectrum intensity in counts e Processed sample spectrum reflectance Plant canopy reflectance CR is calculated using the equation CR Sample Dark White Dark x 100 Equation 7 1 Typical results for radiation reflected from a wheat canopy in comparison with white and dark reference readings and canopy reflectance in two environments irrigated and droughted in NW Mexico are shown in Figures 7 3 and 7 4 respectively White Reference Sample Canopy Reflectance Dark Reference 400 437 473 509 545 580 615 650 684 717 751 783 816 848 879 910 940 970 Wavelength nm Figure 7 3 Radiation reflected from wheat canopy with white and dark reference readings Reflectance 100 90 80 70 60 50 40 Irrigated Droughted 400 437 473 509 545 580 615 650 684 717 751 783 816 848 879 910 940 970 Wavelength nm Figure 7 4 Canopy reflectance of wheat in irrigated and droughted environments in NW Mexico Spectral reflectance indices and pigment measurement 35 Troubleshooting Problem Solution After one hour of sampling the readings white reflectance are showing sat
128. eld porometer Figure 2 1 Take the following equipment to the field e Hand held porometer e Field form and clipboard Advice on taking measurements Remember that stomata are sensitive to physical manipulation so avoid physical stress contact with the leaf as much as possible Make measurements as quickly and accurately as possible as use of the porometer will alter the leaf surface and the boundary layer environment causing a drift in the conductance resistance value Note that stomata are also sensitive to light RH carbon dioxide water stress pathogens and pollutants and that agro chemical products affect stomatal responses c Figure 2 1 Using the Decagon SC 1 A top view showing chamber clamped at the mid point of the sample leaf B side view with the white Teflon disc clearly visible and C data output view showing the stomatal conductance reading of 471 5 mmol m st 16 Physiological Breeding II A Field Guide to Wheat Phenotyping Measurements should be made on the youngest fully emerged leaf receiving sunlight typically the flag leaf once fully expanded Be sure to select leaves which are exposed to the sun and not those in the shadow or shade as these will have very different readings to those leaves in the sun The leaves must be clean dry intact green with no sign of disease or damage Readings should be within 10 or approximately 50 mmol m st of each other if not then a furthe
129. ely depending on the soil type but only a reasonably small amount should be tolerated see Figure 10 8 The DGC action has now been modified with all modifications automatically saved for the next analysis 30 Now open several additional images throughout the trial e g photographs of plots 1 50 100 150 etc to test for consistency of recorded parameters over sampling time accurate color selection and B inaccurate color selection Photosynthesis and light interception 51 Running the DGC action To run the full DGC action on the sample images click on the PLAY SELECTION button at the bottom of the Actions palette This will play through all components of the DGC action and present a black and white image of the sample photograph Automatic batch image processing Once the DGC action is established all sample photographs for a batch i e site and or trial can be automatically processed The processing of images may be interrupted by pressing ESC at any time 31 Create an empty folder in the Adobe Photoshop directory C Program Files Adobe Adobe _ Photoshop_CS3 The program uses this folder to temporarily store images during processing which are then removed by the program 32 Ensure all data is removed from the Measurement log Figure 10 4 to do this click on the SELECT ALL MEASUREMENTS button at the top of the Measurements log palette then se
130. ely express data on a per area basis typically m7 It is often useful to mark the area to be harvested e g using colored spray paint to allow post harvest measurement Careful handling of fertile stems is important to avoid losses of grain from spike shattering and other plant organs especially the leaf lamina The bag should cover the entire sample and culms should be placed inverted into the bag with the spike at the bottom so as to avoid grain losses When cutting samples for biomass measurement it is important to cut stems as close to the ground as possible whilst avoiding inclusion of soil and roots In drought conditions it may be difficult to cut plants as they are easily uprooted in this case it is easier to cut the plants using clippers ensuring to remove any roots before placing the sample in the bag Detailed physiological studies often require partitioning of the canopy into individual organs i e leaf lamina all leaf lamina individual leaf layers leaf sheath stem internode lengths and peduncle and spike for the measurement of biomass and or nutrients content Partitioning is typically based on a sample of 220 fertile culms When sub sampling selecting culms care is needed to ensure that all plant material associated with the culms is included Note that nutrient analysis requires additional considerations see this volume Chapter 15 Direct growth analysis 95 Dry samples in an oven at 60
131. ements observations are important to assist the analysis and interpretation of physiological data and may help identify and explain data anomalies Figures 21 5 Crop Health a healthy experimental crop is essential to ensure quality data which represent the yield potential of the genotypes under trial in that particular environment Record incidence s of disease pests weeds including identification date and severity Development sowing and establishment dates and periodic recording of developmental stages especially leading up to heading anthesis and physiological maturity Effect of stress resulting from imposed stresses in experimental conditions drought heat and their interaction Damage caused by the weather e g frost environment e g drought tipping lodging pests e g aphids birds or diseases e g rust Husbandry applications of fertilizer herbicides pesticides and fungicides may affect crop physiology e g plant gas exchange and so records are essential for the planning of sampling measurement 124 Physiological Breeding II A Field Guide to Wheat Phenotyping Site Location name of site and physical location latitude and longitude coordinates Information soil depth texture toxicities organic matter content moisture distribution nutrient content and physical root barriers should be made before at planting gradients such as slope of the land etc Previous use
132. en leaf area Green normalized difference vegetation index Grain number m Grains per spike Ground cover Growth stage from Zadoks decimal scale Harvest index Infrared Infrared gas analysis Infrared thermometer Canopy coefficient Leaf area index Leaf water potential Normalized difference vegetation index Near infrared Near infrared reflectance spectroscopy Non photochemical quenching Normalized pheophytinization index Normalized water index 1 Normalized water index 2 Normalized water index 3 Normalized water index 4 OA OP PAR PDA PBD PI PRI PS RARS RARS RARS RGR RH RLD R NDVI RUE R S RW RWC RWD SC SIPI SLA SNO SPS SR SRa SRI SRL SS TDR TE TGW VI VPD WI WP WSC WU WUE Osmotic adjustment Osmotic potential Photosynthetically active radiation Palm top computer Peedee Belemnite Pigment related index Photochemical reflectance index Photosystem either or II Quadrat Ratio analysis of reflectance spectra chlorophyll a Ratio analysis of reflectance spectra chlorophyll b Ratio analysis of reflectance spectra carotenoid Relative growth rate Relative humidity Root length density Red normalized difference vegetation index Radiation use efficiency Root to shoot ratio Root dry weight Relative water content Root weight density Stomatal conductance Structural independent pigment index Specific leaf area Spike number m Spikel
133. en the start of heading and mid grain filling For stressed treatments the chlorophyll content is at a maximum earlier in the season the severity of stress and experimental conditions will determine the optimum time for these measurements Take measurements earlier in severely stressed conditions as plants will senesce quickly For the determination of stay green or senescence patterns measurements should start at mid grain filling and continue at regular intervals approximately every 4 7 days until physiological maturity Number of samples per plot Take three averages of five leaves per plot i e 3 x 5 leaves Procedure The following procedure describes taking measurements using the hand held Minolta SPAD 502 chlorophyll meter Figure 9 1 Take the following equipment to the field Hand held chlorophyll meter Field form and clipboard Advice on taking measurements Measurements are typically made on the flag leaf once fully expanded although measurements of lower leaves may be taken to assess canopy chlorophyll profiles The leaves must be clean dry intact green with no sign of disease or damage Consistency is very important Always place the adaxial upper surface facing upwards in the instrument Avoid placing the midrib major veins or particularly thick parts of the leaf in the chamber Typically take measurements a third to half of the way along the leaf from the stem insertion Figure 9 1B Re
134. ence it is possible to estimate the LAI GAI of a plot from observation Scoring i Place a quadrat in the plot to define an area a f z fi shy A z ao nbn B M Sylvester Bradley et al 2008 Credit The Home Grown Cereals Authority 60 Physiological Breeding II A Field Guide to Wheat Phenotyping ii Stand along the side of the plot so that the observer can look down directly over the crop iii Observe this defined area of crop iv Rate the LAI GAI by estimating in increments of 0 1 see Figure 12 1 Take repetitions sequential measurements approximately one week apart For post anthesis measurements it is also useful to take a close inspection of several individual culms to account for senescence in the lower canopy Trial measurements for assessment of senescence Senescence appears as yellowing which turns brown with time Canopy senescence starts with the lower leaves and progresses upwards to the flag leaf Individual leaf senescence in general starts at the tip and progresses towards the base finally reaching the leaf sheath Repeated observations e g every 10 days from mid grain filling to physiological maturity can be made to assess senescence rates For stay green or senescence studies where repeated measurements are to be taken on selected leaves it is highly recommended to mark each culm with colored tape around the peduncle to facilitate their re location 0
135. enotyping work these errors are of lesser importance compared with the large differences sought Site and environmental conditions Samples can be taken under most environmental conditions However it is important that the plant surfaces are not wet from dew irrigation or rain Take measurements in drought trials or when root access to water and or vascular capacity is limited in a high vapor pressure deficit VPD environment In general differences between LWP measurements in irrigated trials may be too small for genotypic discrimination Time of day Two samples should be taken during a 24h period e First from one hour before to two hours after solar noon when the plant is most water stressed 18 Physiological Breeding II A Field Guide to Wheat Phenotyping the results will be commensurate with the level of stress and e Second before dawn late night very early morning when the plant is least water stressed and has had the opportunity to recover the results will indicate the ability of the plant to rehydrate and reach an equilibrium with soil water potential Plant developmental stage Samples can be taken at any developmental stage from mid stem elongation to late grain filling depending on the experimental objectives timing of peak stress For instance in drought trials sampling is performed at early grain filling as an assessment of the stress severity Number of samples per plot Take 2 4 leaves pe
136. environmental conditions For active sensors measurements can be taken under any light conditions for NDVI sensors without a light source take measurements on a clear sunny day Take measurements when there is negligible wind as even a light wind can modify canopy structure It is important that the plant surfaces are dry and not wet from dew irrigation or rain Time of day For active sensors measurements can be taken at any time of day For NDVI sensors without a light source take the majority of measurements as close to solar noon as possible typically from 11 00h to 14 00h Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from the emergence to physiological maturity depending on the experimental objectives timing of peak stress To compare between genotypes do not take measurements during heading and anthesis where differences in phenology may confound results e Early vigor take three measurements at 5 10 and 15 days after emergence DAE to rank genotypes It is recommended to use the same seed source for all genotypes as seed from different environments may present variation in establishment which may confound analysis Figure 8 1 A Greenseeker NDVI portable x sensor B in field use at GS31 and C in field a use during grain filling Spectral reflectance indices and pigment measurement 37 e Biotic and abiotic stress detectio
137. es DW dry weight Laboratory measurements Data and calculations 6 Weigh all sample tubes tube W FW 7 Add 1 cm of distilled water to each tube Figure First obtain the fresh weight FW of the leaf samples 5 1D FW tubeW FW tubeW Equation 5 1 8 Place the sample tubes in a refrigerator at 4 C in Then calculate the leaf RWC darkness for 24h for leaves to reach full turgor Leaf RWC FW DW TW DW x 100 Equation 5 2 Fi 5 1E Figure Where FW fresh weight TW turgid weight DW dry weight Figure 5 1 Measuring the leaf relative water content A weighing empty tubes B select and cut leaves in the field C cut the top and bottom of all leaves together D tube containing leaf samples filled with 1 cm of distilled water E sample tubes in the refrigerator F carefully blot dry the turgid leaf samples and G dried leaf samples 26 Physiological Breeding II A Field Guide to Wheat Phenotyping Worked example Table 5 1 Calculating the leaf relative water content of severely droughted flag leaves during early grain filling Tube Tube weight Plot weight g fresh weight g Fresh weight g Turgid weight g Dry weight g Leaf RWC 1 12 065 12 730 0 665 0 985 0 292 53 8 2 12 111 12 920 0 809 1 322 0 350 47 2 3 12 022 12 833 0 811 1 086 0 345 62 9 Typical values of RWC range between 98 in turgid and transpiring leaves to about 40 in severely desiccated and senescing leaves leaf
138. es or heat drying at a precise temperature and duration Ensure to check the specific procedural requirements of the laboratory Figure 22 1 Drying ovens A large capacity forced draft oven and B small capacity non draft oven suitable for drying open container samples e g soil moisture samples 3 Accurate weighing of samples It is essential that accurate weights are recorded for sampled material Poor weighing technique and or incorrect use of the balance will cause significant data errors either consistent e g due to not removing the bag TARE weight or random e g due to irregularly cooled oven dried samples Note that all balances are sensitive to changes in the environment and that laboratory balances both precision and analytical are more sensitive than field scales i e battery powered bench balance or spring mechanical scale Follow manufacturer s instructions for installation and e Keep level use inbuilt spirit level e Keep ona stable non vibrating surface e g a concrete plinth e Avoid areas near heaters ovens or air conditioners e Avoid direct sun and air flows e Avoid sharing power circuits with high consumption items e g a Microwave oven It is essential to select the type of balance according to the capacity and resolution demanded Table 22 2 Figure 22 2 It is often observed that samples are weighed on inappropriate balances e g weighing stems from the
139. escence Irrigated Stressed F F i Close to 0 83 lt 0 75 psi 0 4 0 5 lt 0 4 NPQ 0 5 3 5 gt 3 5 Non photochemical quenching NPQ estimates the non photochemical quenching from F to F To monitor the apparent rate constant for heat loss from PSII References Fracheboud Y 2006 Using chlorophyll fluorescence to study photosynthesis Institute of Plant Sciences ETH Universitatstrass CH 8092 Zurich Long SP and Bernacchi CJ 2003 Gas exchange measurements what can they tell us about the underlying limitations to photosynthesis Procedures and sources of error Techniques 54 392 2393 2401 Flexas J Diaz Espejo A Berry JA Cifre J Galm s J Kaldenhoff R Medrano H and Ribas Carb M 2007 Analysis of leakage in IRGA s leaf chambers of open gas exchange systems quantification and its effects in photosynthesis parameterization Journal of Experimental Botany 58 6 1533 1543 Rodeghiero M Niinemets U and Cescatti A 2007 Major diffusion leaks of clamp on leaf cuvettes still unaccounted how erroneous are the estimates of Farquhar et al model parameters Plant Cell and Environment 30 8 1006 1022 Useful references Evans JR and Santiago L CSIRO Publishing A guide to measuring gas exchange and performing A PAR and A C curves with the LI COR 6400 Available at http prometheuswiki publish csiro au accessed 30 August 2011 Sharkey TD Bernacchi CJ Farquhar
140. essure is greater than the maxima of the equipment record as lt e g lt 40 Solution Put oil or grease on the thread of the chamber valve connecter to ease opening Ensure that the leaf samples are correctly inserted into the rubber seal Check integrity of rubber seal clean or change as necessary Large difference in values between leaves Cut surface of the leaf is not sufficiently Ensure to choose healthy flag leaves from main culms Ensure that the air inside the chamber is kept moist using moistened gauzes Ensure to cut samples carefully from the plant do not clean or level Useful references PMS Instrument Company 2011 Available at http www omsinstrument com accessed 12 August 2011 Soilmoisture Equipment Corp 2011 Available at http www soilmoisture com accessed 12 August 2011 20 Physiological Breeding II A Field Guide to Wheat Phenotyping re cut leaf sample as this will affect values Turner NC 1988 Measurement of plant water status by the pressure chamber technique Irrigation Science 9 289 308 Turner NC and Long MJ 1980 Errors arising from rapid water loss in the measurement of leaf water potential by the pressure chamber technique Functional Plant Biology 7 527 537 Chapter 4 Osmotic adjustment Carolina Saint Pierre and Vania Tellez Arce Osmotic adjustment OA refers to the net increase in cell solute concentration in order to maint
141. et from dew irrigation or rain Measurements for non destructive sampling can be taken under any environmental conditions Time of day Samples for destructive sampling should be taken in the morning to allow for same day processing Measurements for non destructive sampling can be taken at any time of the day in irrigated crops but in droughted treatments should be taken at the coolest part of the day before leaf wilting affects leaf area Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from emergence to mid grain filling for LAl and GAI and from mid grain filling to physiological maturity for assessment of senescence and stay green depending on the experimental objectives timing of peak stress e Early vigor take three of non destructive assessments i e normalized difference vegetation index photography or visual assessment at 5 10 and 15 days after emergence DAE to rank genotypes It is recommended to use the same seed source for all genotypes as seed from different environments may present variation in establishment which may confound analysis e Canopy expansion take non destructive assessments every 7 10 days between the start of stem extension and the end of booting e Maximum leaf crop green area take a destructive measurement typically using the biomass sample at anthesis 7days e Senescence stay green and duration of grain fillin
142. ets per spike Spectral reflectance Simple ratio a Spectral reflectance indices Specific root length Sub sample Transpiration Time domain reflectrometry Transpiration efficiency Thousand grain weight Vegetation index Vapor pressure deficit Water index Water potential Water soluble carbohydrates Water uptake Water use efficiency SJ9 9WUI U99 Ol LI Al l vi gi o LL 8l 6l 0c Le CG c Vo Ge 9 Le BMZ eee ne i E i r a a F E m
