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1. 34 Introduction to germplasm characterization and evaluation ee 34 Selection of Materials for Characterization kee 34 Plant terio lol E SI ee one A DE De EE DE 34 Morpho Agronomic CharacterizatiON sesse ees ee AA Re ee AR ee ee ee 34 CHAPTER 6 GERMPLASM EXCHANGE see 41 Rec ipt o PP EE e 41 Processing newly received germplasm uv 41 Protocol f r seed CIS MUON ais Sema RR EE RR EG DA EER 42 IRRI s policy on EELER deg Eg N Ke De ci n 44 IRRI s Policy on Intellectual Property Rights AAA 44 Protocol for requesting IMM OM ess 44 CHAPTER 7 DOCUMENTATION AND EXCHANGE OF GERMPLASM INFORMA TON EE EE N EE iii 46 Documentation and exchange of germplasm information sees Re 46 Background ER EE ML EA EE EO 46 System ers ee RE EE EER 46 SUSTO SCI usos 46 System menu SM CUE ies sai ae ER Ge ER ER RE Eva Seine eene 47 Features and capabilities es Ee mime eect suet ewan See 47 IRCGIS AIS SSG NA ES RE alicia Sea 47 Data el EE EE EE AE e 48 Bata END ee EE Boca ete Ee ee es eee 48 MERS ie AP 48 System COC UMS MAU O AE OE EE N 48 CHAPTER 8 MOLECULAR MARKER LABORATORV ees ees ees ee ee see ee 49 Molecular marker ADO Y conoci 49 Protocol for DNA Sample Preparation kee 49 RAPRDPrOtOCOI o A 51 Microsatellite POU Gl suecia EE ke EE RE EE Ee oe EE ee Es 53 Ed ese MERE EE EE EE EE EE 57 Abbreviations AGrONVIMS eeu EE GRENSE ERA iaa 61 CHAPTER 9 CONSERVATION SUPPORT ees ese ee ee ee e
2. 2 Mix gently by flicking the bottom of the tube and spin to collect the solution 3 Aliquot 36 ul of the mixture in each of the 10 properly labeled 0 5 ml microtubes Add a 4 ul DNA sample to each tube Stock and final concentrations per 40 ul mixture Component Stock Conc Final Conc Vol 40 ul AFLP grade HO 24 8 ul Reaction buffer 5x 1x 8 0 ul Enzyme 10 u ug 3 2 ul EcoR1 Mse1 DNA 57 DNA sample 100 ng 250 ng 4 0 ul total volume 40 0 ul 4 Mix gently by flicking the bottom of each tube and spin to collect the solution Overlay with a drop of mineral oil 5 Incubate the mixture for 2 h at 37 C and 15 min at 70 C using a thermal cycler This is now digested DNA 6 After incubation remove the tubes from the thermal cycler Place tubes in ice and spin to collect the solution 7 Transfer 25 ul of each digested DNA to properly labeled 0 5 ml new tubes for ligation of adapters 8 The remaining 15 ul of the digested DNA will be used for digestion check Restriction digestion check 1 ql o N Add 3 ul 10x loading buffer in each of the 15 ul digested DNA Mix gently by flicking the bottom of the each tube and spin to collect the solution This is now ready for loading in the gel Prepare a 1 2 agarose gel Refer to the electrophoresis section in the RAPD protocol for procedure in gel preparation Load 10 ul of 1 Ko DNA ladder on the first well of the gel Load 10 ul of each digested DNA in the succeeding wel
3. Add 25 g chloral hydrate and dissolve for 24 h Add 3 ml glycerin Mix thoroughly and filter through cloth 8 hydroxyquinoline 002 M solution in water 0 29 g a bromo napthalene used as saturated solution in water or 1 aqueous solution of stock solution of 1 ml bromonapthalene dissolved in 100 ml absolute ethanol for 30 min Fixative Fixative should be freshly prepared for each fixation It consists of absolute ethanol chloroform glacial acetic acid or absolute ethanol glacial acetic acid Appendices Appendix 1 1 doc Appendix 1 2 pdf Appendix 2 1 pdf Appendix 2 2 pdf Appendix 2 3 pdf Appendix 3 1 doc Appendix 3 2 doc Appendix 3 3 3 5 doc Appendix 3 4 pdf Appendix 5 1 pdf Appendix 5 2 pdf Appendix 5 3 pdf Appendix 6 1 doc Appendix 6 1A doc Appendix 7 1 pdf Appendix 9 1 pdf 6 parts 3 parts 1 part 3 parts 1 part 6 GERMPLASM EXCHANGE 68 Isozyme Protocol Leaf Sample Preparation 1 for five to ten day old seedlings Germinate the seeds of each accession in petri dishes lined with moist filter paper Place these in a growth cabinet at 30 C for 5 10 days These seedlings will be used for the crude extract 2 for forty day old seedlings Collect needle like leaf samples from 40 day old seedlings Wrap samples in moist paper towel and keep inside an ice chest until ready for extraction Gel Preparation 1 Get 3 gel molds and seal their anodal and cathodal ends with 2 masking ta