143. evelopmental stage It is important to plan sampling schedule in advance to account for this This sub sample should reflect the consistency of the biomass sample Culms should have a stem but not necessarily a spike It is important that all the material associated with each culm is retained when cutting and or sub sampling In the field ensure to check carefully the quadrat area and collect any fallen material after cutting Cut material can be stored at 4 C for up to 4 days before processing do not store samples for WSC analysis It is much easier and quicker to remove the leaf sheath from the stem after drying omass cultivation Macmillan Publishers Australia Available at http plantsinaction science ug edu au edition1 q content 6 1 2 plant biomass accessed 20 December 2011 Monteith JL 1994 Validity of the correlation between intercepted radiation and biomass Agricultural and Forest Meteorology 68 213 220 82 Physiological Breeding II A Field Guide to Wheat Phenotyping Chapter 16 Water soluble carbohydrate content Julian Pietragalla and Alistair Pask Water soluble carbohydrates WSC are sugars such as fructans sucrose glucose and fructose which are accumulated in the stem as reserves WSC accumulate up to and around anthesis and are partitioned to the stem from where they are later available as a reservoir for remobilization to the developing grains These reserves are an important source
144. fer et al 1996 as additional grains are located in more distal florets and or spikelets with lower grain weight potential Wheat has the ability to mutually compensate yield through the sequential development of the components and high yields are often attainable by diametrically opposite routes For instance should the plant number m7 be low e g due to poor establishment then an increased survival of fertile tillers will maintain SNO Site and environmental conditions Samples can be taken under most environmental conditions It is important that the plant surfaces are dry and not wet from dew irrigation or rain Time of day Samples can be taken at any time of the day Grain losses can be reduced by sampling in the morning when the spike moisture content is slightly higher Plant developmental stage Take samples as soon after physiological maturity GS87 as possible Higher culm spike moisture content compared with the harvest ripe stage GS92 will reduce losses of biomass e g leaf lamina or grain due to brittleness and shattering Number of samples per plot Harvest either a large area of the plot Methods A and B or a smaller defined area 21 m Method C Procedure Advice on taking measurements Remove buffer rows and the ends 50 cm of the plot prior to harvesting Methods A and B Ensure to accurately measure the harvested area length and width count number of harvest rows in order to accurat
145. g duration take non destructive assessments twice weekly between mid anthesis for stressed crops mid grain filling GS75 for favorable conditions and physiological maturity Number of samples per plot Take either one sample of 20 fertile stems or one observation per plot see individual measurements below A Destructive measurements with an automatic planimeter The following procedure describes the determination of LAI and GAI from in season biomass samples taken at anthesis 7days using an automatic planimeter See Schematic 12 1 Procedure The following procedure describes the determination of leaf and green area using a sub sample from the in season biomass sample as detailed in this volume Chapter 15 The following equipment is required e Secateurs knife e Automatic planimeter e Calibration discs Advice on taking measurements When using the automatic planimeter take care to ensure that the plant material passes through the sensors of the machine and is flat i e not folded twisted For non flat surfaces such as stems and spikes take the planar area rather than the total area of green surface this is better correlated with light interception If an automatic planimeter is not available a standard computer scanner and appropriate software can be used or the individual plant component can be measured for both length and width which are strongly associated with area If it is necessar
146. g a TARE container is to i e Samples for precision weighing may be kept ina subtract the average DW weight of the containers desiccator after drying only appropriate for use 10 empty containers to do this or ii to weigh small quantities individual containers as for the aluminum pots in the e Distribute the weight of the sample evenly across the determination of soil moisture content this volume balance plate Chapter 17 e For small samples lt 20 g carefully empty the sample from the container i e the bag envelope etc into 4 Typical ranges and units a specific weighing container and remember to It is recommended to keep all measurements in the subtract the container weight from the total weight same unit system typically on the decimal scale e For samples gt 20 g keep the sample in its container to REAA ane a avoid losses and remember to TARE the container weight Table 22 3 Useful units of measurement Removing the container weight by using a TARE Multiple Area length Weight When weighing samples in containers e g a bag 1 000 000 s Ton t envelope tube etc remember to first TARE this weight 10 000 Hectare ha so that the weight of this container is deducted from the 1 000 Kilogram kg gross weight to give the sample weight This is typically il Meter m m Gram g appropriate for samples gt 20 g 0 01 Centimeter cm To do this 0 001 Millimeter mm Milligram mg
147. harvest Determination of the plant number m7 A count of the number of plants m should be made after the maximum number of plants has emerged and before tillering occurs typically 5 days after the end of emergence Occasionally the plant number m may decrease through the season e g winter kill in which case a second count is advisable at GS31 Plant number m typically varies between 50 and 300 plants m It has a broad optimum which varies with variety conditions and environment In the field 1 Randomly select two representative areas of the plot 2 Place a 0 25 m quadrat in each area and count the number of plants within the quadrat Table 18 2 Formulas for calculating yield biomass and harvest index using the three different harvesting methods Method Yield g m A Total biomass harvest FW_HA_G x DW_HA_SS_G FW_HA_SS_G DW_SS_G HA FW_HA x DW_SS FW_SS HA yield biomass Biomass g m Harvest index B Sub sample harvest FW_HA_G x DW_HA_SS_G FW_HA_SS_G DW_GB_G HA yield HI DW_GB_G DW_GB C Reduced threshing harvest Biomass x HI FW_HA x DW_SS FW_SS HA DW_SS G DW_SS Where FW fresh weight DW dry weight SS sub sample GB grab sample G grain HA harvested area m7 Formulas assume that grain is dried to 0 moisture Table 18 3 Example data for the three presented methods for yield biomass and harvest index determination Method A To
148. he cylinder to discharge slowly 2 Calibrate the IRGA to zero and once empty it can be safely removed o The chamber must be empty and closed 1 After turning the gas exchange photosynthesis system o Fresh CO and H O desiccants must be in full on the instrument should be allowed to warm up for bypass setting around 20 minutes o Wait until the reference CO is close to 5 umol molt Check the following parameters and the reference H O is close to 0 3 mmol mol o Pressure set to 100 kPa exact pressure varies o If CO RorCO Sare gt 5 orH O RorH O Sare according to altitude check user manual gt 0 3 go to CALIBRATION MENU gt ZERO IRGA o Light check that this is working and that the LEDs are and follow the instructions Wait until the values are stable first zero H O and wait for 1 minute to stabilize and then zero CO and again wait for 1 minute to stabilize o Return to MAIN MENU and select MATCH IRGA so that both IRGAs sample and reference are calibrated with the same values not damaged Thermocouple check that this working by touching the sensor with a finger Then disconnect thermocouple to check that the leaf temperature is equal to the block temperature T2Leaf T2block if not adjust accordingly te P B 5 j gt i i Figure 13 5 Sensor head of a portable photosynthesis system LICOR LI 6400XT showing A vii leaf chlorophyll fluorometer LCF optional extra
149. heath attached and spike using the automatic planimeter After area measurement the material can be further processed e g for dry weight nutrient content etc as detailed in this volume Chapter 15 Remember to return to the sample any yellow dead material which has been removed A Ean Be are Le Figure 12 1 Visual estimation of green area index GAI showing GAls of A 0 9 B 2 0 and C 4 0 Images reproduced from B Non destructive measurements The following procedure describes the visual assessment of crop LAI and or GAI and senescence For measurements using techniques based onlight interception see this volume Chapters 8 NDVI and 11 Light interception Procedure Take the following equipment to the field e Scale for LAI GAI Figure 12 1 or leaf senescence scoring Figure 12 2 e Field form and clipboard Advice on taking measurements As observations are subjective so it is important that ratings are consistent e Ensure that the ratings of new observers are calibrated with those of an experienced observer who is familiar with assessing ground cover so that values are standardized e If several people within the group will be making observations it is recommended that all observers meet to calibrate their readings before starting and regularly thereafter e Ensure that only one person makes observations within a replicate Trial measurements for LAI and or GAI With experi
150. heck that the camera is set to take 640 x 480 resolution photographs Higher resolution will increase file size and slow computer processing Do not use the zoom on the camera maintain in the no zoom position Do not include feet and shadows in photographs When sampling from a large trial separate each row by taking a photograph of the sky at the end of each row this will facilitate orientation and minimize errors if plots are accidentally skipped or repeated Once the whole trial has been photographed take three photographs of the sky to indicate the end Trial measurements 2 Stand along the side of the plot and take photographs looking down over the plot Maintain a consistent height above the ground typically 1 m which captures the maximum amount of the plot without including any neighboring plots in the photograph Take photographs in a vertical direction along the plot and ensure the camera is held centrally above the crop bed Figure 10 2 Download photographs onto a computer in preparation for digital processing Figure 10 1 Visual ground cover ratings and corresponding percentage cover A 1 10 cover B 3 30 cover C 5 50 cover and D 9 90 cover Photosynthesis and light interception 47 Processing of digital ground cover photographs using Adobe Photoshop Photographs can now be analyzed to obtain a score for digital ground cover DGC Software Use A
151. humidity pen allow the instrument to equilibrate with the ambient temperature for at least three minutes or as much time required for the readings to stabilize then record the air temperature and RH During this time ensure that the instrument is kept in the shade and not exposed directly to the sun Trial measurements 3 Take two canopy temperature readings of each plot Final measurements and completion 4 After measuring all plots record the finish time and re record the air temperature and RH Box 1 1 Calibrations There is no need to calibrate the IRT However as readings are subject to in field user judgment i e to accept or reject the reading it is sometimes useful to have an idea of the upper and lower CT thresholds between which the crop CT readings should lie This can be done by spraying i a transpiration inhibitor and ii water onto two different areas of a BORDER plot of the trial you are testing waiting for three minutes and then measuring their CT The two readings serve as reference readings for no transpiration transpiration inhibitor upper CT and maximum transpiration water lower CT Data and calculations CT readings depend on the environment in which the measurements were taken there are as many responses in CT as there are environments It is therefore a relative measurement Generally the good genotypes are those which have relatively cooler canopies than gen
152. ide Prepare labeled paper bags for oven drying large bags for 50 culm sub sampling medium bags for 20 culm spike sub sampling and small bags for partitioning Punch holes in the bags to increase oven drying efficiency e g using a hole punch and ensuring you have a similar hole pattern in every bag Figure 15 1D toes a d i l Po eer r ka pn ig w 7 i Ea a x n s a i Mag i 7 Ly Ads m P fi i 4 T Ei Field measurements 3 Select a representative length of the plot avoiding borders see Figures 15 1A and B Use the quadrat to cut the exact area of crop from the plot Immediately put the cut culms into the black plastic bag check label with plot number ensure that all the biomass is carefully collected and be careful not to include soil or roots in the sample Immediately place bagged samples in the shade do not allow samples to sweat in the sun this may cause water to condense on the inside of the plastic bag and plants may lose moisture unevenly Once you have finished sampling the plots start laboratory processing as soon as possible Figure 15 1 Sampling for in season biomass A cutting a 50 cm quadrat sample at anthesis 7days B using a quadrat to cut a sample at mid grain filling in a drought treatment C samples are immediately placed into the labeled black plastic bag and D paper bags with hole punches to increase oven drying efficiency Direct g
153. ikes per plot at least 20 spikes per plot and aim for 60 100 spikes per treatment Solution Take samples when spike moisture content is higher such as soon after physiological maturity and or in the morning Adjust air flow through the thresher Dry spikes more thoroughly to reduce moisture content to make them easier to thresh either by harvesting at grain ripe stage during a dry day and or in the afternoon Adjust rotor and or cylinder speed of thresher References Slafer GA Calderini DF and Miralles DJ 1996 Generation of yield components and compensation in wheat Opportunities for further increasing yield potential In Reynolds MP Rajaram S and McNab A Eds Increasing Yield Potential in Wheat Breaking the Barriers pp 101 133 CIMMYT International Symposium Mexico D F CIMMYT Useful references Slafer GA 2003 Genetic basis of yield as viewed from a crop physiologist s perspective Annals of Applied Biology 142 117 128 Dolferus R Ji X and Richards RA 2011 Abiotic stress and control of grain number in cereals Plant Science 181 4 331 341 103 Direct growth analysis Crop observations Chapter 19 Crop morphological traits Araceli Torres and Julian Pietragalla Crop morphological characteristics can be quickly easily cheaply and non destructively observed or measured in the field to give quantitative trait data which can be related to yield yield potential
154. il 2 3 4 0 10 20 30 40 50 Observe whole canopy senescence either by e Making a general observation by standing at a 45 angle alongside the plot or e Randomly selecting 10 main stems per plot aim for 30 per treatment and counting the number of green partially green leaves from the flag leaf downwards e g 3 5 Scoring i Observe senescence of individual leaves typically the flag leaf or on different leaf layers within the canopy i e flag leaf leaf two etc ii Rate using a scale from 0 0 senescence to 10 100 senescence using the guide in increments of 10 Figure 12 2 Data and calculations LAI total leaf lamina area for 20 stems x number of stems per m 20 Equation 12 1 GAI total green area for 20 stems x number of stems per m 20 Equation 12 2 SLA Leaf dry weight LAI Equation 12 3 Typical values for LAI and GAI at anthesis 7days in non stressed conditions are 4 5 and 6 0 and in stressed conditions are 2 0 and 2 5 respectively Specific leaf area SLA g m7 is typically around 1 g of leaf DW per m of leaf green area 6 7 8 J 10 60 70 80 90 100 Figure 12 2 Flag leaf senescence scale indicates approximate senescence Credit The John Innes Centre and The University of Nottingham Photosynthesis and light interception Troubleshooting Problem The automatic planimeter is counting area before plant material is
155. in very sandy soils Ensure that the paper wrapping the roots is kept moist during processing Chapter 18 Grain yield and yield components Julian Pietragalla and Alistair Pask The grain yield yield is the ultimate expression of the many individual physiological processes which have interacted with the weather and environment during the crop s growth cycle Its accurate measurement is required to demonstrate significant association between physiological characteristics and productivity The determination of grain yield and its components spike number m SNO plant number m x fertile tillers per plant grain number m GNO spike number m x grains per spike spikelets per spike SPS x grains per spikelet and thousand grain weight TGW g is therefore essential for all breeding and physiology trials Although determination is typically performed on destructively harvested samples some in field assessments are also possible both approaches are discussed in this chapter An understanding of yield components and yield compensation strategies of a wheat crop in a particular environment is the key for a successful breeding program The three components of yield are developed sequentially during crop development first SNO then GNO and finally grain weight The number and potential weight of grains determines the sink size of the crop Generally a negative relationship is observed between GNO and TGW e g Sla
156. ing that a 500 plot trial can be screened in around 90 or 45 minutes respectively A Visual assessment Trial measurements With experience it is possible to estimate the crop cover of a plot from observation As observations are subjective it is important that ratings are consistent e Ensure that the ratings of new observers are calibrated with those of an experienced observer who is familiar with assessing ground cover so that values are standardized e If several people within the group will be making observations it is recommended that all observers meet to calibrate their readings before starting and regularly thereafter e Ensure that only one person makes observations within a replicate Scoring i Stand along the side of the plot so that the observer can look down directly over the crop ii Observe the crop It is sometimes useful to look at the crop through a circle formed by the thumb and index finger held 10 cm from the eye iii Rate the crop cover using a scale from 0 0 to 10 100 by estimating the percentage cover in increments of 10 see Figure 10 1 B Digital assessment Preparations 1 Before taking photographs record the full details of the trial and include the name of the person taking the photographs e g include this information on a photograph which is grouped with the sample photographs e Ensure that the camera batteries are charged and take spare batteries C
157. irectly downwards over the white reference panel at a set distance usually 60 200 cm and set the integration time manually to keep the peak of white reference reflectance reading between 75 and 85 of the maximum so that reflectance value is not saturated at any wavelength when taking a white reference reading yet is not too low for data interpretation During sampling regularly re take white reference measurements every 15 30 plots see details below Trial measurements 4 Take a dark reading to establish the lower reference point for the device Figure 7 2A Completely cover the end of the fiber optic so that no light is captured by the spectrometer This reference line will then be a straight line at 0 i e zero reflectance The frequency of dark readings will depend on the length of time that the instrument has been running When the instrument is initially turned on the dark reading should be taken every 10 minutes as the base line measurements will change as the instrument warms to operating temperature Once this is reached a dark reading should be re taken at the same time as the white reading Figure 7 2 Taking readings with a field spectrometer A dark reading B white reading and C canopy reading 5 Take a white reading to establish the upper reference point for the device Figure 7 2B e Hold the probe vertically above the center of the white reference plate