4. Store in dark bottle Acrylamide Bis acrylamide upH20O volume to Store in dark bottle Ammonium persulfate UPHO volume to Amount 150 mI 4209 200 ml 1000 ml 190g 10g 500 ml 1g 10 ml 93 Binding solution Developer solution for Promega APC film Store in dark bottle EtOH 95 1 ml Acetic ccid 5 ul Bind silane 3 ul Kodak GBX developer 200 ml dH20 800 ml Store in dark bottle Table 8 9 con t Solution Developer solution for polyacrylamide gel Promega kit dNTP mix 100 mM dNTP mix 25 mM dNTP mix 5mM Fixing solution for Promega APC film Chemical Composition Amount Nao CO 60 g Dissolve in 2000 ml upH20 and cool at 4 C Add before use Na2S205HO 10 mg ml 400 ul dATP 100 mM 100 ul dTTP 100 mM 100 ul dCTP 100 mM 100 ul dGTP 100 mM 100 ul Mix together and dispense into aliquot dNTP mix 100 mM 125 ul supH O 375 ul Mix together and dispense into aliquot dNTP mix 25 mM 100 ul supH20 400 ul Mix together and dispense into aliquot Kodak GBX Fixer 200 ml 94 Fix stop solution for polyacrylamide gel upH20 Store in dark bottle Glacial acetic acid upH20 800 ml 200 ml 1800 ml Table 8 9 con t Solution KCI 2M Mot 500 mM MgCl2 15 mM NaOH 5M Silver stain solution Promega kit STR loading buffer 3x TBE buffer 10x Chemical Composition KCI upH20 volume to MgCl
5. The system automates several tasks for example Generation of Batch ID and Temporary ID for easy identification of incoming samples prior to assigning of IRG accession number Checking of incoming samples for probable duplicates based mainly on the sources country and soundex code of variety name Selection of materials for seed increase and rejuvenation based mainly on the seed stocks and viability Selection of materials for morpho agronomic characterization based mainly on the availability of the characterization data Generation of plot number unique within a cropyear for each material selected for planting and generation of the corresponding field books datasheets and labels This facilitates the identification and monitoring of the materials planted in different locations Identification of qualified incoming samples for long term storage Generation of on date summary reports on the status of different genebank activities Selection of materials to be designated under the auspices of FAO Screening of qualified accessions for seed distribution Inquiry on the status of the seed request Generation of needed document agreement regarding germplasm exchange Selection of seed sources for distribution planting and for viability monitoring Generation of different summary reports on all germplasm dispatched and stored There are other tasks automated by the system These are all explained and illustrated in detailed in the IR
6. 104 ECH EE N RE EE EE EER OE OE RE 74 NN 15 E TT EE 74 100 EE AAA E E A E ES baa a ATA aad ant Ee hed naan awa 74 el ER AE once Howat ca Siew OO TR OE EE hi heh as 15 ESE RE ER RE HE RR OE OE a EE 85 GESE E peer ee E E EE Ee Ee ee 85 GESMEE AA N ER AO E OR EN EE OO 85 NN 68 GBP Hai A EE 85 Gawel ER Ee AR GE GE ee ee A a Baa Bead aaa AA aes 56 BEA ARA dd EE SE AT evans tea ws RE RE aes ant RS 85 el deel RE N ME EE EE N 77 Gel electrophoresis EE ED ad ait 60 Gel Preparation cs ii sede ge Pe teste ea a ia anne 77 EE AA RE ED LE OE OE OE EE AT RO N 77 Gelati e ii EE RE ta tadas dto 60 Genebank ooocccccccccccccccccncnononononononononononononononononononos 5 8 11 13 28 30 31 39 46 47 52 53 54 ASSIS EN RE EE EE NN AE OE ON AE N ER OE EN N 52 53 IMAI kc sis te