158. is very obviously not representative Do not restrict sampling to one part of the plot Distribute samples around the plot to include as much of the plot as possible e g in a two row raised bed design it would be advisable to sample equally from all rows both beds Figure 21 1 Random stem plant sampling within different planting systems A one raised bed with two rows of plants B two raised beds each with two rows of plants and C flat broadcast planting with eight rows of plants 121 General recommendations f least 50 cm r a l l l m A B Plot border Sample area E 1 a Selected sample area Figure 21 2 Random and systematic quadrat sampling within different planting systems A one raised bed with two rows of plants B two raised beds each with two rows of plants and C flat broadcast planting with eight rows of plants A Quadrat sampling Figure 21 3 Sampling approaches A quadrat sampling B grab sampling and C area sampling 122 Physiological Breeding II A Field Guide to Wheat Phenotyping 3 Taking measurements and observations The following points should be taken into consideration for accurate and representative measurements observations and results It is important to maintain uniform approach Do take samples measurements as accurately and consistently as possible to reduce experimental error increase comparability between data and r
159. ise control of environmental variables such as CO and H O concentrations temperature and light Easier and faster than gas exchange photosynthesis systems Excellent for large screenings and plant response to stresses Relatively quick and easy to observe Integrative absolute measurement Sampling can be combined with biomass sampling simple approach Direct measurement of soil water content and crop water uptake Allows assessment of field grown crops Integrative absolute measurement Relates yield to physiological processes through growth Quick easy and cheap to measure no instruments required non destructive Observations only no requirement for instruments Disadvantages of tool Precision phenotyping only not suitable for large screenings operators need to be highly trained Training is essential Observations are subjective training is essential Time consuming and laborious requires large Capacity drying ovens Stem carbohydrates are rapidly respired samples need to be processed quickly sample analysis is typically outsourced Hand coring is laborious soils are heterogeneous and require multiple sampling As above and root washing and preparation for scanning is laborious Laborious harvesting and threshing machinery required Laborious Observations are subjective so training is essential Observations are Subjective so training is
160. ithin a plot taking into account all harvested rows until a defined number of culms plants or weight is reached This method reduces the in field sample volume 130 Physiological Breeding II A Field Guide to Wheat Phenotyping Harvest index is the ratio of grain yield to above ground biomass Minimum tillage with a limited number of passes of machinery it aims to achieve some soil disturbance and physical weed control but to leave much of the crop residues on the surface of the soil or in the surface layers Phenology is the occurrence of events during the life cycle of the plant e g the date of the initiation of flowering Phenotype is the sum of the observable characteristics of a wheat plant crop such as its morphology development biochemical and physiological properties It is an expression of both the genotype and environment Photosynthetically active radiation is the proportion of the light spectrum that can be used by plants for photosynthesis it has wavelengths between 400 blue and 700 nm red Plant water status is a description of the water content of a plant leaf in relation to that required for optimal growth Population is a collection of wheats for breeding or experimental purposes usually from common parentage e g F1 population Senescence is the loss of greenness in photosynthetic tissues normally brought about by aging but also by disease or stress Sink potential is the capaci
161. ke inset can be further partitioned At anthesis Anther The part of the flower that produces the pollen Carpel The part of the flower containing the ovule which develops into the seed Floret An individual flower within the spike enclosed by the lemma and palea Glumes The pair of bracts located at the base of a spikelet in the head Rachis The main axis of the spike Spikelet The flower of a grass consisting of a pair of glumes and one or more enclosed florets At harvest Chaff All the spike structures except grain Grain The seed also called kernels spikelet 6 spikelet 5 spikelet 4 spikelet 3 spikelet 2 spikelet 1 Figure 23 1 Parts of the wheat plant showing the main culm and its component organs 132 Physiological Breeding II A Field Guide to Wheat Phenotyping Abbreviations A Photosynthesis CCI Chlorophyll concentration index 0 99 9 CGR Crop growth rate CHL Chlorophyll CID Carbon isotope discrimination CIMMYT International Maize and Wheat CT DAE DAS DAA DGC DIM DW ETR FW GAI GB GLA G NDVI GNO GPS GC GS HI IRGA IRT LAI LWP NDVI NIR NIRS NPQ NPQI NWI 1 NWI 2 NWI 3 NWI 4 Improvement Center Canopy temperature Days after emergence Days after sowing Days after anthesis Digital ground cover Days to maturity Dry weight Electron transport rate Light radiation intercepted Fresh weight Green area index Grab sample Gre
162. l Extension Service Zuber U Winzeler H Messmer MM Keller M Keller B Schmid JE and Stamp P 1999 Morphological traits associated with lodging resistance of spring wheat Triticum aestivum L Journal of Agronomy and Crop Science 182 17 24 Crop observations 117 General recommendations Chapter 21 General recommendations for good field practice Alistair Pask and Julian Pietragalla It is important for researchers to have clearly defined plan carefully taking into account the time and experimental objectives in order to correctly select resources available for accurate and repeatable field the most appropriate experimental design sampling measurements method and choice of measurements Ensure to 1 Experimental design for the physiological characterization of germplasm Choice of target environment that is appropriate to the objectives of the experiment i e temperature profile daily radiation rainfall latitude soil type etc and gives appropriate treatments i e sowing dates crop management etc It is advisable to replicate trials across a number of locations within the target environment Choice of germplasm considerations when selecting material should include i general adaptation to the target environment ii acceptable range of phenology iii acceptable agronomic type iv pest and disease resistance v genetic and trait diversity vi not contrasting in genes for height
163. l now be displayed check that this is representative of the color image 17 In the menu bar select ANALYSIS gt SELECT DATA POINTS gt CUSTOM 18 Select ONLY DOCUMENT and GREY VALUE MEAN from the list of Data Points remove all other options and click OK 19 On the menu bar select SELECT gt ALL 20 On the menu bar select ANALYSIS gt RECORD MEASUREMENTS Completion of creating an action 21 Stop recording the action by clicking the STOP PLAYING RECORDING button at the bottom of the Actions palette Figure 10 4 Lolor Haire Figure 10 6 Example color range selection 50 Physiological Breeding II A Field Guide to Wheat Phenotyping 22 The list of actions which have been performed to record DGC will now be able to be viewed from the drop down menu in the Actions palette Figure 10 7 Testing and adjusting the DGC action Prior to the automated processing of sample photographs it is important to first test the accuracy of the DGC action on several representative photographs and make adjustments In particular the Hue Saturation color adjustment and the Color Range selection process variables may need to be calibrated for different environments this calibration becomes quicker and easier with experience 23 View the components of the DGC action in the Actions palette this is divided into 7 steps Figure 10
164. le making sure that these are all well connected CO and H O desiccants to full bypass and check with no leaks that the flow rate does not change then turn the e Check that the instruments have sufficient memory to CO and H O desiccant to full scrub and check again that the flow does not change If the flow rate does change more than 1 2 units then check that the air mufflers in the chemical tubes are not blocked or broken Now set the flow rate to zero and switch off the leaf fan If the flow value at this stage is not close to zero go to the calibration menu and re zero the flow meter save all measurements Ensure that the drierite water desiccant and soda lime CO absorbant scrubber are fresh and reactive These chemicals typically have color indicators to show their condition drierite turns from blue to pink and soda lime turns from white to lilac when no longer useful o Check that there are no leaks breathe near the chamber CO and H O desiccant connection tubes and console Check that CO values do not increase more than 2 ppm If so try to localize the leak by breathing through a plastic straw e Remove the previous carbon dioxide cylinder and attach a new one Check the status of the O rings replace with new ones if these are swollen Be careful to never remove a full cylinder from the console as the gas will be released at high pressure which can be dangerous It is advisable to allow t
165. lect DELETE SELECTED MEASUREMENTS 1 33 Close any open images 34 On the menu bar select FILE gt AUTOMATE gt BATCH 35 In the Batch window enter the options shown in Box 10 1 click OK The program will now start to process all of the images in the selected folder and display data in the Measurements log window BOX 10 1 Settings for automatic processing Set Action Play Folder Choose Source Data processing The data recorded in the Measurements log now need to be imported into a MS Excel spreadsheet so that it can be used to calculate the actual percentage ground cover 1 Open the Measurements log window 2 Click on the SELECT ALL MEASUREMENTS button 3 Click on the EXPORT SELECTED MEASUREMENTS button Z 4 Use the SAVE WINDOW to save the data as a TXT Tab delimited file 5 Open a MS EXCEL spreadsheet 6 Import the TXT document using the Text delimited option The percentage ground cover is calculated as a proportion of the Mean Grey Value compared to Mean Grey Value if the image were completely white 255 given that the Mean Grey Value of a completely white image is 255 100 cover and the grey value fora completely black image is 0 0 cover 7 The spreadsheet should consist of two columns of data Column 1 is the image name and Column 2 contains the Mean Grey Value of the image 8 Ina third column
166. ling 2 Using a pair of scissors cut the newest fully expanded leaf from each of 10 20 plants 3 Put the cut leaves into a pre labeled paper bag Grain sampling Or collect approximately 2 5 g of grain per plot To provide a well mixed sample this should be taken from the bulk grain after plot harvest Laboratory measurements Preparation of samples for analysis 4 Oven dry leaf samples at 75 C for 48h as soon as possible after the samples have been collected grain samples may already be sufficiently dry but can be also oven dried 5 Grind the leaf grain sample e g using a sample mill with a 0 5 mm screen Ensure to clean the mill carefully between samples using a compressed air hose 6 Place ground sample into a labeled envelope 7 Store samples at room temperature in a dry place Carbon isotope analysis by mass spectrometry Mass spectrometry analysis of samples is typically out sourced and performed by a specialist laboratory In brief a small homogenized and accurately measured quantity of the solid sample 1 5 mg is heated to high temperatures 1 400 1 800 C to produce CO and N gases The isotopic forms of carbon and nitrogen are measured by the isotope ratio mass spectrometer Ensure to check the specific procedural requirements of the laboratory Data and calculations Calculation of carbon isotope composition 673C Mass spectrometers generate differential values of stable carbon isotope c
167. little resultant effect on the yield whilst late frosts to the same crop between the onset of stem extension and flowering may damage the spike either the florets causing sterility or the grains causing shriveling causing a reduction in yield Three types of in season damage are discussed i Spike tipping appears as the premature senescence of the upper half of the spike typically occurring at around spike emergence in stressed environments or after adverse weather conditions e g frosts It is a common feature in drought environments where it may act as an escape mechanism by reducing the grain number and therefore the spike demand during grain filling However should the drought pass then the permanent tipping effect reduces the yield potential ii Lodging is the permanent displacement of plant stems from the vertical resulting in stems leaning or lying horizontal on the ground It is typically caused by strong winds and or excess water causing a very wet soil either from precipitation or irrigation in combination with tall and thin stems and or root or stem rots which weaken the plant base Lodging is an undesired trait and it is usually expressed under high yielding or favorable conditions during late grain filling iii Vegetative damage caused by adverse weather conditions pests and or diseases which may damage all of the above ground parts of the plant throughout the growth cycle It is important to r
168. ltivation Agriculture Conservation AGRIS CATEGORY CODES F01 Crop Husbandry F30 Plant Genetics and Breeding F63 Plant Physiology Reproduction Dewey Decimal Classification 631 531 PAS ISBN 978 970 648 182 5 Design and layout Marcelo Ortiz S Eliot Sanchez P and Miguel Mellado Front cover photographs in order from top left Measuring canopy temperature with an infrared thermometer Alistair Pask Measuring stomatal conductance with a hand held porometer Mary Attaway Measuring leaf chlorophyll content with a Minolta SPAD 502 chlorophyll meter Julian Pietragalla Taking light interception measurements with a hand held ceptometer Julian Pietragalla Soil coring using a tractor mounted hydraulic corer Alistair Pask Breeder in a wheat field in NW Mexico Petr Kosina Back cover photograph Measuring leaf chlorophyll content at the Ayub Agricultural Research Institute Faisalabad Pakistan Muhammad Shahbaz Rafique Index Introduction 2 Introduction Matthew Reynolds Alistair Pask and Julian Pietragalla Canopy temperature stomatal conductance and water relation traits 10 15 18 21 25 28 Chapter 1 Canopy temperature Julian Pietragalla Chapter 2 Stomatal conductance Julian Pietragalla and Alistair Pask Chapter 3 Leaf water potential Carolina Saint Pierre and Jos Luis Barrios Gonz lez Chapter 4 Osmotic adjustment Carolina Saint Pierre and Vania Tellez Arce Chapter 5 Leaf relative wa
169. lume canopy temperature and stomatal conductance correlated with photosynthetic rate Chapters 1 and 2 carbon isotope discrimination to integrate stomatal aperture over the lifetime of the crop Chapter 6 vegetative indices which correlate with the size of the photosynthetic canopy Chapter 7 chlorophyll content for photosynthetic potential Chapter 9 senescence stay green loss maintenance photosynthetic capacity Chapter 12 biomass for cumulative photosynthetic activity over the lifetime of the crop Chapter 15 and water soluble carbohydrates for the accumulation of photosynthates Chapter 16 Site and environmental conditions Take measurements when the sky is clear and the leaves are well illuminated Measurements can still be taken under cloudy skies with self illuminating instruments and when there is wind however photosynthetic parameters require more time to attain stability It is important that the plant surfaces are dry and not wet from dew irrigation or rain Time of day Take the majority of measurements as close to solar noon as possible typically from 11 00h to 14 00h For dark measurements dark chlorophyll fluorescence and dark respiration take measurements at night or during the day with adapted leaves see details of how to adapt leaves below Plant development stage Measurements can be taken at any developmental stage from mid seedling development to mid grain filling depending on
170. m of the screen Figure 11 4 If the external sensor is attached both the Carefully clean the light probe before and after taking measurements using the recommended cleaning solution above and below canopy values will be taken each time 1 After turning on the ceptometer allow the the down arrow is pressed instrument to equilibrate with the ambient Pressing ENTER saves these values to memory pressing temperature for around 10 minutes ESC deletes the values 2 Use the MENU button to select the PAR option If you need to exit a function use the ESC button Data and calculations 3 Connect the external sensor Figure 11 1C Depending on the instrument set up either take note of the values calculated by the device during sampling Trial measurements or save the data and download them with the software 4 To make above canopy PAR measurements press supplied with the instrument Data are typically the up arrow key while in the PAR LAI menu downloaded as a comma delimited text file and ease imported into MS Excel 5 To make within canopy measurements press the down arrow 57 or the green circular key in the In its simplest form when taking separate above and upper right corner of the keypad below canopy measurements Light interception A B C A B x 100 Equation 11 1 Where A above canopy PAR B reflected PAR and C below canopy PAR see Figure 11 3 A
171. mass leaf chlorophyll and canopy temperature in wheat Crop Science 46 1046 1057 Condon AG Richards RA Rebetzke GJ and Farquhar GD 2004 Breeding for high water use efficiency Journal of Experimental Botany 55 2447 2460 Condon AG Reynolds MP Rebetzke GJ van Ginkel M Richards R and Farquhar G 2008 Stomatal aperture related traits as early generation selection criteria for high yield potential in bread wheat In Reynolds MP Pietraglla J and Braun H Eds International Symposium on Wheat Yield Potential Challenges to International Wheat Breeding Mexico D F CIMMYT 4 Physiological Breeding II A Field Guide to Wheat Phenotyping Fischer RA 2007 Understanding the physiological basis of yield potential in wheat Journal of Agricultural Science 145 99 113 Fischer RA Rees D Sayre KD Lu Z M Condon AG and Saavedra AL 1998 Wheat yield progress associated with higher stomatal conductance and photosynthetic rate and cooler canopies Crop Science 38 1467 1475 Guttierrez Rodriguez M Reynolds MP and Klatt AR 2010 Association of water spectral indices with plant and soil water relations in contrasting wheat genotypes Journal of Experimental Botany 61 3291 3303 Lopes MS and Reynolds MP 2010 Partitioning of assimilates to deeper roots is associated with cooler canopies and increased yield under drought in wheat Functional Plant Biology 37 2 147 156 Lope
172. measurements retaken where necessary 8 Select AVERAGE and record the average reading Figure 9 1C Troubleshooting Problem The SPAD chlorophyll meter is not giving a CCI reading but is making a series of beeps when the pinchers are closed There is a large variation in the CCI readings within a plot There is damage to the surface of the crop e g from frost disease etc Useful references 9 Now delete all readings otherwise they will be included in the next average reading 10 Repeat to provide 3 average readings per plot Data and calculations Data are recorded directly on the field form unless the instrument has a data memory Data are used to calculate a mean CCI for each plot either of peak chlorophyll content or of sequential sampling intervals CCI values are typically 40 60 for a healthy green flag leaf at anthesis Solution The chlorophyll meter is unable to make a reading possibly the chamber and or chamber seal is dirty or the leaf has not been inserted correctly into the chamber Re take this reading It is important to maintain a consistent point of measurement along the leaf Lower readings may be due to the fact that the leaf being measured is damaged diseased or dirty or that the tip of the leaf has been inserted The SPAD samples only point measurements of individual leaves so it is difficult to infer results to the whole canopy For integrative me