Sie ss vd aw aa ds ato o 28 MANAGE RE O ER EE EE OR EO Uiu aa ranun aa raaa 52 Genebank Activities ee ee ees ee ee ee ee ee ee ee Re Re Re Re ee ee ee ee Re Re ee Re ee ee ee ee ee Re Re Re Re ee ee ee ee ee Re ee ee 54 ACCESS tanda A dad 54 Generale AE EE N EE OE OT ER N 14 TR RA AA AE LE OE TO OE RE RE E N 14 Generate iieldbookK ii gege as el di 14 Genetic conservation air ad Gee Re RENEE Ee Re Ee EER EE ee os ee ee Gee T de bee ee be eb ge 8 need DE EER EE RE DE GR EE EE GE E RD EE EE Ee funnest 8 eed RESOU OO AE AO EE RE N N OE OE Hane ais 11 49 52 System wide Information Network 11 The System wide Information Network eie ee ee
7. Leersia perrieri 1 Leersia tisseranti 3 Luziola leiocarpa 1 Porteresia coarctata 1 Potamophila parviflora 1 Rhynchoryza subulata 1 Zizaniopsis villanensis 1 4 Total 370 1 543 90 348 In addition more than 10 000 incoming samples are to be registered Germplasm acduisition and conservation e The role of the IRRI genebank is to ensure the conservation and continued availability of genetic resources for rice improvement Germplasm from the IRRI genebank is freely available on request IRRI will continue to restore valuable germplasm which has been lost in the country of origin and will remain an important conduit for germplasm exchange between countries In October 1994 IRRI signed an agreement to place in trust germplasm collection under the auspices of FAO in an International Network of Ex Situ Collections e IRRI holds rice germplasm in trust for the rice producing and rice consuming nations of the world It will not seek Intellectual Property Protection on germplasm in accordance with the Policy on Intellectual Property Rights approved by IRRI s Board of Trustees in September 1994 see Appendix 1 1 e Security back up or blackbox storage of germplasm is provided at the National Seed Storage Laboratory NSSL Fort Collins Colorado USA under the terms of an agreement signed in 1993 with the United States Department of Agriculture Agricultural Research Service see Appendix 1 2 e Germplasm stored comes from more than 110 cou
8. Rhynchoryza 1 S America t Maltebrunia 5 TropicalandS T Africa Prosphytochloa 1 S Africa t Potamophila 1 Australia t T Table 1 3 The composition of the rice germplasm collection in the International Rice Genebank at IRRI Only samples with an accession number are included in the table Number of accessions Species name Wild O glaberrima O sativa O alta 6 O australiensis 36 O barthii 216 O brachyantha 19 O eichingeri 29 O glaberrima 1 543 O glumaepatula 54 O grandiglumis 10 O granulata 24 O latifolia 40 O longiglumis 6 O longistaminata 204 O meridionalis 53 O meyeriana 11 O minuta 64 O neocaledonica 1 76 O nivara 1 251 O officinalis 278 O punctata 60 O rhizomatis 19 O ridleyi 15 O rufipogon 1 022 O schlechteri 1 O sativa 90 348 Hybrids 35 9 Chikusichloa aquatica 1 Hygroryza aristata 4 Leersia hexandra 2 Leersia perrieri 1 Leersia tisseranti 3 Luziola leiocarpa 1 Porteresia coarctata 1 Potamophila parviflora 1 Rhynchoryza subulata 1 Zizaniopsis villanensis 1 4 Total 370 1 543 90 348 In addition more than 10 000 incoming samples are to be registered Table 8 1 Buffer systems for electrophoresis Gel buffer Electrode buffer Enzyme Buffer Chemical Amt 9 Chemical Amt 9 system composition 0 51 composition 0 51 Amp Cat Trizma base 10 4 Trizma base 24 23 Enp Est Icd Sdh Histidine HCI 9 6 Citric acid 11 08 Distilled water Distill
9. harvest for their retention of greenness Four classes are recognized 1 late and slow senescence two or more leaves retain their green color at maturity 5 intermediate 9 early and fast senescence leaves are dead when the grains have become fully ripened and 999 mixture Spikelet fertility Spikelet fertility readings are obtained from counts of well developed spikelets in proportion to total number of spikelets on five panicles Six classes are recognized 1 highly fertile 90 3 fertile 75 90 5 partly sterile 50 74 7 highly sterile 50 to trace 9 completely sterile 0 and 999 mixture Panicle shattering The extent to which grains have shattered from the panicle at maturity is described as 1 very low less than 1 3 low 1 5 5 moderate 6 25 7 moderately high 26 50 and 9 high more than 50 and 999 mixture This is recorded at maturity just before getting the panicle samples for post harvest characterization Panicle threshability 1 9 999 The matured panicle is grasped by the hand and a slight rolling pressure is applied with the palm and the fingers Based on the extent of grain removal four categories are recognized 1 difficult few or no grains removed 5 intermediate 25 50 of grains removed 9 easy more than 50 of grains removed and 999 mixture Lemma and palea pubescence Pubescence of the hull is classified as 1 glabrous 2 hairs on lemma keel
10. ridleyi complexes which are best maintained in partial shade When purelines are to be developed only 1 plant per pot is maintained and spaced widely preferably alternating species of different complexes If a bulk population of seeds is required 2 3 seedlings per pot are transplanted and all the plants are maintained Granular insecticide e g Carbofuran is applied 7 and 14 DAT to protect the plants against hoppers and defoliators Top dressing is recommended at 30 DAT and 45 DAT with 5 g ammonium sulfate per pot For O meyeriana complex 2 g of ammonium sulfate is applied weekly for 3 weeks 30 DAT The plants are watered daily Plant health is monitored regularly Appropriate control measures are applied to specific pest and diseases once symptoms appear Maintaining the cleanliness of plants also helps in preventing the spread of diseases At the late vegetative stage about 60 DAT the tillers are tied loosely with abaca twine to a bamboo stake 2 inch x 2m to prevent plants from encroaching from one pot to another and later at the late reproductive stage to facilitate panicle bagging Panicle bagging is necessary for handling wild rices to minimize outcrossing to prevent seed loss due to shattering and to prevent mixtures at harvesting Panicles are bagged a week after full panicle emergence using nylon net bags which provides ample ventilation to facilitate anther dehiscence and prevent mold formation on glumes For speci
11. 3 hairs on upper portion 4 short hairs 5 long hairs velvety and 999 mixture Sterile lemma shape Five classes are observed 0 absent 1 linear long and slender 2 linear lanceolate tapering to a point at the apex or sometimes at the base 3 subulate or setaceous linear and tapering to a fine point set with or consisting of bristles 4 very small and triangular and 999 mixture Sterile lemma length Measurement is made on each of the two sterile lemmas Six classes are recognized on the basis of 5 grain samples 0 absent 1 short not longer than 1 5 mm 3 medium 1 6 2 5 mm 5 long longer than 2 5 mm but shorter than the lemma 7 extra long equal to or longer than the lemma 9 asymmetrical and 999 mixture 100 grain weight A random sample of I00 well developed whole grains dried to 13 moisture content is weighed on a precision balance to give the 100 grain weight 10 grain weight Ten grain samples are taken specifically for wild rices due to its low seed production Grain length Ten grain length is measured in mm as the distance from the base of the lowermost sterile lemma to the tip apiculus of the fertile lemma or palea whichever is 38 longer In the case of awned varieties the grain is measured to a point comparable to the tip of the apiculus Grain width Ten grain width is measured in mm as the distance across the fertile lemma and the palea at the widest point A modified p