173. meters 54 Physiological Breeding II A Field Guide to Wheat Phenotyping Site and environmental conditions Measurements should be taken when the sky is clear and sunny and there is negligible wind Light conditions must remain constant throughout the trial sampling period measurements may be taken when the sky is overcast i e there is continuous cloud cover although this is not recommended due to a disproportional increase in the amount of diffuse radiation It is important that the plant surfaces are dry and not wet from dew irrigation or rain Time of day Take the majority of measurements as close to solar noon as possible typically from 11 00h to 14 00h Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from mid seedling development to mid anthesis e For canopy light interception during growth take measurements periodically from the start of stem elongation to full cover anthesis to estimate change in light interception over time e g for the calculation of RUE e For maximum canopy light interception take measurements at anthesis 7days For stressed treatments the peak LI is at a maximum earlier in the season the severity of stress and experimental conditions will determine the optimum time for these measurements Take measurements earlier in severely stressed conditions as plants will senesce quickly Number of samples per plot Take
174. method for WSC concentration This is a quantitative colorimetric estimation for the carbohydrate content of a solution A green color is produced when carbohydrates are heated with anthrone in acid solution for details see Yemm and Willis 1954 Near infrared reflectance spectroscopy using calibration curves Near infrared reflectance spectroscopy NIRS can be used to estimate WSC concentration using predictive Figure 16 1 Sampling for WSC content A taking 20 stems in field B removing by hand the leaf lamina and leaf sheaves from dry stems and C grinding dry stem sample using a cyclone mill Data and calculations Data is typically given as WSC in dry matter This can be used to calculate the WSC content per stem g stem or per unit area g m WSC g stem WSC x DW_20stems 20 Equation 16 1 WSC g m WSC g stemt x stems m Equation 16 2 In optimal conditions peak WSC concentration ranges between 10 25 WSC content per 2 g stem is 0 2 0 5 g stemt and WSC content per m at a stem density of 300 m is 60 100 g m equations developed and cross validated using the results of chemical analyses by the Anthrone method Samples are scanned at 1585 1595 and 1900 2498 nm A different calibration curve is required for different developmental stages and environments Note that when NIRS is used it is recommended to replicate 5 of samples analyzed by the Anthrone method to check the calibrati
175. mill near infrared Outsourced All carbohydrates reflectance spectroscope at 0 50 NIRS or 5 Antherone per sample Soil coring for moisture Hand corer electric 500 2 000 by hand All content percussion hammer 15 000 15 000 tractor hydraulic tractor corer respectively Soil coring for root content As above As above As above All Yield Plot combine harvester 80 000 180 000 All Yield components Plot stationary thresher 20 000 30 000 All small bundle thresher seed 7 000 10 000 counter automatic manual 5 000 7 000 200 Crop morphological traits None None None All In season damage None None None All Key Time is divided into lt 30 seconds lt 2 mins lt 5 mins lt 10 mins gt 10 mins and none not applicable ID identification NIRS near infrared reflectance spectroscopy Introduction 7 8 Table 3 A timetable for phenotyping measurements based on visible developmental stages Seedling Stem Early Late Measurement development Tillering elongation Booting Heading Flowering grain filling grain filling Ripening Osmotic adjustment EEE ELLE EEL Leaf relative water content Canopy temperature Stomatal conductance CID for potential TE leaf tissue CID for water uptake grain Spectral reflectance cert il NDVI for senescence Slot ne SERRE NDVI for pigments Crop ground cove
176. most used chlorophyll fluorescence parameters for adaptation parameters a light adaptation Pps quantum yield of photosystem e Use dark adaptation leaf clips either provided with PS Il photochemistry i e the number of fluorescent events for each photon absorbed F minimal fluorescence F maximal fluorescence F variable fluorescence F F PS Il maximum efficiency and b dark adaptation F minimal fluorescence F maximal fluorescence F variable fluorescence and F F maximum quantum efficiency of PS II photochemistry i e the maximum efficiency at which light absorbed by PS Il is used for reduction of Plastoquinone A Q For more details see Lopes Molero and Nogues Volume 1 The following procedure describes measurements using a Fluorpen FP 100 chlorophyll fluorometer Figure 13 2 instrument or self made using aluminum foil Figure 13 3 or laminated carton and a paper clip e Do not allow illumination of the dark adapted leaf during measurement if dark adapting with a self made dark adaptation leaf clip then ensure to use a blackout cloth covering the plant instrument and operator during measurement Take the following equipment to the field e Hand held chlorophyll fluorometer e Dark adaptation leaf clips Advice on taking measurements It is important that both the light and dark adapted measurements are taken on the same leaf It is highly recommended to use
177. mponent of the DGC action It is critical that the green pixels in the image are accurately selected before automatic processing Note that this process may take several attempts if the sample selection is not satisfactory select the Regular Eye dropper tool and click on the sample image once to reset the color range selection process Start again to select pixels Transforming the image into black and white 9 Watch the Selection Image in the Color Range window as the color range accumulates this will begin to represent a black and white image of the original sample image 10 Stop selecting green pixels when the image represents the actual ground cover in the sample image Figure 10 6 11 Check the green pixels now outlined in the sample image this indicates the pixels which are within the color range selection 12 In the Navigator palette resize the image to 100 and ensure that the green leaf area has been accurately selected Measuring the black white ratio of the image 13 On the menu bar select EDIT gt FILL then in the CONTENTS menu select USE WHITE ensure the following settings Mode Normal and Opacity 100 and click OK 14 0n the menu bar select SELECT gt INVERSE 15 On the menu bar select EDIT gt FILL then in the CONTENTS menu select USE BLACK and click OK 16 A black and white image wil
178. n see this volume Chapter 3 and the second on day 2 is taken in the morning Plant developmental stage Measurements can be taken at any developmental stage from the start of tillering to mid grain filling depending on the experimental objectives timing of peak stress For instance in drought trials sampling is performed at early grain filling as an assessment of adaptation to terminal drought stress Number of samples per plot Cut one leaf sample from each plant within a pot Or in the field cut four leaves from different plants within a plot Procedure This procedure describes the rehydration method in greenhouse grown plants with notes for in field measurements The following equipment is required e Large clear plastic bags that cover the plant and the pot e 2 ml Eppendorf tubes e Latex gloves e Scissors e Paper towel e Thermal vacuum flask Canopy temperature stomatal conductance and water relation traits 21 e Ice to conserve the samples e Freezer 15 C e Vapor pressure osmometer e Calibration standard solutions e Paper sample discs e Glass rod e Pipette Advice on taking measurements The rehydration method requires a control well watered and stressed water withheld or droughted treatment Figure 4 1A Use large pots 5 10L each with a plant from four to six different genotypes Arrange pots in either a lattice design of four to six genotypes per sub block or in an
179. n take measurements before during and after the stress event period Effects on NDVI e g for estimation of Advice on taking measurements Whilst taking measurements ensure to hold the sensor head green biomass will allow discrimination of sensitive and stress tolerant resistant genotypes e Biomass accumulation and crop growth rate take measurements periodically from emergence to the end of anthesis to estimate biomass accumulation over time for the calculation of crop growth rate e Senescence stay green and grain filling duration take measurements weekly from anthesis to physiological maturity Genotypes which maintain Canopy green area greenness and duration are associated with higher yield Number of samples per plot Take one measurement per plot of a fixed duration depending on plot size e g approximately 5 seconds for a 5m plot Procedure The following procedure describes taking in field measurements using a hand held Ntech Greenseeker NDVI meter an active sensor Take the following equipment to the field e Field portable NDVI sensor 47 42 43 44 028 38 a e Leveled horizontally so that the field of view is directly over the crop e Consistently aligned over the plot typically centered over the middle row Ideally the field of view should cover two or more rows Figures 8 1C and 8 2B e Ata distance of 60 120 cm above the crop within the optimal distance range the readings are
180. n with performance and moderate to high heritability allows the elimination of poor material from a breeding program EGS allows testing of large amounts of material in early generations whilst saving time and resources to select that with the most potential Conventional tillage inverting the soil surface layer incorporating crop residues and vegetation and breaking up the surface to a fine tilth Developmental phase the development of the wheat plant is divided into three key phases i vegetative from germination to the appearance of the terminal spikelet ii reproductive from the appearance of the terminal spikelet to the end of anthesis and iii grain filling from the end of anthesis to physiological maturity Developmental stage or growth stage the development of the wheat plant is divided into ten key stages which mark important changes in the crop s life cycle see The Zadoks scale this volume Chapter 14 Dry weight refers to the constant weight reached after drying for plant material typically at 60 75 C for 48h ina well ventilated forced draft oven Fertile culms those culms expected to produce spikes during the period GS30 50 or bearing a spike after GS50 Genotype is a specific genetic identity of a wheat plant crop usually with reference to a specific character under consideration and or parentage Grab sample is taken in the field by grabbing sample material at random from w
181. ncrease lodging and reduce HI Typical range is 25 to 60 cm Figure 19 1B Measurement e Measure from the uppermost last node on the stem to the spike collar record to the nearest centimeter e Note that the peduncle continues to lengthen until the end of anthesis Awn length The awn is a long slender extension of the lemma in wheat It is an important photosynthetic and transpiratory organ on the spike and also provides some protection for the grain Awns increase the total surface area of the spike and are located at the top of the canopy giving high light exposure Awns can significantly contribute to spike photosynthesis are maintained well into the later stages of grain filling and with high water use efficiency Typical range is O to 75 mm Figure 19 1C Measurement e Measure from the tip of the spike to the tip of the longest awn record to the nearest millimeter e Awn color green to brown and awnedness scale 0 10 can also be recorded al measurements A peduncle length B flag leaf length C awn length and D plant height Crop observations Plant height Plant height is typically 70 120 cm with current CIMMYT elite varieties 80 100 cm although some dwarf varieties can be lt 50 cm Plant height is strongly controlled by genes in particular the Rht genes height reducing genes and it is therefore highly heritable Plant height shows a strong correlation with peduncle length
182. nitially yellow in color and turn white 14 4C Typically the date of mid anthesis GS65 is with age Figure 14 4 anthers stigmas stamen Sr filaments Figure 14 4 Anthesis A mid anthesis GS65 showing both newly extruded yellow anthers and older white anthers Photograph Xochiquetzal Fonseca CIMMYT B a carpel and associated anthers Credit wheatbp net and C a schematic of anthesis showing GS61 GS65 and GS69 74 Physiological Breeding II A Field Guide to Wheat Phenotyping Grain filling GS71 85 Grain development passes through water milk soft and hard dough stages Grain growth for the 7 14 days after fertilization is mainly of the maternal pericarp the ovary wall containing a watery fluid GS71 Only then does starch deposition begin GS73 GS77 Dough development begins when no liquid remains and the grain moisture content decreases from 45 at GS83 30 at GS85 to lt 20 at GS92 Hard dough represents the attainment of maximum grain dry weight Typically measurements are taken at mid grain filling GS75 determined when 50 of the grain on 50 of the spikes have reached the medium milk stage Assessment is typically achieved by squeezing grains between the forefinger and thumb to exude the developing endosperm It starts as milky fluid that increases in solidity as the grain progresses through the milk and dough stage and becomes hard as the water content decreases Figure 14 5
183. nternode upper internodes for remobilization studies or lower internodes for lodging stem sawfly studies e Rate stem solidness using a scale from O hollow to 10 solid using the guide Figure 19 2 e Specific stem weight dry weight per unit length may also be calculated Figure 19 2 Stem solidness scale from hollow 0 to solid 10 In the diagram dark green signifies stem wall and light green signifies pith T j i f E F s j 1 F j hed i i he wi F g i lt 7 if d 1 oo n 4 y i 4 s M a 4 A i al i nail s x a Figure 19 3 Examples of stem solidness A hollow score of 0 and thick pith score of 8 108 Physiological Breeding II A Field Guide to Wheat Phenotyping Trait observations adaxial surface of the leaf ii for the peduncle spike it starts on the peduncle then the spike moving from the Leaf and spike glaucousness base upwards Figures 19 4 and 19 5 Glaucousness appears as a grayish white substance on Scoring the surface of the plant although transparent waxes also occur which are not apparent to the naked eye Surface waxes can be easily rubbed off between forefingers and this can be used to estimate the quantity thickness of glaucous covering In general waxiness progresses in sequence i for the flag leaf sheath it starts on the leaf ii Observe glaucousness on the peduncle and or spike sheath then the abaxial su
184. o its maximal water holding capacity at full turgidity RWC provides a measurement of the water deficit of the leaf and may indicate a degree of stress expressed under drought and heat stress RWC integrates leaf water potential Ww another useful estimate of plant water status with the effect of osmotic adjustment a powerful mechanism of conserving cellular hydration as a measurement of plant water status A genotype with the ability to minimize stress by maintaining turgid leaves in stressed environments will have physiological advantages e g this allows turgor dependent processes such as growth and stomatal activity and to protect and maintain the photosystem complex Leaf RWC is easily and simply measured without the need for expensive specialized instruments Fresh leaf samples of field grown crops are first weighed then placed in water chilled overnight and re weighed before being oven dried and weighed a final time The relative difference in the water content of the leaf samples provides a quantitative measure of their in field hydration status Trials can be rapidly screened for genotypes which maintain high leaf RWC values during water deficit stress and vice versa Sources of error in the estimation of RWC can be summarized as i change in dry weight mainly due to respiratory losses ii increases in water content in excess of full turgidity and iii water accumulation in intercellular spaces Barrs and Wea
185. omposition 6 C expressed negative values in parts per thousand o Farquhar et al 1989 61C o0 Reampie Rstandara 2 x 1000 Equation 6 1 Where the ratio of heavy to light isotope R C 7C for the sample is in comparison to Peedee Belemnite PDB carbonate limestone standard For example a tC value of 28 o means that the C 7C ratio of the sample is 28 ppt lower than the PDB standard Approximate 67 C values for C3 plants range from 35 to 20 o and for C4 plants range from 17 to 9 o Calculation of carbon isotope discrimination A3C Following Farquhar et al 1989 rather than using carbon isotope composition values 57 C positive values for CID AC can be calculated for easier statistical analysis as A C 5 6 1 6 1000 Equation 6 2 Where 6 and 6 refer to the stable carbon isotope composition of the atmosphere and plant sample respectively On the PDB scale free atmospheric CO 6 has a current composition of approximately 8 o Farquhar et al 1989 although this value may vary across different sites 9 to 7 5 o and is becoming more negative each year ca 0 02 to 0 03 o per year due to the effects of deforestation and use of fossil fuels Therefore in order to compare data across sites environments greenhouse or growth chamber values of 10 to 13 o and years it is useful to measure the actual free air carbon isotope ratio of each experiment F
186. on see Figure 16 2 y 0 97x 4 26 R 0 76 WSC NIRS 0 5 10 15 20 25 30 35 WSC standard Figure 16 2 Calibration curve to estimate WSC concentration from near infrared reflectance spectroscopy values at anthesis adapted from Pinto et al 2006 Direct growth analysis 85 Troubleshooting Problem Solution Large error variance in data Check that the mill is consistently grinding to 0 5 mm and sieve carefully to ensure good particle distribution within sample When grinding samples it is important that the mill is thoroughly cleaned between samples to avoid cross contamination Ensure to re dry samples before NIRS analysis to removed any reabsorbed moisture which may affect readings References Kiniry JR 1993 Nonstructural carbohydrate utilisation by wheat shaded during grain growth Agronomy Journal 85 844 849 Lopes MS and Reynolds MP 2010 Partitioning of assimilates to deeper roots is associated with cooler canopies and increased yield under drought in wheat Functional Plant Biology 37 147 156 Pinto S Gonzalez H Saint Pierre C Pe a J and Reynolds MP 2006 Obtenci n de un modelo matem tico para la estimacion de carbohidratos solubles en paja de trigo Triticum aestivum mediante reflectancia espectral cercana al infrarojo NIRS 6500 VI Congreso Nacional de la Asociacion Nacional de Biotecnologia Agropecuaria y Forestal ANABAF A C ITSON Cd Obregon Son
187. ons check experimental soils for correct values Soil bulk density SBD assumed to be 1 3 at all depths in this example water content DW soil x 100 Gravimetric water content x soil bulk density 10 GWC 100 x SBD x core section length soil water content at tOt Total water uptake number of days between irrigation and sampling soil water content at t1 t Soil water content at t0 assumed to be 113 5 mm at all depths in this example but should be measured after each irrigation event Number of days between irrigation and sampling 15 in this example Core length in cm Soil root calculations Rooting depth The maximum depth reached by the roots It is an important trait as it determines the amount of soil profile that the plant can explore It depends on the cultivar soil type and below ground resource availability Typical rooting depths spring wheats 80 120 cm and winter wheats 140 200 cm Root to shoot ratio R S This relates the biomass of the above ground plant to that below ground Root dry weight RW This is the total RW and distribution through the profile Total RW is observed to increase exponentially to anthesis when it reaches its maximum with a small decrease to harvest due to a decrease in the RW in upper parts of the profile Typical total RW values spring wheats 75 110 g m Root weight density RWD This describes the RW per unit of soil volume and its distribution through
188. oportion of each of the spikes which are affected using a scale from 0 0 to 10 100 using the guide in increments of 10 Figure 20 2 Figure 20 1 Spike tipping due to A drought score of 4 40 of spike is damaged and B frost score of 1 10 of spike is damaged with a bleached white appearance chlorosis 3 days after the event Figure 20 2 Spike tipping scale 10 100 Lodging Two forms of lodging are recognized i stem lodging where the roots are held firmly in the soil and the wind force causes failure at the lower internodes of the stem and ii root lodging where the root anchorage strength is reduced by a weak soil and or poorly developed root anchorage and failure occurs at the root soil connection Lodging is most likely in the post anthesis period influenced by the increasing weight of the spike Lodging typically reduces crop yield 1 per day that a crop is lodged after anthesis and quality and causes the crop to dry slowly Figure 20 3 Scoring e Observations should be taken as soon as possible after the lodging event since the angle of the crop may change with time e Continue to re assess the lodged crop at least every 7 10 days as it is now more susceptible to diseases e Record the type of lodging i e stem or root i Rate the proportion of culms within the plot which are affected by lodging using a scale from 0 0 to 10 100 in inc
189. or example if the 64 C value is 28 AC 8 28 1 28 1000 20 0 972 20 58 Leaves from plants with higher TE grown under well watered conditions show lower A C i e lower discrimination Plants grown under water stress generally produce grain with lower A SC which is negatively related to WU and positively related to TE Canopy temperature stomatal conductance and water relation traits 29 Troubleshooting Problem Anomalous data due to sample contamination which typically gives very high peaks of C detected and can greatly change the isotope ratio in the sample Very low AC in leaf samples References Condon AG Farquhar GD and Richards RA 1990 Genotypic variation in carbon isotope discrimination and transpiration efficiency in wheat Leaf gas exchange and whole plant studies Australian Journal of Plant Physiology 14 9 22 Farquhar D Ehleringer JR and Hubick KT 1989 Carbon isotope discrimination and photosynthesis Annual Review of Plant Physiology and Plant Molecular Biology 40 503 537 Rebetzke GJ Condon AG Richards RA and Farquahr GD 2002 Selection for reduced carbon isotope discrimination increases aerial biomass and grain yield of rain fed bread wheat Crop Science 42 739 745 Sayre KD Acevedo E and Austin RB 1995 Carbon isotope discrimination and grain yield for three bread wheat germplasm groups grown at different levels of water stress Field
190. ora 22 25 Octubre 2006 Reynolds MP Manes Y Izanloo A and Langridge P 2009 Phenotyping approaches for physiological breeding and gene discovery in wheat Annals of Applied Biology 155 309 320 Yemm EW and Willis AJ 1954 The estimation of carbohydrates in plant extracts by anthrone The Biochemical Journal 57 508 514 86 Physiological Breeding II A Field Guide to Wheat Phenotyping Useful references Blum A 1998 Improving wheat grain filling under stress by stem reserve mobilization Euphytica 100 77 83 Dreccer MF van Herwaarden AF and Chapman SC 2009 Grain number and grain weight in wheat lines contrasting for stem water soluble carbohydrate concentration Field Crops Research 112 43 54 Pollock CJ 1986 Fructans and the metabolism of sucrose in vascular plants New Phytologist 104 1 24 Rebetzke GJ Van Herwaarden AF Jenkins C Weiss M Lewis D Ruuska S Tabe L Fettell NA and Richards RA 2008 Quantitative trait loci for water soluble carbohydrates and associations with agronomic traits in wheat Australian Journal of Agricultural Research 59 891 905 Ruuska S Rebetzke GJ Van Herwaarden AF Richards RA Fettell N Tabe L and Jenkins C 2006 Genotypic variation for water soluble carbohydrate accumulation in wheat Functional Plant Biology 33 799 809 Van Herwaarden AF Farquhar GD Angus JF Richards RA and Howe GN 1998 Haying off
191. ots before placing the sample in the bag Biomass samples should be kept cool and in the shade until processing Samples taken for WSC analysis should be kept cool and processed dried rapidly within 2 hours of cutting to avoid respiratory losses of carbohydrates Do not cut these stems into pieces instead bend if necessary see this volume Chapter 16 Detailed physiological studies often require partitioning of the canopy into individual organs i e leaf lamina all leaf lamina individual leaf layers leaf sheath stem internode lengths and peduncle and spike for the measurement of biomass and or nutrients content Partitioning is typically based on a sample of gt 20 fertile culms When sub sampling selecting culms care is needed to ensure that all plant material associated with the culms is included Note that nutrient analysis requires additional considerations In most cases determinations of dry mass are made on representative sub samples to reduce oven space requirement take additional measurements e g fertile culm count etc Organize sampling to optimize use of the oven in particular to avoid mixing fresh samples with dry samples Preparations 1 Prepare field sample bags with clearly visible labels detailing the name of the trial date of sampling and plot number e g black plastic bags shown in Figure 15 1C Use two labels per bag one attached to the outside of the bag and the other placed ins
192. otypes with warmer canopies typically by 1 2 C Calculation of canopy temperature depression CTD air temperature CT is not recommended due to the errors associated with measuring air and canopy temperatures with different types of instruments thermo couple air and infrared canopy and the additional experimental error of measuring two values Instead it is recommended that CT values for genotypes are compared and environmental variables accounted for within statistical analysis e g using spatial analysis CT measurements are comparable within a developmental phase i e pre heading or grain filling as in particular the spike has a large effect on CT readings Three measurements are taken at each developmental phase approximately one week apart This allows for a mean CT for each plot in each phase to be calculated Specifications to keep in mind when buying an IRT for CT e Sensor with an 8 to 14 um spectral range e Adjustable adjusted emissivity between 0 95 and 0 98 e Measurement range between 0 C and 60 C with at least 0 1 C of resolution e 10 1 to 50 1 of Distance Spot D S ratio e An averaging mode used to average multiple temperature readings over the sampling time Canopy temperature stomatal conductance and water relation traits 13 Troubleshooting Solution Ensure to select the AVG option check that LOCK and MAX MIN options have not been sele
193. ove tillering node biomass is most easily sampled by uprooting plants and removing the roots plants may also need to be carefully washed and well dried as soil particles may adhere to the lower leaves These data 78 Physiological Breeding II A Field Guide to Wheat Phenotyping can also be used to calculate plant density Between stem elongation and grain filling stages GS32 77 biomass is sampled as described in this chapter At physiological maturity ripening biomass is sampled as described in this volume Chapter 18 Number of samples per plot Take one quadrat sample per plot typically of gt 0 25 m2 from a representative part of the plot Procedure The following procedure describes biomass sampling at anthesis 7days including sub sampling for the calculation of flag leaf or total leaf area biomass partitioning tiller density spike index and determination of WSC and or nutrient content See Schematic 15 1 Take the following equipment to the field e Pre labeled bags e Quadrat open ended U shaped for ease of use e Sickle or large cutting implement Advice on taking measurements When cutting samples for biomass measurement it is important to cut the culms as close to the ground as possible whilst avoiding including soil and roots In drought conditions it may be difficult to cut plants as they are easily uprooted In this case it is easier to cut the plants using scissors ensuring to remove any ro
194. palette see Figure 10 4 Enter the Action name Digital Ground Cover and leave all other options as Hue Saturation default 3 Click RECORD the icon in the Actions palette will turn red Color adjustment of image to improve the resolution of green leaf area 4 Onthe menu bar select IMAGE gt ADJUSTMENTS gt HUE SATURATION LIGHTNESS 5 Adjust values to Hue 0 Saturation 60 Lightness 20 and click OK Figure 10 5 Figure 10 5 Hue saturation settings Aigbe Pholeshon CS Lerteaded A ATIE JPG ee CRE BY Ti P y Pathe one Shala How it z gii Werte e Ea Navigator Palete LY l A i l Aoa 4 K ii A i al ig a A E i AET K j a a j fi P a f L fh B s A AENG j T i a i Fh f i o wo JE Fil ae j i y Select all measurements w Pe b ALAALA 4 k b apu Tarasp layer d J Export selected measurements i i i Chamin Colar pean ora ora Digital Ground Cavar Actio ae nano rr Measurement Log Palette f y A yy 7 Delete selected measurements kur x J NEN op te MN Pea Arana Pears Log i Play section Begin recording k Stop pleyingirerording begs PONS Erne See RRS NAP DLE nee ee Figure 10 4 Workspace configuration indicating important features Photosynthesis and light interception 49 This process is the most crucial co
195. r Light Interception ronnie tT tt tt te EE EE EE area index Gas exchange and canopy fluorescence In season biomass Water soluble carbohydrates Soil coring for root content Soil coring for moisture content Yield and yield components Crop morphological traits CID carbon isotope discrimination NDVI Normalized difference vegetation index TE transpiration efficiency Key Most typical time to take Measurements taken related Not recommended for measurements taken on measurements to objectives the same day where phenology range gt 5 days or during senescence Physiological Breeding II A Field Guide to Wheat Phenotyping Canopy temperature stomatal conductance and water relation traits Chapter 1 Canopy temperature Julian Pietragalla The surface temperature of the canopy is related to the amount of transpiration resulting in evaporative cooling A hand held infrared thermometer IRT allows canopy temperature CT to be directly and easily measured remotely and without interfering with the crop Studies have shown that CT is correlated with many physiological factors stomatal conductance transpiration rate plant water status water use leaf area index and crop yield Genotypes with cooler canopy temperatures can be used to indicate a better hydration status It is used routinely particularly for stress diagnostic and breeding selection of stress adapted genotypes i under drought
196. r plot Procedure Samples may be taken from plants either in the field or the greenhouse Take the following equipment to the field e Scholander pressure chamber e Cylinder of compressed air e Tools for connecting the pressure chamber to the cylinder e 2 x scissors e Magnifying glass e Water bottle with spray nozzle e 5 x cotton gauzes large enough to wrap the entire flag leaf e Mobile work table and chairs e Field form and clipboard For the greenhouse also take e Large black plastic bags large enough to cover both the plant and its pot Advice on taking measurements It is very important to ensure that the top of the pressure chamber is correctly and securely fitted in order to withstand the high pressures inside the chamber gt 40 bar 4 0 MPa Failure to do so may result in serious injury to the operator It is very important to release the pressure slowly and completely after taking measurements Removing the top of the pressure chamber before all the pressure is released may result in serious injury to the operator Care is needed to minimize the time between cutting the sample and its measurement Typically two people work together one finds and cuts the samples whilst the other operates the pressure chamber To reduce the time per plot it is possible to take and test two leaves together from the same plot see Figure 3 2E although this requires additional care Apply pressure slowly at 1
197. r reading should be taken Measurements are typically made on the upper adaxial surface of the leaf In wheat the ratio of stomatal frequency on the upper and lower leaf surface approaches 1 0 but the stomata on the upper surface show a greater degree of difference between genotypes in mid day closure when the temperature and radiation increases Ensure that the leaf is consistently placed into the clamp in the same way with the upper surface always facing upwards When using the SC 1 porometer it is of paramount importance that at no point do you touch the white porous Teflon filter disk as this will cause inaccurate readings and the disk may need to be replaced Do not breathe near the disk leaf or chamber as this effects the humidity and carbon dioxide concentration gradient within the sensor head do not take measurements when there is smoke in the air e g from fires cigarettes or pollution and do not bring the sensor into contact with any sort of chemical vapor e g glue alcohol or gasoline Preparations Check that the batteries are fully charged and that the chamber seal and gaskets and sensor are free of dust pollen etc 1 After turning on the porometer allow the instrument to equilibrate with the ambient temperature for around 10 minutes Press the MENU button choose the CONFIG MENU screen and use the arrows and ENTER button to make necessary changes 2 Check that the MODE i
198. ration easier Standardize the time for each sample to 15 minutes Dry carefully with absorbent paper to remove all the excess water and ensure that there are no bubbles of air or water on the roots ili Scanning To scan the roots the following is required prepared root samples scanner and U lead Photo Express 3 software a Create and name a new album e g Root trial 1 Root trial 2 etc Click GET gt SCANNER to open the scanner Click ACQUIRE and in the SETTINGS window select LINE ART the roots appear as lines AMPLIFICATION 100 to show the actual size of roots 600 dpi and HIGH QUALITY Click PREVIEW to see the scan make adjustments as necessary e g change the scan area Figure 17 5 Root scanning A root scanner WinRHIZO STD 1600 Regent Instruments Inc Quebec Canada and B scan of barley roots Photographs Pedro Carvalho The University of Nottingham 92 Physiological Breeding II A Field Guide to Wheat Phenotyping Click SCAN Right click the mouse and choose RENAME e g use plot name 1B 30 60 etc g Click SAVE as a TIFF file iv Analysis of the root scans To analyze the root scans the following is required Delta T SCAN software and TIFF files of the root scans a Open the DT SCAN Application b Open FILE and LOAD IMAGE FILE Data and
199. re is a diminishing increase in the proportion of radiation intercepted as the green area increases for wheat crops with a GAI of 5 typical of a wheat crop at heading more than 95 of the incident PAR will usually be intercepted The most important attribute affecting the geometry of the canopy is leaf angle but it is also affected by leaf surface properties such as thickness size and shape and the vertical stratification of the leaf area There are substantial differences in the extent of light penetration into the canopy with leaf angle canopies with more erect leaves will intercept less PAR per GAI resulting in less saturation of the upper leaves and more PAR being available to the lower leaves Over the season the total amount of light intercepted by a crop canopy is a function of its size longevity optical properties and structure As a physiological driver of yield values for the amount of intercepted PAR can be used to calculate radiation use efficiency RUE i e efficiency of conversion of the intercepted light radiation into above ground crop dry matter and light interception LI can influence water use efficiency WUE and can indicate differences in canopy architecture and growth between genotypes Ceptometer readings can also be used to estimate the GAI and leaf area index LAI by using the above and below canopy PAR readings combined with other variables such as the zenith angle and the leaf distribution para
200. re that samples are correctly loaded into the osmometer samples greater than 11 ul can contaminate the thermocouple Remove air bubbles on the sample disc before proceeding a bubble bursting inside the sample chamber will contaminate the thermocouple Ensure that the sample holder is clean and undamaged e g do not use metal forceps to remove wet sample discs Use deionized water to clean equipment References Useful references Babu RC Pathan MS Blum A and Nguyen HT 1999 Morgan J 1983 Osmoregulation as a selection criterion for drought Comparison of measurement methods of osmotic adjustment in tolerance in wheat Australian Journal of Agricultural Research 34 rice cultivars Crop Science 39 150 158 607 614 Moinuddin Fischer RA Sayre KD and Reynolds MP 2005 Munns R 1988 Why measure osmotic adjustment Australian Osmotic adjustment in wheat in relation to grain yield under Journal of Plant Physiology 15 717 726 water deficit environments Agronomy Journal 97 1062 1071 Zhang J Nguyen HT and Blum A 1999 Genetic analysis of osmotic adjustment in crop plants Journal of Experimental Botany 50 291 302 24 Physiological Breeding II A Field Guide to Wheat Phenotyping Chapter 5 Leaf relative water content Daniel Mullan and Julian Pietragalla The relative water content RWC or relative turgidity of a leaf is a measurement of its hydration status actual water content relative t
201. reen area with visible spikes using an automatic planimeter see this volume Chapter 12 Ensure to return all the leaf material to the 20 culm sample separate labeled bags for dry mass determination 12 Place the FW_SS50 into the labeled large paper bag for oven drying and dry weight determination check label with plot number Record fresh weight potty j n Se i Q Put in black plaster bag Cut quadrat sample PARTITIONING Record fresh weight Take 5S0 culm subsample OS PN re ain 50 culm Take 20 culmi absari cub sample in bag Leaf lamina stem with 7 MA leaf sheath Ory to constant Record weight wi dry weight s i ii Schematic 15 1 Measuring in season biomass with optional partitioning of culm into individual components 80 Physiological Breeding II A Field Guide to Wheat Phenotyping Determination of dry weight 18 Place the stems spikes and other partitioned plant components into separate labeled small or medium paper bags check label with plot number It is not necessary to weigh the FW of these plant component sub samples 19 Place all sub samples into a well ventilated forced draft oven at 60 75 C until they reach constant weight typically for at least 48h Include clean empty paper bags to use as TARE 20 Remove sub samples from the oven and allow to cool to ambient temperature but do not allow time to absorb moisture from the air Keep the
202. rements of 10 ii Rate the average angle of the stems in relation to the vertical For this use a scale of 0 no lodging 1 stems leaning to 45 from the vertical to 2 stems between 45 and 90 from the vertical A lodging score LS can be calculated by Lodging score proportion of the plot affected x degree of lodging Equation 20 1 e g if 50 of the plot is affected with a 30 lodging 0 50 x 30 LS 15 eS IN 4 Sess oe i A oe Vegetative damage Damage to the vegetative parts of the plant can be caused by adverse weather conditions e g frosts or pests and or diseases Vegetative damage may affect physiological processes e g light interception thereby reducing growth biomass and ultimately yield with effects to the spike typically causing the largest reduction in yield It is important to record the plant part s affected the extent of the damage and the probable cause of the damage Figure 20 4 Scoring e Observations should be made as soon after the damage event as possible and repeated after 7 10 days as effects often become pronounced with time i Rate the proportion of culms within the plot which are damaged using a scale from 0 0 to 10 100 in increments of 10 ii Rate the proportion of each plant part s or total culm which is affected using a scale from 0 0 to 10 100 using the guides in increments of 10 Figures 12 2 20 2 and 20 5 Figur
203. retical and Applied Genetics 120 6 1107 1117 Chapter 2 Stomatal conductance Julian Pietragalla and Alistair Pask Stomatal conductance estimates the rate of gas exchange i e carbon dioxide uptake and transpiration i e water loss through the leaf stomata as determined by the degree of stomatal aperture and therefore the physical resistances to the movement of gases between the air and the interior of the leaf Hence it is a function of the density size and degree of opening of the stomata with more open stomata allowing greater conductance and consequently indicating that photosynthesis and transpiration rates are potentially higher The hand held porometer provides rapid measurement of leaf stomatal conductance in irrigated trials though it is not a recommended measurement under water stress unless very mild as the stomata are generally closed A relatively rapid drop in pressure fast gas flow rate or a rapidly changing relative humidity RH gradient through the instrument indicates that the resistance to gas conductance are relatively small and that the stomatal conductance is high Results can be used as a proxy for measuring photosynthetic rate The heritability of stomatal conductance is reasonably high and gives high correlation with yield greater leaf conductance under warmer temperatures has been associated with cooler canopy temperatures Research at CIMMYT has shown that increased yield of CIMMYT wheat