12. 48 BBCC O officinalis 24 48 CC BBCC O punctata 24 48 BB BBCC O rhizomatis 24 CC Ser Australienses O australiensis 24 EE Sect Brachyantha Ser Brachyanthae O brachyantha 24 FF Sect Padia Ser Meyerianae O granulata 24 GG O meyeriana 24 GG O neocaledonica 24 2 Ser Ridleyanae O longiglumis 48 HHJJ Distribution Sub Saharan Africa West Africa South Central America Sub Saharan Africa Tropical Australia Tropical Subtropical Asia Tropical Subtropical Asia Tropical Australia Worldwide South Central America South Asia East Africa South Central America South Central America Philippines Papua New Guinea Tropical Subtropical Asia Sub Saharan Africa Sri Lanka Tropical Australia Sub Saharan Africa South Southeast Asia Southeast Asia New Caledonia Indonesia Irian Jaya Papua New 75 Guinea O ridleyi 48 HHJJ Southeast Asia Ser Schlechterianae O schlechteri 48 HHKK Indonesia Irian Jaya Papua New Guinea Table 1 2 Genera number of species distribution chromosome number and spikelet structure in the subfamily Oryzeae adapted from Chang and Vaughan 1991 Genera No of Distribution Tropical species T temperate t Oryza 22 Pan tropical T Leersia 17 Worldwide t T Chikusiochloa 3 China Japan t Hygroryza 1 Asia t T Porteresia 1 South Asia T Zizania 3 Europe Asia N t T America Luziola 11 N and S t T America Zizaniopsis 5 N and S t T America
13. 5 ul of each of the diluted ligated DNA to properly labeled new 0 5 ml tubes Store the remaining 95 ul diluted ligated DNA portion at 20 C 2 Prepare the following in a 1 5 ml microtube enough for 10 reactions 400 ul pre amplification primer mix 50 ul 10x PCR buffer for AFLP plus Mg 10 ul Taq polymerase 460 ul total volume 3 Mix gently by flicking the bottom of each tube and spin to collect the solution Add 46 ul of the mixture to each of the 5 ul diluted ligated DNA 5 Mix gently by flicking the bottom of each tube and spin to collect the solution Overlay with 1 drop mineral oil 6 Place the tubes in a thermal cycler Amplify using the following temperature profile e Time No of cycles Temperature C 94 30 sec 1 56 1 min 72 1 min Hold temperature 4 C 7 After pre amplification remove the tubes from the thermal cycler This is now pre amplified DNA 8 Divide the pre amplified DNA in the following manner a Transfer 3 ul of each of the pre amplified DNA to properly labeled new 0 5 ml tubes Add 147 ul TE buffer to make a 1 50 dilution Mix gently by flicking the bottom of each tube and spin to collect the solution This is diluted pre amplified DNA This will be used in the selective amplification b Transfer 33 ul of each of the pre amplified DNA to properly labeled new 0 5 ml tubes and store at 20 C c Add 3 ul 10x loading buffer to the remaining 15 ul pre amplified DNA Mix gently by flicking
14. AE AA RE N 85 MI 20H20 EE RR OR A lle Gal ean eel 85 ER ER OE A EN EE OR EN 58 60 68 85 dl Le RE EE OE ae a en OE t 39 la RE EE A EE EE N 56 MnGl24H2 i ceive eege ae Ek lee dk AER vane eee Ee ge ok as Wee 104 MnSO4 4H20 ni EE ER EE EE OE adds 104 Mede KC EE 77 Molecular marker laboratory AANEREN 56 Mediale N EE EE EE 17 Molluscide application EERS RES ata rar reia 17 let La d ar RE RE RE lb abad 52 UE et EE EE N EE O 52 MOnODASIC EE ER EE OE EE RA EE ed 68 85 Monohydrate RE da rentado 104 Moor Plantation RA OE EE OE EE N ER tht cas 79 Morpho agronomie asiano ita 11 52 Characterization AR AO OE OE ernie 52 Morpho agronomic Characterization ee ed ee ee Re AA Re ee Re cnn ee ee nana 39 53 ER EE 79 Most JONEDANKS ER EE EE EE ER EE OE EN 13 de inr elle OE RE RE EE NEE 79 EE EE EK HIE OE AE EE OE OE N 31 79 VEE N EE ER dates N OE KO EE N 64 UIER EE ER EE EE IE a AA Ia aid 47 49 MT A OE ea ad A OE EO Site 68 85 Multiplication rejuvenation 00 0 2 ccc eee AR Re AA ee AR Re AA ee cnn ee ee ee Ad ee ee ee ed ee ee 52 Mi EE de AR EE OE AE EEN 56 EE RE OR LE EN AAK OR EE DE EE hee 68 Mes ales te EE EE OR aan 104 N N 7 13 15 17 39 49 68 83 85 ER EE and AR EE EE AE EE DEE 17 AE ERA EN EE AA EL EO OR OIE 85 Na2CO3 EE 68 121 Na2HPO7H20 RE ene iinet ATi a cae ah N ME NE IE N 68 N 2S205HO SE EE 68 85 NAS GOS site Cox EE OR EE MOE LA EE EE EG 85 MERE BE re RE DEE EE N EE EE ENE OR eens 85 N a benzoyl DL a
15. EE 52 SERE EE HE OR IE AK ER ED N DE A EE 79 Bandar Seri Begawan 20Pp ee AA Ge RA AA AA Ge AA ee GR AA ee ee AR Re ee Ge de ee Ge ee ee ee Re ee 79 Bangkok 109001 EE SG DA oi 79 ae Ek N EE EE OR EE DE 79 Bangladesh Rice Research Institute A 79 BARO E RE ET EE ER N EO EE EE OE 79 Barrier Foil Products CO iii ES IS a SE ea ida 31 Baldini ia 7 8 39 83 SLEE EE OE N OE OON N EE HE OE 83 Base SE at Seva ed GE ERC ev Saeed ae ee GE Dee 23 25 Base Collection EE EE De aid vie EE SE De Ge ees EEN 8 10 13 23 25 28 31 Based EE EE EE ER N EG EE RE SG RE Ge GE aa aiid Seance aed GE a dab Ee EG nae eee a 39 Batch ID oii scien dees EE EE Sante via wav teks a ae esa areata eaten 53 B BEE E E dee Ee oe ore ke 7 83 BB dee geste ue eg GR AE Ge ee ge ag EE N AE li ge 7 83 BODA Dia EE EE GE GE AAA ED Ee A AE E EG Ee ED A 39 Bearind ed do 39 LE EER OE EE daa 39 Beltsville ti A GE De GE N ee Ge De Ee 79 BT RE A RE EE OR EE EE EE a 79 Elle EE 85 Blois tdo reinado eebe 56 ARE UIE UR EE N EE iaa 11 73 Birds e ER E td at Aa 17 Bis aerylamide sa EE ti aed ER Re Ee Maes a adel 85 IEN ue Es ve ida ee E E ee GE Ge EE Ge ee BOE 10 Blackish Brown 39 Edele DEE oie ttt atin he A 79 Block Jin rt faust ata cat roves EE Ee Ee De 79 BlockS ss EERS EE Ee darias a iv EE Ee Se De 17 2 BOOM at 17 ENEE aa AE en ee oe aah oe Go GE ee Ge GR EE ee De Ge 68 85 Banany nc a edad hich 85 ales de AE N OR OR EE EE EO ER EE OE es 85 SE ie ill e ls N ER EE OE ED E N O
16. EE EE HR EE LE EE AA ee 68 TeMpe arture RE EE EA EE EE OE OE AE 60 Temporary ID EE EE EE ER N EE OE A 39 53 NE ED AE N ED OR EI saa OE OE KEN N 14 79 The International Rice Genebank Collection Information System ie ee ee ee 52 The System wide Information Network 52 Genetic E EE 52 Their authorized chaninel reveiran elec caval da dd di di 79 Thermophilus aquaticus 00 00 0020 eee cece AR Re Re AA GR Re AA ee ee AR ed AR ee AA ee ee AR ee ee ee ee 68 Thiamine Hina RE E EO ai OE OE 104 LEET RE RE IR ME ER MT aai 68 85 Le ET 68 THOMPSON EA N RE A N IE EES 56 Through Quarantine Station ee ee RR AA Re Ge AA Ge AA Ge RA Ge ee AA ee ee AA ee ee ee ee Re ee 79 di AE ON e N EE av RE N RO ME LEEK 49 System wide Information Network 49 dd geil 52 ESRA MR RE ME RE oleae saab RE N EA 31 TOGO AR N N RE OE RE EE N RE OE HO ER EE 79 TRACKING AE EE N Ne 77 Dye Line Characteristics After 4 hour Run 77 CEET RO EE ita 5 TRANSAMIMNASE Zeg OE AE EA AO NE veel a 85 Transter 29 Wh RE EE EE EE EE EE ER tes 64 ME EEUE a EE ER RE ER EE 64 UCI die RE ii EE EE N N 14 Esel eu MA AE RE EE ER EE lice 14 Transplantingireplantinid EE 17 EER EE EE EE OE OE abr ita 77 JESS RR RE EE OE EE IE EE ENE 68 85 Tris Dorate EDTA RE EE OE EE EO OE RE OE canes 68 Ee RE EE EE EE OE EE N OE EO T 85 SAUK EER EE EER EE EE EE OE EE OG 68 AE il Tei TEE 60 85 RE EL EL OE TE OE OE TE NE 85 Ed ES ER EE EE EE OE IE EE OE TA EN EE 85 Tropical Subtropicah Asia
17. EES A AI AAA ee ee 7 83 Tropical Australa ORE N OO RE DE HE EE EE EO 7 83 Trustees EE EE 10 49 o EA AE MR EE EE HE OE iey 49 PRR E iS ES ER SEE EG E 10 IER 73 Kou AA ET AE AR RR EE LR saan 73 Tubesiplate EE RE EDE he eaves o 60 TU Le EE EE ME EE ER ER N N rer eee 17 ae AE EER EE EE RE HE ER N ER OE EE OO EE OR ER EE 85 131 U 60 Upg DNA EE EE EE ME A A aa 64 Uli OR ER EE eed SN eee 58 die RE EE NE RE a EE AN 58 UR EE EE ES NE OE OE EE IE 31 URE EE EE EE OER OE EE EE N EN EI OR 64 Under IRRI s Policy on Intellectual Property Rights AA 49 di EE EE A EK ER ERG 47 United States Department cece ee A 10 Agriculture Es EE EO Ee Ee RE EE 10 Universities institutionS in EE EE ERGE see ee be af Eed 47 