204. rface of the leaf and finally the e Rate glaucousness using a scale from 0 none to 10 total cover i Observe glaucousness on the flag leaf sheath adaxial and abaxial surface of the leaf lamina e Rate glaucousness using a scale from 0 none to 10 total cover using the guide Figure 19 6 ME c M S fi Pid aa a mf Figure 19 4 Glaucous and non glaucous genotypes A glaucousness on the flag leaf peduncle and spike B the peduncle and spike of a non glacuous plant and C in field glaucous and non glaucous genotypes E No glaucousness J Glaucousness Figure 19 5 Flag leaf and leaf sheath glaucousness scale indicates approximate glaucousness cover Crop observations 109 Leaf and spike pubescence Pubescence appears as silvery hairs on the surface of the plant typically no more than 1 mm in length The density and location of hair varies In addition to a visual assessment it is often also useful to feel the amount of pubescence on the leaf or spike This can be done by running your finger along the organ in a backwards direction hairier organs will feel more resistant in a forwards direction hairier organs will feel softer Figure 19 6 Scoring i Observe pubescence on adaxial upper and or abaxial lower surface of the flag leaf lamina ii Observe pubescence on the glumes and raquis of the spike e Rate pubescence using a scale from O no hair gslabrous
205. rowth analysis Laboratory measurements Partitioning 13 From the quadrat sample randomly select a 20 culm sub sample of fertile culms ensuring that all culms have a well formed spike 8 TARE the balance by placing an empty large plastic bag with 2 x labels see this volume Chapter 22 9 Immediately weigh the total quadrat sample fresh weight FW_Q of all samples Ensure that the bags containing samples are accurately placed on the 15 Re count to ensure that there are 20 culms and 20 balance spikes 16 From the 20 culms remove leaf lamina and separate into leaf layers flag leaf leaf two etc or bulk all leaf lamina cut the stem into internodes as required or bend stems for determination of WSC to avoid losses of WSC from cut ends Note that the leaf sheath is most easily removed from the stems once dried 14 Cut the spike from the culm at the spike collar Determination of number of fertile culms 10 From the quadrat sample randomly select a 50 culm sub sample of green culms i e the newest leaf is green and weigh FW_SS50 It is not important that these culms are fertile i e with spikes but it is important to have a representative mix of all culm classes Determination of flag leaf or total leaf area 11 Within the 50 culm sub sample of green culms 17 From the leaf lamina separate the green tissue from count the number of culms either clearly booting or the yellow dead material and determine g
206. ruments available for indirectly estimating water and nutrient uptake and root architecture direct measurement via soil coring remains the most accurate approach for obtaining this information Soil samples to a depth of 120 cm can be obtained either manually with a hand corer or hydraulically with a tractor mounted hydraulic soil corer and either dried analyzed and or washed for the measurement of water nutrient and or root content respectively However it should be noted that taking and processing field soil cores is a labor intensive and time consuming process especially in dry and or compacted soils and is therefore not a suitable rapid screening method for large trials Site and environmental conditions Measurements can be taken in most environmental conditions However it is important that the soil is not extremely wet as this restricts and makes extremely difficult the movement of the equipment in the field Time of day Measurements can be taken at any time of the day although where possible samples should be taken in the morning to allow for same day processing Plant developmental stage Measurements can be taken at any developmental stage and or at regular intervals from the start of tillering to physiological maturity depending on the experimental objectives timing of peak stress Sampling is typically performed just after biomass sampling which also avoids adverse effects of root damage on plant productivit
207. ry facilities required osmometer required for measurement requires control of soil water content Semi analytical balance required to 3 d p Complicated data interpretation sample analysis outsourced Complicated data interpretation sensors can be relatively expensive Passive sensors are limited to good light conditions resolved by active sensors Point measurement only data needs to be integrated across whole green canopy Digital ground cover requires software and image processing skills Sensitive to environmental fluxes interaction with time of day and phenology Typically measured by destructive sampling leaf area meters can be slow Introduction 5 Measurement 13 Gas exchange for photosynthesis 13 Chlorophyll fluorescence 14 Determining key developmental stages 15 In season biomass 16 Water soluble carbohydrates 17 Soil coring for moisture content 17 Soil coring for root content 18 Yield 18 Yield components 19 Crop morphological traits 20 In season damage Physiological trait s Leaf plant and spike photosynthesis and respiration F F quantum yield of PSII non photochemical quenching light curve response electron transport rate Crop development stage Crop growth and crop growth rate Accumulation of carbohydrates sugars in the stem Soil moisture content water uptake Root chara
208. ry inaccurate Scientist fal Area measured It is necessary to control for phenology in populations with diverse anthesis dates as plants under different stages of development have different architectures and present differences in the source sink relationships and these may confound the analysis This Figure 1 5 Recommendations for CT field measurements sown in can be corrected by splitting the population A beds readings taken along rows or B flat planting readings into early and late lines and therefore making taken diagonally across the plot different populations to be screened A range of up to 10 days in anthesis date is quite reasonable Advanced generations and some advanced line screening trials with 300 to 1 000 lines or families may be established in the field trial without replications In this case it is useful to include a known high and a low CT check genotype every 10 20 plots within the experimental design CT values can be compared with these check values in the analysis to improve the ranking of lines Figure 1 6 Shows the use of the IRT during in field measurements at the pre heading phase on bed plots with two rows The dotted red lines illustrate the field of view of the IRT and the arrow illustrates movement of IRT out and back over the crop remembering to avoid the ends of the row which act as the border A B Direction of IRT movement along plot Direction of IRT movemen
209. s MS and Reynolds MP 2012 Stay green in spring wheat can be determined by spectral reflectance measurements normalized difference vegetation index independently from phenology Journal of Experimental Botany in review Morgan JM and Condon AC 1986 Water use grain yield and osmoregulation in wheat Australian Journal of Plant Physiology 13 523 532 Mullan DJ and Reynolds MP 2010 Quantifying genetic effects of ground cover on soil water evaporation using digital imaging Functional Plant Biology 37 703 712 Parry MAJ Reynolds MP Salvucci ME Raines C Andralojc PJ Zhu XG Price GD Condon AG and Furbank RT 2011 Raising yield potential of wheat Il Increasing photosynthetic Capacity and efficiency Journal of Experimental Botany 62 2 453 467 Pinto RS Reynolds MP Mathews KL McIntyre CL Olivares Villegas JJ and Chapman SC 2010 Heat and drought adaptive QTL in a wheat population designed to minimize confounding agronomic effects Theoretical and Applied Genetics 121 1001 1021 Reynolds MP Manes Y Izanloo A and Langridge P 2009 Phenotyping for physiological breeding and gene discovery in wheat Annals of Applied Biology 155 309 320 Table 1 An overview of wheat phenotyping techniques Measurement Physiological trait s 1 Canopy Evaporative cooling temperature from the canopy surface 2 Stomatal Stomatal aperture conductance 3 Leaf water Leaf w
210. s The mention of trade names and commercial products are for information purposes only and do not Table 22 5 Suggested models of instruments websites accessed August 2011 Instrument Brand Model s Measurement level Website Ceptometer Delta T Devices SunScan System and SS1 Canopy htto www delta t co uk Decagon Devices AccuPAR LP 80 Canopy http www decagon com Chlorophyll Opti Sciences OS1 FL and OS 30p Leaf htto www optisci com fluorometer Qubit Systems Z990 FluorPen Leaf http www qubitsystems com WALZ PAM 2500 MINI PAM Leaf http www walz com Hansatech Intruments FMS 2 Pocket PEA Leaf http www hansatech instruments com Chlorophyll meter Minolta SPAD 502 Plus Leaf http www specmeters com Field Scout CM 1000 Canopy http www specmeters com Opti Sciences CCM 200 Leaf http www optisci com Hansatech Intruments CL 01 Leaf http www hansatech instruments com Apogee CCM 200 Leaf http www apogeeinstruments com FT Green LLC At Leaf Leaf http www atleaf com Qubit Systems Z955 Nitrogen Pen Leaf http www qubitsystems com Infrared Sixth Sense LT300 Canopy htto www instrumart com thermometer Mikron MI N14 Canopy htto www mikroninfrared com Extech 42540 Canopy htto www extech com instruments Leaf area meter Licor LI 3100C and LI 3000C Leaf http www licor com CID Bio Science Cl 202 and Cl 203 Leaf http www cid inc com Delta T Devices WinDIAS 3 Leaf htto
211. s by hand iii Collect the roots into a heat proof plastic tube 4 Now open the plastic bag and fill a numbered iv Repeat the washing process at least three aluminum pot with a well mixed sub sample times as roots may remain in the soil at the af thesoil bottom of the tray 5 Carefully replace the lid of the pot and clean the v Adda solution of 15 alcohol to tube to utide preserve the root sample 6 Weigh the pot lid and soil sub sample to 2 d p vi Store roots at 4 6 C pot fresh soil vii Hand clean the root samples using forceps 7 Dry the soil sub samples in an oven at 105 C for tweezers Note that roots are fragile and must be 48h with the lids ajar to allow for evaporation cleaned very carefully Remove all of the material Figure 17 2C that is not live roots especially dead roots which can be identified from their darker color and 8 Remove samples from the oven and allow to cool heir lack orelactiti d flexibili hich i to ambient temperature but do not allow time to AAE A A E em eno ir omie an characteristic of living roots a keneen eaa Cache de soll viii Dry root samples in tubes at 60 75 C for 24h ix Allow to cool and weigh to 3 d p plastic bag and C soil sub samples drying lids slightly ajar to allow moisture to evaporate Direct growth analysis 89 Take soll sample 0 30 cm Cut core into sections 30 60 cm 60 90 cm 90 120em j MOISTURE CONTENT Put
212. s as a summary of all events up to and a little beyond anthesis combining the effects of management and climate on plants m2 spikes plant spikelets spike and grains spikelet into a single term The number of grains m determines the sink size of the crop and under many conditions it is strongly correlated with yield Under optimal conditions 15 000 25 000 grains m could be expected to maximize yield potential Grains m GNO yield g m TGW g x 1000 Equation 18 7 Determination of the spike number m Spike number m as described above can also be estimated from measured values Spikes m SNO biomass g m DW_fertile culm g Equation 18 8 Troubleshooting Problem Shattering of spikes during harvest causing losses of grain Combine harvester or plot thresher loses grain through the chaff duct Combine harvester or plot thresher loses unthreshed spikes in the chaff duct Determination of the number of grains per spike Spike fertility is a function of fertile spikelets per spike and fertile florets per spikelet Values typically range between 10 40 in stressed environments i e heat and drought and 40 100 in favorable conditions It has a broad optimum which varies with variety conditions and environment Grains per spike GSP grains m spikes m Equation 18 9 Alternatively the number of grains per spike can be measured independently by threshing a known number of randomly selected sp
213. s important that all plots have been well irrigated from establishment to sampling this ensures that the measurement of TE is not confounded by differences in genotypic response to a drying soil profile Time of day Samples can be taken at any time of the day Plant developmental stage Take leaf samples during seedling development after the three leaf stage GS13 Take grain samples after physiological maturity GS87 Number of samples per plot For leaf sampling randomly select 10 20 different plants per plot avoiding borders For grain sampling take a single sample from a well mixed dry bulk grain after plot harvest Procedure Take the following equipment to the field for leaf sampling e Pre labeled bags e Scissors Advice on taking measurements Note that herbicide or pesticide applications potentially affect plant gas exchange which may confound results Careful records are therefore important for the interpretation of CID data It is recommended to include several double up samples within the isotope analysis to check for consistency typically repeat around 10 of samples Preparations 1 Prepare labeled paper bags for oven drying For leaf samples use medium sized bags with holes punched in them to increase oven drying efficiency use a hole punch and ensure you have a similar hole pattern in each bag For grain samples use small bags or envelopes Trial measurements Leaf samp
214. s set to manual not automatic and that the UNITS are set to mmol ms this ensures that measurements are made in units of conductance as the other two units m s molt and s mt are of resistance Return to the MAIN MENU Trial measurements 3 Choose a flag leaf that is clean dry free of disease and receiving sunlight to the adaxial surface 4 Place the leaf into the chamber at the mid point of the leaf and ensure that the selected area of the leaf completely covers the aperture of the sensor During the measurement take care to keep the white filter facing upwards and in full sun do not allow other plants to shade the filter 5 To start taking measurements press ENTER Once the readings have equilibrated press ENTER again to hold the reading The reading can then either be recorded manually or saved to the instrument It should take approximately 30 120 seconds to take the measurement If the reading takes longer than 3 minutes to equilibrate then discard this sample Troubleshooting Problem Solution Values are low lt 200 mmol m s 6 There are three options on the screen SAVE to save the data DISCARD to discard this measurement or ANNOTATE and press ENTER to name this data file After you have annotated and given your data a file name subsequent measurements can just be SAVED 7 Between measurements the porometer will request that
215. section sub samples inte aliminium pots Record S fresh weight a Remove debris r s Dry to constant weight Root sample Record dry weight Schematic 17 1 Determination of soil moisture content and or root content from a field soil core 90 Physiological Breeding II A Field Guide to Wheat Phenotyping D Figure 17 3 Root washing and cleaning A soil and water mixture is decanted through a sieve B clean water is used to repeatedly clean the sample C final mixture of roots and organic debris D root sample cleaned by eye E root sample ready for hand cleaning using forceps tweezers and F a completed sample F 2 Rapid root analysis This method is very quick and iii Repeat the measurement 5 times per soil core provides information on root content for genotype section e g 0 30 cm 30 60 cm etc comparison It is best utilized when it is expected Note that as observations are subjective it is that there will be large differences between cultivars important that ratings are consistent and or when measurement time is limited The method uses visual observation of the soil cores with little processing required This can be done either in the field or laboratory however ensure that soil moisture is not lost if samples are also to be assessed for moisture content A sample can be e If several people within the group will be processed every 5 10 minutes making observations it is recommended that all
216. sensor Take a note of the plot number and mistake and delete the unintentional value during data processing Confounding effects of plant height within the trial This can be corrected by grouping phenotypically similar lines within the population Oaklahoma State Univeristy 2011 Nitrogen use efficiency Available at http www nue okstate edu accessed 13 August 2011 Raun WR Solie JB Johnson GV Stone ML Lukina EV Thomason WE and Schepers JS 2001 In season prediction of potential grain yield in winter wheat using canopy reflectance Agronomy Journal 93 1 131 138 Verhulst N and Govaerts B 2010a The normalized difference vegetation index NDVI GreenSeekerTM handheld sensor Toward the integrated evaluation of crop management Part A Concepts and case studies CIMMYT Mexico D F Verhulst N and Govaerts B 2010b The normalized difference vegetation index NDVI GreenSeekerTM handheld sensor Toward the integrated evaluation of crop management Part B User guide CIMMYT Mexico D F Chapter 9 Chlorophyll content Debra Mullan and Daniel Mullan Chlorophyll is the green photosynthetic pigment which absorbs sunlight mainly in the blue and red portions of the electromagnetic spectrum and transfers this energy to the reaction center of the photosystems The chlorophyll content of leaves and other green tissues can be quickly and non destructively measured using a hand held battery porta
217. sful experimental program Many different scales exist for the evaluation of the developmental stage of a plant growth stages GS and the quick and non destructive Zadoks scale decimal code based on ten major stages Table 14 1 is the most commonly used Zadoks et al 1974 Tottman and Broad 1987 The precise determination of the crop development stage is important in physiological studies as key development stages emergence GS10 terminal spikelet GS30 first node at 1 cm above tillering node GS31 heading GS51 anthesis flowering GS61 and maturity GS87 mark important changes in the crop s life cycle Applications of fertilizer irrigation pesticides insecticide and fungicide and the impact of diseases insects and stresses e g frost heat drought are also best related to crop GS rather than to calendar date The optimum timing of sampling for physiological studies is best determined by crop GS and data expressed in relation to thermal time Cd days x mean daily temperature which in addition to the day length and amount of vernalizing cold drives the rate of growth and development The response of development to heat units is approximately linear above a minimum base temperature usually taken as 0 C up to a maximum mean daily temperature of about 25 C Typically the thermal time taken to complete a given period of development is constant for a given genotype On average the thermal
218. showing GS30 GS31 and GS32 Credit wheatbp net and C magnified developing spikes at GS30 showing terminal spikelet Magnification x 40 photograph Ariel Ferrante University of Lleida Direct growth analysis Heading spike emergence GS55 59 This is when 50 of the spike is emerged i e middle of the spike at the flag leaf ligule on 50 of all stems GS55 however often it is recorded as when the base of 50 of the spikes have emerged from the flag leaf sheath equivalent to full heading GS59 Measurement is typically by visual assessment of the whole plot by the same observer judging all treatments within a trial Alternatively assessment can be made by assessing 50 or 100 culms per plot Figure 14 3 icm Anthesis GS61 65 This stage takes around tem icm 3 5 days from appearance of the first A anther to completion for individual spikes depending on ambient temperature The start of anthesis GS61 is defined as the date at which 50 of spikes have extruded B C D Figure 14 3 The sequence of spike emergence the end of booting A GS47 and B GS49 and the start of spike heading C GS51 and D GS57 Credit wheatbp net at least one anther note that anthers first appear recorded which is defined as the date at which from florets in the middle of the spike and are then 50 of spikes have extruded 50 of their anthers extruded both above and below the center Figure Anthers are i