Unpolymerized acrylamide AAA 60 do ER RE OER EE EE ENE EE N 85 dle EE EE EE N EE LE OE OE ON 60 68 85 EE TE AE E 60 PEER EN AE RE A DE 60 Upland Site EE tae eat tia la ee ER Ed ete EG tt a ea la 8 VER EE RE OE RE RE E 10 30 52 79 USDA A A Ci ate en EE n oe 79 USDA ARS se ag GE it ae ata altel ala Ge be se ar eo ie 30 VERE IE EE EA ED RE OD OT 28 39 64 73 LE EER AE OR OE OE RE N EE OE ere 73 el EE RE ER N RE OR RE EG 28 eu EE OE EE RE EE EE EE AR N OE EE NE A 39 pre amplificationy ER OE EE OR OE N N ERG 64 User SEE Ee ee ees EG N ee De EE aia AE us nate twas sale Re Ge ERGE ea 52 53 MODROM RR dia 52 VER sae AR eis ve bee ale a RO OE EA ER 53 URE RE EE N OE AE EE ER EE HO OE 56 58 64 68 V EU 39 detmplasm SEEN EE he
18. Ge AA ee AA Rae ee AE AA ERNA ee ee AG Ge Ge ee ee ee ee 79 United States Department 10 Ne ARE ON EE AE BE EE RE EE OR OE AN 53 e Die ul EEUE RA ER ME i RR OE EE 53 Air Conditioning Section ee ee AR GRA AA AR RR ee AA ee ee ee Re ee ee ee de ee ee ee 31 ACKOtOG liese AE EE EA HE NE ME OE RE 85 ils e ue EEN 85 GE 68 85 Alcohol vlt e GIE 85 Alfredo Mazarredo RE ERK EE OE a EE 31 Aliduot36 UI st ere SR RD EE ED GR EE AI EE ED EE EL RE De IG 64 104 LE MAR RE ER EK N EE EE enti aia EN 17 ER EE EE EE NE AE AE EF 7 8 83 ER EE IE EE EE ME AE EE N se 83 AMEBTICA AA AE EO NE OE N AO ER HE EE 7 83 lie Ee AE RA ca MR ENE OE O EE EK 7 ed DE EE ER EE EE OE 68 AMING Pe LE AR RE AA N OR OAR EA vase ba 68 Aminopeptidase alanine AAA 68 Aminopeptidase arginine ee se ee ee AR Re AR ee ee Re ed AR rr 68 Aminopeptidase leucine AAA 68 Ammonium molybdate A bvdrate AAA 104 A OE EE EE ER EE OE EN 85 Ale EE E 104 Ale Un SOOM AE EE IE KO RE EE ON 85 Amp Gat AAA A EE GE A AS 85 Amplified Fragment Length Polvmorphtsm sees see ee se ee ee Re ee ee ee no Re ee ee ee ke ee ee ke ee ee 56 ui EE EE IE OE AE OE MEE OM EE OE EE EG AE Ra 85 Analysis ANE ERAN Ges EE Ee A en EEN 17 A MAP My AE EA RE EE EE OE OR NA EN 85 il Ma 35 39 46 And or EER EE RE eee nd i ee N 47 A eee ie N eh ie a 79 Anthesis EE EE oN AE EE Gal in Dy dg We TN 17 39 ae EE EER AE EE RE HEEN N N a ee 60 ARE RA EE OR E E EET 60 es EE HE EE DE HE 60 ADO EE ER ME EA E E EO N 79 Api UUS Esco
19. N OE N AE ee 47 49 79 A RR ME AE AE RN 47 FHC CH sss EE ER oe DE GE EE DE Ee EE GE acta bac Re n oe OE 60 85 de EE ER ON RE OO ES 60 AO EE ER EE AE OE OE eee ee 85 Hexadecyltrimethyl Es se dd id 68 AE DR EE ET ME EE ER ON 7 83 HK SE ind ve a EE N wa a a N EN N 7 83 ell EI VE 7 Hispid scabrouS 3025 0h0 hain hn Ge ee ee aie nee 39 Histidine ele RO AR a E 85 HM 305 CTE Constant Twin Element 31 Hoeye r Se ed civ SE De ai DR A ge avons 74 ao RR EE OE Adin Ma A EO OE RE 73 9 73 Homemade Tag Poivmerase AAA 60 ale ER EE EE EE HD 79 lede RR RR OR RE leia 104 House 39 ate es ME EE EO AA ER EE EE 79 However O 15 Hume Martin EE 31 Hybaid OmniGene fhermalcvcler cence ceneeeseaeeeeeaeeseeeeseeeesaeeeseeeesseneeaas 58 le else Add EE A aE EE EE OE di 68 e Viele OE ET EE RE E 7 35 83 e Viele GE UIE AR ER EE ERE 8 83 e Vive lU ii EE RE EE EE RE 73 l let EE a OE LE LT N EE EO ee ae a eee 79 cd EE DE EE tad He tee Do ee ee ee eed an BOD ELE RE Ee ie tee seen ae ee 68 85 le AE OE EE iia tio 14 46 52 RE ER EE OO MEE as 52 ll 77 85 11177 85 INOIS 60620 Elo leas 31 Import Permit EE ER amare is EE ee 47 79 A 22 230 EE OO AA AE EE ined 47 Import el IEN 79 e Tu EO RR RE EE OE ER EE HO N 17 LESER TE ER RR OE ORE OE tree 17 Incubate Mila iia RE KEN 85 Incubate 20 MM ro RE EE Re RD ai isla dora onto 85 ll TE WEE 47 79 Indian Agricultural ROS EE OE RE EE EE RE OE ER 79 115 NdICA MEE IE ER RE Shei E E E 7 23 25