219. sociated with yield under heat and drought stress and both physiological Lopes and Reynolds 2010 and genetic Pinto et al 2010 evidence suggests this to be associated with a root vascular capacity However CT is also sensitive to the environment and requires clear skies and low winds for a reliable reading CID measured on non stressed leaf tissue during early development has been used to select for transpiration efficiency TE in environments where a conservative use of water early in the cycle is necessary to compensate for extremely limited water availability during grain filling Condon et al 2004 In fact the CID signal of any given tissue reflects the average internal carbon dioxide concentration during its growth Therefore when CID is measured on grain of different cultivars especially that of water stressed crops it is more likely to indicate their relative access to water rather than water use efficiency per se Hence the interpretation of CID data must always consider the growing environment and genetic effects that influence the amount of water available to a cultivar and therefore the stomatal response However CID is more expensive to measure than CT or stomatal conductance and requires access to a mass spectrometer Stomatal conductance has been proposed as a selection tool and when measured on multiple plants in a canopy is equally effective as CID or CT Condon et al 2008 However the instrumentation is
220. spike of which gt 90 can be fertile in optimal conditions or lt 50 in stressed conditions e g drought heat etc The plant reduces the number fertile spikelets as a stress escape mechanism to ensure that at least some viable grain is Figure 18 1 Harvesting for yield and yield components A plot combine harvester B large stationary thresher C small stationary thresher D hand threshing a grab sample E a threshed grab sample requiring final cleaning and F a Contador seed counter Pfeuffer GmbH Kitzengen Germany Direct growth analysis 101 produced However as this reduction in spike fertility is irreversible the plant is unable to restore lost fertility if the stress event should pass In the field 1 Randomly sample 6 10 spikes per plot by selecting at the base of the culm aim for 20 30 spikes per treatment 2 Count the total number of spikelets pair by pair from the base to the tip 3 Of these count the number which are infertile i e containing no grain Measurements from harvest samples Determination of the thousand grain weight TGW Values are typically 20 50 g i e 20 50 mg per grain and tend to be characteristic of a variety there are large differences between varieties even under good conditions Reduction in TGW may be caused by weather e g higher grain filing temperature or biological e g pathogen stress during grain filling or in field effects e g
221. ss wax spikes presence size and density awns density length and color It is therefore important to group genotypes by similar stage of phenological development e Amount and angle of incident radiation this is continuously changing throughout the day due to the movement of the sun and the passing of clouds These affect reflectance and calculated indexes to differing degrees It is therefore important to repeat the white and dark reference readings every 15 30 plots Preparations Ensure the spectrometer is securely connected to the control computer and that batteries for both devices are fully charged 1 After turning on the spectrometer allow the instrument to equilibrate with the ambient temperature for around 10 minutes Always turn the spectrometer on first before turning the controlling computer on in order to reliably establish a secure connection although this may be dependent on the specific device 2 Open the data capture software on the computer Set up the data capture file including the date of measurement and trial information 34 Physiological Breeding II A Field Guide to Wheat Phenotyping Initial measurements oP Calibration is required before taking the first measurement Adjust the configuration of the device including the foreoptic in use the integration time and the number of readings averaged per data point Point the sensor foreoptic downwards in the nadir position i e d
222. st This method is recommended when a large scale threshing machine is not available or when working with hard to thresh materials e g wheat wild relatives or synthetic wheat as only the sub sample is threshed Both sampling and processing are quicker than Methods A and B but care needs to be taken to ensure that a representative sample is cut The harvested area is smaller typically with an area 21 m defined using a quadrat or of a specific number and length of rows A sample is taken from the plot and a sub sample of a specific number of fertile culms is taken and dried to allow calculation of biomass and to allow expression of data on a per culm or per spike basis The sub sample is threshed and the grain weighed to allow calculation of HI and for TGW measurement The plot yield is calculated from biomass x HI See Schematic 18 3 Cut quadrat sample Record gr in dry weight Thresh sub sample Schematic 18 3 Reduced threshing harvest 1 a 4 In the field Select and carefully measure a representative area to be harvested with an area 21 m7 avoiding border rows and ends of the plot Cut all above ground biomass within this defined area and weigh FW_HA Put the harvested area biomass into a labeled paper or textile bag ensuring not to lose biomass In the laboratory Grab sub samples from the harvested area biomass i e by grabbing biomass at randomly chosen and represent
223. strument user manual Do ensure that the operator is familiar with each instrument functionality correct approach to take data and expected readings before going into the field it is worth receiving advice and training from an experienced user and reading the user guide Do take measurements consistently this is very important In particular ensure that the instrument is calibrated correctly before and sometimes again during use Keep a careful eye on the data during measurement to guard against erroneous data and large variations within a plot Do maintain batteries of correct type size and polarity Recharge batteries fully before use note that this may require overnight charging Take spare batteries to the field to ensure that measurements are not interrupted Do ensure to take the whole repetition with the same instrument If more than one instrument is available cross compare between instruments to check that they are giving similar data Do always make data easy to interpret process at a later date For example when taking readings with a data logger which records only basic information at the end of each section take two blank readings without a sample in the sensor chamber as an end marker 2 Drying of samples It is important that samples are dried to absolute dry weight DW i e 0 moisture The DW refers to the sample weight reached after drying in a well ventilated forced draft oven
224. surement recommended and record the values Repeat gas exchange measurements for 2 4 leaves per plot Once all measurements have been taken close the file Press ESCAPE to return to the New Measurements mode then press 1 and select CLOSE_FILE F3 Final measurements and completion 10 LA 12 With the chamber empty and closed and the system still on turn the drierite screw to the full scrub position and increase the flow to maximum then wait until the relative humidity falls below 10 Turn off the system Leave the CO cylinder attached so that any remaining CO is released slowly Ensure that the screws of the chamber and desiccants are loose when the system is not in use to avoid damaging the chemical tubes Saved data can be downloaded with the software supplied with the instrument Data is typically downloaded as a comma delimited text file and imported into MS Excel Data and calculations For most measurements calculations are given directly by the instruments Typical values for the most used gas exchange and chlorophyll fluorescence parameters for wheat in irrigated or stressed environments are shown in Table 13 1 Table 13 1 Typical data for gas exchange and chlorophyll fluorescence measurements in irrigated and stressed environments Gas exchange Irrigated Stressed Ara 15 30 umol m st 5 20 umol m st g 300 700 mmol m st lt 300 mmol m s4 Chlorophyll fluor
225. t PI Rs Regn spectra carotenoid NPQI Normalized Normal chlorophyll PI Ras Rags RaistRaas pheophytinization index degradation can be used to estimate phenology pest and diseases SIPI Structural independent Senescence related to stress PI Rs00 R435 R415 R435 pigment index PRI Photochemical reflectance index Dissipation of excess radiation PI R531 Rs70 R531 Rs70 WI Water index Plant water status WI Roz Rago NWI 1 Normalized water index 1 Plant water status WI Ro79 Rooo Rozo Roool NWI 2 Normalized water index 2 Plant water status WI Ro79 Rgsol RozotResol NWI 3 Normalized water index 3 Plant water status WI Ro7o Regol RozotRegol NWI 4 Normalized water index 4 Plant water status WI Ro79 Ro29 Ro7o Ro201 32 Physiological Breeding II A Field Guide to Wheat Phenotyping Figure 7 1 Measuring spectral reflectance from a crop canopy Site and environmental conditions Measurements should be taken e Ona clear sunny day as cloud cover or overcast conditions will increase the amount of diffuse indirect radiation incident on the canopy increasing canopy light penetration and the amount of radiation absorbed by photosynthetic pigments The estimation of vegetation indices will be overestimated under these conditions e When there is negligible wind as even a light wind can modify canopy structure and may distort the calculation of spectral indices e When there is no dew or moisture on the surface of the leaves
226. t along plot a i RO A YN Figure 1 4 Ensure that the IRT is held at an appropriate angle so that measurements are taken from A the crop canopy and B not from the soil e g where there are establishment problems or low biomass Before starting to take measurements it is recommended to first examine the entire trial decide the angle and distance from the crop that maximizes the green area interception and hold the IRT at this angle and distance for all plots within the trial 12 Physiological Breeding II A Field Guide to Wheat Phenotyping Specific advice for using the Sixth Sense LT300 Mode press this to change the function between MAX DIF AVG PRB set this to AVG to give average of all temperature readings while the trigger is held C F to change between C and F set this to C or F depending on units desired EMIS do not change this should be 0 95 Lock this is the function for permanent readings and should be deactivated Trigger 4 activates and deactivates the laser Trigger 7 activates and deactivates the light on the screen Preparations 1 After turning on the IRT allow the instrument to equilibrate with the ambient temperature for around 10 minutes Check that the average mode is selected AVG is shown on the screen and ensure that the lock function is deactivated LOCK is not shown on the screen see Figure 1 2 2 After turning on the temperature and
227. t they must be taken at high sun angles to avoid confounding effects Leaf chlorophyll content can be measured directly by several dedicated devices the most common of which is the simple to use hand held SPAD meter Spectrum Technologies Inc Plainfield IL USA The SPAD and GreenSeeker have built in light sources active sensors and thus can be used under any conditions Section 3 Photosynthesis and light interception Photosynthetic rate is the principal driver of yield in agronomically adapted crops Direct measurement of gas exchange using infrared gas analysis IRGA can be performed in the field to quantify photosynthetic rate at the leaf level However the measurements are time consuming require expensive instrumentation and because they are typically measured one leaf organ at a time are not integrative see Lopes Molero and Nogues Volume 1 Although expression of light saturated flag leaf photosynthetic rate has been associated with yield other easier to measure traits like CT and stomatal conductance show equally good association Fischer et al 1998 Chlorophyll fluorescence is faster to measure than gas exchange and has been shown to explain genetic variation in crop performance Araus et al 1998 although it has not been adopted as a routine procedure in breeding programs because the protocol is not straightforward In the absence of other constraints i e in relatively high yielding environments
228. ta on a per culm or per spike basis A sub sample of grain is taken and dried to allow 5 calculation of grain DW and for TGW measurement See Schematic 18 1 In the field culm classes and count the number of fertile culms continue until the sub sample contains 50 or 100 fertile culms and weigh FW_SS Thresh all harvested area biomass when dry using a large stationary thresher Figure 18 1A remove chaff and weigh grain FW_ HA_G Remember that the grain from the sub sample of harvested biomass is separate Take a sub sample of harvested area grain and weigh approx 50 g and put in labeled paper envelope FW_HA SS G In the laboratory 1 Carefully measure the area to be harvested a E a A AEE G A excluding border rows and ends of the plot AG Raa 2 Cut all above ground biomass within the ls oe a Net aan ie a mained harvested area and weigh FW_HA a siki 3 Grab sub samples from the harvested area i Taresh me sub sample iror the Narvested biomass using a small stationary thresher biomass i e by grabbing biomass at randomly i i i or by hand Figures 18 1 C D and E remove chosen and representative places in the biomass i Wa i chaff and weigh grain DW_SS G sample and gaining a representative mix of Cut all biomass in harvested area and Measure area Dry to constant weight Take 50 100 Thresh culm sub sample Wi sub sarmple 7 Record dry Record grair weight dry w
229. tal biomass harvest B Sub sample harvest C Reduced threshing harvest FW_HA g 9600 1500 FW_SS g 500 0 500 0 FW_HA G g 3640 3640 FW_HA_SS_ G FW_HA GB G g 50 00 50 00 DW_HA SS _G DW_HA GB G g 47 50 47 50 DW_SS DW_GB g 475 0 475 0 475 0 DW_SS G DW_GB G g 190 0 190 0 190 0 Yield g m 3640 x 47 50 50 00 190 0 6 4 1425 x 0 40 570 570 Biomass g m 9600 x 475 0 500 0 6 4 570 0 40 1500 x 475 0 500 0 1 1425 1425 1425 Harvest index 570 1425 190 0 475 0 190 0 475 0 0 40 0 40 0 40 Where FW fresh weight DW dry weight HA harvested area SS sub sample GB grab sample G grain In this example there are 100 fertile culms in the sub sample grab sample of biomass Formulas assume that grain is dried to 0 moisture Grain yield at x moisture g m yield x 100 100 x The grain from the SS GB of biomass is separate from the FW of harvested area grain 100 Physiological Breeding II A Field Guide to Wheat Phenotyping Determination of the spike number m The number of spikes m i e fertile culm number m is determined by events occurring between sowing to flowering and is dependent on variety management and environment When combined with the plant number m2 it can be used to assess the number of fertile tillers per plant typically 1 10 It can be easily and non destructively measured during grain filling i e before physiological maturity
230. ter content Daniel Mullan and Julian Pietragalla Chapter 6 Carbon isotope discrimination Marta Lopes and Daniel Mullan Spectral reflectance indices and pigment measurement 32 37 41 Chapter 7 Spectral reflectance Julian Pietragalla Daniel Mullan and Raymundo Sereno Mendoza Chapter 8 Normalized difference vegetation index Julian Pietragalla and Arturo Madrigal Vega Chapter 9 Chlorophyll content Debra Mullan and Daniel Mullan Photosynthesis and light interception 46 54 58 63 Chapter 10 Crop ground cover Daniel Mullan and Mayra Barcelo Garcia Chapter 11 Light interception Daniel Mullan and Julian Pietragalla Chapter 12 Leaf area green crop area and senescence Alistair Pask and Julian Pietragalla Chapter 13 Gas exchange and chlorophyll fluorescence Gemma Molero and Marta Lopes Direct growth analysis 72 78 83 87 95 Chapter 14 Determining key developmental stages Alistair Pask Chapter 15 In season biomass Julian Pietragalla Debra Mullan and Eugenio Perez Dorame Chapter 16 Water soluble carbohydrate content Julian Pietragalla and Alistair Pask Chapter 17 Sampling soil for moisture nutrient and root content Marta Lopes J Israel Peraza Olivas and Manuel L pez Arce Chapter 18 Grain yield and yield components Julian Pietragalla and Alistair Pask Index Crop observations 106 Chapter 19 Crop morphological traits Araceli Torres and Julian Pietragalla 113 Chapter 20
231. ter relations in contrasting wheat genotypes Journal of Experimental Botany 61 12 3291 3303 Osborne SL Schepers JS Francis DD and Schlemmer MR 2002 Use of spectral radiance to estimate in season biomass and grain yield in nitrogen and water stressed corn Crop Science 42 165 171 36 Physiological Breeding II A Field Guide to Wheat Phenotyping Pe uelas J Filella l Biel C Serrano L and Save R 1993 The reflectance at the 950 970 nm region as an indicator of plant water status International Journal of Remote Sensing 14 1887 1905 Pefiuelas J Filella and Gamon JA 1995 Assessment of photosynthetic radiation use efficiency with spectral reflectance New Phytologist 131 3 291 296 Prasad B Carver BF Stone ML Babar MA Raun WR and Klatt AR 2007 Potential use of spectral reflectance indices as a selection tool for grain yield in winter wheat under Great Plains conditions Crop Science 47 1426 1440 Wiltshire J Clark WS Riding A Steven M Holmes G and Moore M 2002 Spectral reflectance as a basis for in field sensing of crop canopies for precision husbandry of winter wheat HGCA Project Report No 288 Home Grown Cereals Authority Caledonia House London UK Zhao C Wang J Huang W and Zhou Q 2009 Spectral indices sensitively discriminating wheat genotypes of different canopy architectures Precision Agriculture 11 557 567 Chapter 8 Normalized
232. the bag ensure to break up and mix the soil thoroughly before opening the bag to avoid losing moisture Incorrect oven drying temperature check using an auxiliary thermometer Do not use higher temperatures to reduce drying times as this may destroy some of the soil constituents and bias results Re absorption of moisture after drying ensure to weigh dried samples once they have cooled sufficiently whilst not allowing time for moisture re absorption Root content Contamination with previous crops Take test soil samples across the field where you plan to measure roots to check for the in the field A lot of soil remains with roots during sieving Rapid root analysis Difficulties while cutting the core transversally The soil is very dry and the core is crumbling Root analysis using a digital scanner Roots dry up in the refrigerator 94 Physiological Breeding II A Field Guide to Wheat Phenotyping presence of roots at different depths This can be done by visual observation of the cores Mix water soil and roots by hand very gently whilst destroying any existing soil aggregates Wait about 10 minutes without disturbing the mixture to allow the soil to drop to the bottom of the tray then decant gently Use a sharp instrument to cut the core e g spatula knife or guitar string and use oil if necessary to avoid the two parts sticking to each other after cutting Cutting the core may be difficult especially
233. the chamber is opened to ventilate any residual humidity Data and calculations Depending on the instrument set up either take note of the values given during sampling or save the data to be downloaded with the software supplied with the instrument Data is typically downloaded as a comma delimited text file and imported into MS Excel Typical values for irrigated trials are 300 700 mmol m s and for mildly water stressed trials are 830 300 mmol m s7 The soil is too dry and stomata have closed Only take measurements in reasonably well watered trials irrigate and then repeat measurements Ensure to minimize physical manipulation of leaves as stomata are sensitive Large error variance in data Uniform the leaf selection criteria e g same position age orientation etc Erratic values from porometer Irregular soil moisture across the field possibly due to patchy drying of soil irrigate and then repeat measurements Clouds passing in front of the sun measurements are best taken with cloudless skies Anomalous values from steady state dynamic Avoid exposing sensor head to solvent fumes e g alcohol acetone diffusion or null balance porometers gasoline If this occurs re calibrate the sensor Do not use solvents to clean sensor head Useful references Decagon Devices 2011 Available at http www decagon com accessed 11 August 2011 Fischer RA Rees D Sayre
234. therley 1962 Site and environmental conditions Samples can be taken under most environmental conditions However it is important that the plant surfaces are not wet from dew irrigation or rain Time of day The optimal time for sampling is at solar noon 2 hours as this is the most stable time of day with respect to irradiance and temperature and their effect on leaf water relations A daily curve of leaf RWC can be obtained by taking measurements throughout the day Plant developmental stage Samples can be taken at any developmental stage and or at regular intervals from the start of tillering to late grain filling depending on the experimental objectives timing of peak stress For instance in terminal drought and or heat trials sampling is performed at early grain filling as an assessment of stress adaptation Note that in severely stressed conditions plants will senesce quickly and measurements should be taken earlier Sequential measurements throughout this period will allow assessment of changing leaf RWC Number of samples per plot Take six leaf samples from different plants in each plot Procedure The following procedure describes a whole leaf technique modified from Stocker 1929 Alternatively a leaf disc technique may be used Take the following equipment to the field e Scissors e Labeled sample tubes one per plot e Cool box And required in the laboratory e Semi analytical balance to 3
235. thetic potential effects of stress nutrient deficiency stay green Early interception of radiation early estimate of reduction in soil moisture evaporation Allows calculation of green area index GAI and K canopy extinction coefficient links to canopy architecture Relates to light interception and photosynthetic performance surfaces for transpiration crop biomass Advantages of tool Integrative quick easy and cheap to measure non destructive remote Relatively quick measurement of stomatal activity non destructive Definitive measurement of leaf water energy Small number of samples required relatively simple technique Simple and cheap to measure low technology approach Leaf samples give early estimate of TE grain sampling is quick and easy and gives integrative measurement All indices available from a single repeated measurement integrative non destructive Quick easy and cheap to measure integrative non destructive Quick easy and cheap to measure non destructive Quick easy and cheap to measure integrative non destructive Quick to measure non destructive Easy to measure absolute measurement Disadvantages of tool Sensitive to environmental fluxes interaction with time of day and phenology Stomata are sensitive to manipulation point measurement only High pressures required sequential day and night measurements required Laborato
236. three readings at each sample level within the crop canopy Procedure The following procedure describes taking measurements with the Decagon AccuPAR LP 80 ceptometer which allows simultaneous measurement of PAR above using an external sensor and below using a probe the canopy Take the following equipment to the field e Ceptometer and external above canopy sensor e Weather station solarimeter continuous PAR for RUE calculations Advice on taking measurements Before taking any measurements ensure that the time date and location options are set correctly as this will determine zenith angle and consequently LAI Once you have set these parameters for your location they will remain saved and these will only need to be reset for sampling at a different location When placing the probe below the canopy take care that it does not become dirty If it does it is important to carefully clean the probe with an appropriate solution e g recommended by the manufacturer before taking more measurements Readings may be taken at defined levels within the crop canopy e g below the spike below the flag leaf at the soil level etc Hold the probe within the canopy ensure that it is level and held in a representative orientation e g in a two row plot hold the probe diagonally across both rows see Figures 11 1 and 11 2 Hold connect to the above canopy sensor ensure that that it is also level use bubble spirit level
237. time to produce a mature crop is 1550 Cd e g 15 C above base temperature for 103 days for spring wheats and 2200 Cd for winter wheats Site and environmental conditions Measurements can be taken under any environmental conditions Time of day Measurements can be taken at any time of the day Plant developmental stage Key development stages emergence terminal spikelet first node at 1 cm above tillering node heading anthesis mid grain filling and maturity are the most 72 Physiological Breeding II A Field Guide to Wheat Phenotyping informative Anthesis 7days sampling is considered strategically important for physiology studies as it is the moment where the structure of the spike reaches its maximum dry weight the grain weight is insignificant and the water soluble carbohydrate WSC reserves in stem are at their peak Key sensitive stages the date of heading is particularly useful under stress conditions as it is clearly observed Under extreme drought anthesis may occur before spike emergence and pollination can occur when the spike is still in the boot and under heat the spike will emerge but anther extrusion may not occur In these cases to determine the date of anthesis either the flag leaf sheath can be opened to reveal the spike floret which can be opened to reveal the anthers or the date of anthesis can be determined retrospectively based on length of the developing grain which takes 7 10 da
238. tself is lighter and cheaper It is therefore a good option for measuring most types of plant stress and monitoring plant health However chlorophyll fluorescence is not a straightforward protocol leaves must be dark adapted the fluorescence signal shows highly dynamic kinetics and relationships with performance have not proven to be especially dependable The decision matrix shown in Figure 13 1 will help select the appropriate technique s for individual target environments Target environment Stressed environment Large population Drought Large and small population Low Small population Resources available Yield potential Large population Small population Resources available High Figure 13 1 Decision matrix to select either both gas exchange and chlorophyll fluorescence measurements or only chlorophyll fluorescence measurements for individual target environments Note that variation in dark fluorescence has been found only under very severe stress and should therefore not be used under moderate drought or heat stress Resources available refers in particular to time and money Photosynthesis and light interception 63 However gas exchange and chlorophyll fluorescence are not typically used to screen large numbers of genotypes in breeding programs Instead breeders use quicker and cheaper proxy measurements correlated with photosynthetic performance such as in this vo
239. ty of the grains to use assimilates from photosynthesis Solar noon is the moment when the sun appears at the highest point in the sky during the day The angle of the sun with respect to the horizon 90 is termed the zenith angle required for calculation of certain canopy structure parameters e g leaf area index it is also important to record the longitude latitude date and time of day Source potential is the capacity of the plant crop to produce photosynthetic assimilates Stem elongation stage is the period when the stem elongates by extending the regions between the stem nodes The first nodes joints become visible and progressively larger after the terminal spikelet has formed on the microscopic spike Stomata are pores openings on the surface of the leaf and stem which are used for gas exchange i e carbon dioxide and oxygen Stress is a negative pressure on the yield of a crop e g drought heat Stress adaptation is the ability of a plant crop to reduce and or resist the negative effects of a particular stress Sub sample is a proportion of a field sample taken in the laboratory This method allows processing and weighing in laboratory with greater accuracy Tiller is a side shoot thus the tillers of a plant do not include the main culm Trait is a specific characteristic of a plant crop e g deep rooting Transpiration is the loss of water from the surface of a plant typic
240. typing studies Direct measurements of photosynthesis from gas exchange are performed with an infrared gas analyzer IRGA which measures the carbon dioxide flux within a sealed chamber containing a leaf sample Chlorophyll fluorescence measurements using a fluorometer provide an indirect estimation of the different functional levels of photosynthesis processes at the pigment level primary light reactions thylakoid electron transport reactions dark enzymatic stroma reactions and slow regulatory processes Fracheboud 2006 Both measurements are made at the single leaf level for precision phenotyping of small populations i e lt 100 genotypes when other measurements are not sufficiently precise to detect genetic differences e g to detect the initial stages of stress on photosynthetic metabolism or are not informative Photosynthesis measurements have been successfully used to demonstrate genetic diversity in performance and to explain physiological responses to environmental effects e g light temperature carbon dioxide concentration relative humidity ozone etc and crop inputs e g herbicides However gas exchange measurements in the field are laborious and expensive require detailed expertise and provide complex data of only a limited number of plants In comparison chlorophyll fluorescence measurements can be taken much more quickly lt 30 seconds per plant vs at least 2 minutes per plant and the instrument i
241. unreplicated design with a common check genotype in each pot Grow each group of genotypes in the same pot to ensure common soil WP It is important that measurements are taken on well developed leaves Ensure that the leaf is clean Remove dust from surface of leaf samples using a moist paper towel and then dry well before placing the sample in the container Always use latex gloves to avoid contamination of sample with salts from sampler s hands Perform the measurements when the stressed plants show wilted leaves in the afternoon approximately WP lt 1 2 MPa 12 bar or RWC 60 Preparations Label the Eppendorf tubes with the name of the trial genotype identification number and pot number Figure 4 1 Taking osmotic adjustment readings showing A droughted left versus fully irrigated plants right B overnight rehydration treatment C cutting a leaf sample D placing the leaf sample into the deep freeze E crushing leaf tissue in the Eppendorf tube to extract a drop of cell sap and F taking a reading using a vapor pressure osmometer 22 Physiological Breeding II A Field Guide to Wheat Phenotyping Greenhouse measurements Day 1 Preparations Before dawn measure the LWP of two or three fully expanded leaves from each pot using the Scholander pressure chamber see this volume Chapter 3 to define the level of stress In the afternoon irrigate all pots to saturation and cover with
242. unter manual Seedburo Placement Trays Grain sample htto www seedburo com Soil corer set electric Eijkelkamp Agrisearch Percussion drilling set with Soil root http www eijkelkamp com percussion hammer Equipment light electrical percussion hammer Soil corer tractor Giddings Soil Sampling Co 15 Soil root http www soilsample com mounted Spectrometer Spectral Evolution PSR 2500 Canopy leaf http www spectralevolution com Ocean Optics JAZ Canopy leaf http www oceanoptics com PP Systems UniSpec SC and UniSpec DC Canopy leaf http www ppsystems com CID Bio Science Cl 700 leaf clip ready Leaf http www cid inc com Spectroradiometer ASD Inc FieldSpec 3 AgriSpec and Canopy leaf http www asdi com HandHeld 2 Spectral Evolution PSR 2500 and PSR 1100 Canopy leaf htto www spectralevolution com Thresher Almaco SBT and LPT Plot bundle sample htto www almaco com Vapor pressure EliTech Group Wescor VAPRO 5600 Tissue sap htto www wescor com osmometer General recommendations 129 Appendix Glossary and abbreviations Anthesis or flowering is the period when the plant produces pollen and sets grains Each floret s lemma and palea are forced apart by swelling of their lodicules which allows the anthers to protrude Cultivar is a type of wheat with desirable characteristics which has been commercially released and is grown and cultivated Early generation selection EGS for traits expressing good associatio
243. uration White reference panel of Ensure to set the integration time manually to keep the peak of white reference reflectance reading between 75 and 85 in the initial measurements This can be made from a mix of barium sulphate and Spectralon is very expensive white latex paint There are several important considerations to be made when performing remote There is inconsistency in readings across the trial sensing measurements and interpreting spectral results The reflectance of electromagnetic radiation from a canopy may be influenced by numerous factors including the following Canopy structure and morphology Degree of canopy cover Geometry of incident radiation Degree of shading Presence of clouds Presence of nearby objects Useful references Aparicio N Villegas D Araus JL Casadesus J and Royo C 2002 Relationship between growth traits and spectral vegetation indices in durum wheat Crop Science 42 1547 1555 Babar MA van Ginkel M Reynolds MP Prasad B and Klatt AR 2007 Heritability correlated response and indirect selection involving spectral reflectance indices and grain yield in wheat Australian Journal of Agricultural Research 58 432 442 Blackburn GA 2006 Hyperspectral remote sensing of plant pigments Journal of Experimental Botany 58 855 867 Gutierrez M Reynolds MP and Klatt AR 2010 Association of water spectral indices with plant and soil wa
244. ust visible 98 Secondary dormancy 39 Flag leaf ligule collar just visible 99 Secondary dormancy lost 76 Physiological Breeding II A Field Guide to Wheat Phenotyping Troubleshooting Problem How should the main tiller be identified after tillering In field identification of the terminal spikelet for GS30 determination is difficult and or time consuming In drought stressed trials the spike has not emerged from the boot before anthesis Comparison of developmental data across genotypes and sites does not show a clear relationship with time in days References Tottman DR and Broad H 1987 The decimal code for the growth stages of cereals with illustrations Annals of Applied Biology 110 441 454 University of Bristol 2011 Wheat The big picture Bristol Wheat Genomics Available at http www wheatbp net accessed 11 January 2012 Zadoks JC Chang TT and Konzak CF 1974 A decimal code for growth stages of cereals Weed Research 14 415 421 Solution The main tiller can be identified as the longest and most advanced i e with most number of developed leaves To do this arrange all tillers from a single plant with their basal nodes together and select the culm which is the longest from the base of the stem to the tip of the newest fully expanded leaf For in field identification of multiple plots it is more useful to identify the first detectable node at 1 cm above tillering node GS31 In
245. vapor pressure osmometer 6 Check that the thermocouple of the osmometer is clean before assaying samples according to the user manual 7 Calibrate the osmometer with known concentrations e g sodium chloride solution of increasing concentration 100 290 and 1000 mmol kgt depending on model or brand of the instrument pa Crush the tissue in the tube using a glass rod 9 Extract a drop of cell sap using a pipette Figure 4 1E Always change the pipette tip between samples 10 Put the drop of cell sap onto a paper sample disc placed on the sampling cuvette of the osmometer The optimum sample volume 10 ul should fully saturate the sample disc 11 Read the value Figure 4 1F 12 Clean the cuvette of the osmometer using deionized water Data and calculations The OP values obtained from the osmometer are in mmol kgt which need to be converted to MPa pressure unit according to the equation OP MPa R x T x osmometer reading 1000 Equation 4 1 Where R is the gas constant 0 008314 and T is the laboratory temperature measured on the Kelvin scale in this example T 298K i e 25 C OA is calculated as the difference in OP between the non stressed control well watered and stressed treatment water withheld or droughted both of them at full hydration turgor status OA OP icnistreseed OP sieesea Equation 4 2 For example using data from table 4 1 OA 0 409 0 817
246. y see this volume Chapter 15 For total root biomass take samples from anthesis 7days to mid grain filling Number of samples per plot Take 4 6 soil cores per plot However as soils are extremely heterogeneous soil moisture and root data can be very variable within a plot and it is advisable to increase repetition where possible Procedure Take the following equipment to the field e Hand soil corer e g 25 mm in diameter tractor with hydraulic soil corer e g 42 mm in diameter Figure 17 1 x 120 cm and associated tools e Lubricant e g used motor oil e Pre labeled plastic bags e Tape measure to measure 30 cm sections of the core e Spare plastic bags and marker pen And required in the laboratory e Balance to 2 d p e Numbered aluminum pots with lids or aluminum foil e Tweezers e Oven to 105 C not force draft ventilated so the soil is not blown away Advice on taking measurements Typically soil samples are taken after biomass sampling to avoid damage disturbance to the crop and to link data with crop growth or choose locations at random to avoid bias and from all experimental rows of the plot For the determination of crop water uptake soil moisture samples must be taken after each irrigation event to measure the soil water content at tO i e time zero Where possible avoid sampling soil in the vicinity of soil cracking as this affects the soil dynamics and any other obvious obje
247. y to store samples before measurement this can be done by keeping the plant material in a cool moist atmosphere for up to four days e g sealed in a plastic bag and between moistened tissue paper Take 20 culm sub sample rials M l Spikes Stem with Leaf leat sheath lamina Remove veliow dead material Measure area with an automatic planimeter Schematic 12 1 Determination of leaf and green area from a sub sample from the in season biomass sample using an automatic planimeter Photosynthesis and light interception 59 Preparations 1 After turning the automatic planimeter on the instrument should be allowed to warm up for around 10 minutes during this time the area count should remain at zero 2 Use the calibration discs provided or make paper shapes of known area preferably resembling the shape of the material to be tested Laboratory measurements 3 From the quadrat sample randomly select a sub sample of 20 fertile culms ensuring that all culms have a well formed spike Cut the spike from the stem at the spike collar Remove all the leaf lamina from each culm and either bulk together all leaf lamina or separate into leaf layers i e flag leaf leaf two leaf three and below etc 6 Remove the yellow dead material from the green tissue do not discard this material 7 Measure the green area of each component i e all leaf lamina leaf layers stem with leaf s
248. ys after pollination to reach its full length depending on the environment Number of samples per plot Take one observation and or an assessment of 10 plants or 50 or 100 culms per plot see individual measurements below Procedure Take the following equipment to the field e The Zadoks scale Table 14 1 e Field form and clipboard Advice on taking measurements Continual assessment of crop development during the growth cycle is important It is necessary to assess and record the developmental stage of individual plots every 2 or 3 days in the period leading up to the desired sampling stage of development The rate of crop development is affected by genotype therefore plots within a trial may reach key developmental stages at different dates It may therefore be necessary to take samples over a period of several days to ensure comparability between genotypes Breeders and scientists may wish to split populations into early and late lines to avoid confounding effects on data analysis e g see Canopy temperature this volume Chapter 1 A developmental stage is assigned when 50 of the main culms in a plot are at the stage up to and including GS31 and 50 of all culms thereafter Data are usually presented as days after sowing DAS 1 DAS is the day of sowing for emergence and days after emergence DAE 1 DAE is the day of emergence for following the developmental stages Seedling emergence GS1
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