20. Nicotinamide adenine dinucleotide 6 150 0 150 reduced form NADH Nicotinamide adenine dinucleotide 5 125 0 125 phosphate NADP Phenazine methosulfate PMS 1 25 0 025 Phosphogluconic acid 10 250 0 250 Shikimic acid 25 625 0 625 Stock solution Qty Qty 50 mI dH O mg ml mg g dH20 84 DL alanyl B naphthylamide 10 500 0 500 L arginyl B naphthylamide 5 250 0 250 L leucyl B naphthylamide 5 250 0 250 N a benzoyl DL arginine B 5 250 0 250 naphthylamide combine Store all stock solutions at 4 C Table 8 4 Stain buffers and other stock solutions Solution Chemical Amount 500ml composition Acetate buffer 1M pH 4 65 Sodium hydroxide 8g pellets NaOH Acetic acid glacial 30 ml Distilled water Malate buffer 1M pH6 0 DL Malic acid 679 Sodium carbonate 53g Na3CO3 Distilled water Phosphate buffer 0 1M pH6 5 Sodium phosphate 1 529 dibasic 7H20 NasHPO 4 7H20 Potassium 3 88 Y phosphate Monobasic KH2PO4 Distilled water Tris HCl buffer 0 5M pH8 5 Trizma base 30 3 g Hydrochloric acid 6 75 ml HCI Distilled water 85 Tris Maleate buffer 3 3 0 2M pH Trizma base 6 05 g Maleic acid 5 8 Y Sodium hydroxide 0 8 g NaOH Distilled water Calcium chloride 0 1 M Calcium chloride 5 55g CaClz Distilled water Table 8 4 con t Solution Chemical Amount 500ml composition S Hydrogen peroxide 0 7 Magnesium chloride solution 0 1 M Potassium
21. OO E VT 56 Switzerland EE EE Ee aa ee ee ee a ale ee Ge ee Ee ee Ee ER Ee GE gee ee eed ee ee 79 SYSIeM AAA A A ee TEN A lle ca alee dation ee dees 77 Ad RE A eee EE OE N 77 System accessibility EE 52 System COCUMENTALION RS RS EO ER R s 54 System EE EE ER RE EE OE OE ON EE EE 77 Au EE EG 77 System M RE EE LE OR EE ER OE EE EE att 77 Did EE EE N ERG 77 System menu structur Ed cece GEE GER at GEE ge Rek RE da 52 System oblectiVe Si Ee EKG GER EER Ee DOEK EE EE Ge ER Ge IA ee AENEA eege bee ie ay 52 System wide Information Network 11 49 Genetic RESOURCES RA EE OR N EE EE ba 11 AR EE EE RT NE RE AE OE OE NO 49 T T 7 31 83 KE de ligase EE EE EE EE OE OD OO ended eee ER 64 Tables ii ds ER RE EE dt tal 7 49 60 73 77 Table D aa ad 83 Table 1 27 Gedisa o nto o Ve ee Ee Ee EE 83 Tablei a Tiana ET 83 Tablerb 1 CON tacita atadas al ao A 79 Table 61 COUNT ninio 79 A O atau OE AE IE NG 77 85 Table RA LE ME ORE KEEL IM Es 77 Tablon ER EE ERA AE LE MAR EA EE 56 ebe AR AE LE MERE ORE KEN daa a EE EG 56 Table yen oa ee ee Ie 56 58 Table Odin EL MEER ORE KEN dun OE N moons 58 AE le RO EE RE ERA AL EE RE DIE 60 Table SET GR ET EE Ee as 104 Tables lina ios 77 Take photomicrographs A 74 130 AP NE EE RE OO OE OO RA EE ORE De 58 64 68 ECK MA AE OR EE DIE IE EE RE EE EN 7 47 AR EA EE EA EE N EE 56 68 85 EA AE AE EE EO OE EE OE OE EE 64 68 85 Add 90 ll cnica rt ties 64 RE 85 AE EE AL EA AE NE AE EE EE 31 TEMED EE KO
22. OR HE ee ER ER ER eee od 73 PI17 Peko EL ME RE A EE NG 17 dm ER EE RE EE Res Oh EE OK 56 ei EGO EE ER OR EER OR AE N N EO EK 56 Pipette AE ER EE OE AE EE EE EE OR EE O 58 64 dele KARRE ME A AE OE N lees 56 oe EE RE WER OE EE N N A 56 SO ER EE EE EE EO ENE EO EN cents 79 di N EE eds OE EE OE EE EE ees 60 PG ARE EE 60 Place abaca EE DE GR REED GR ao ER Se Ee Gee ee ee ee ee n ed 17 Place PER EE ESE SR GE o ee GE ahs 58 64 Place tipes pilale EE erer EERS DEE EER Ee ER ER salad be SEE Madde DE Ge Ge GER ee ED Ge EE lev yids DER Ee bids 60 AIR MA EA RE ER EE N AR EE EE N KO EE EI ON 79 Director Betreier 79 124 Plant Establishment EE 39 PlantiGenetic ReSQUICOS coi ee EE ee ge gege De ee EG Re Ee en eene Ee 79 National BUTEA ss oe ee GE as 79 Plant Germplasm Quarantine Center iese ee ee RA AA AA Re ee Ge AA Ge ee Re ee ee ee Ge ee eke 79 dode le ER OE EE N ee ebe 10 47 Bure as RR EE 10 PLANE Oh EE EA EE EE EN NE EA EE IE EE 56 Plant Ed are O ss ES AEA EA s 79 BCEE 79 Plant Protection GENOES h sie Ee ese ie sass GEE be AE Re EE EENS de Ge kes Ee od 79 Plant aarantin iS ES De Ge aaa 28 Plant uarantine ele ss GE ese deet Eed ER Pe ee de Di Ge SE Een 79 Plant uarantin StatiO ss ese bee os e dde gus EE Ge be SR See EE ee VG ee EE Gee Gee ee Ga EN ee od 79 Planted harvested ies ie Ee EE EE AR aaa 47 W EE 77 Plexiglass se doves chi nie eg ER WE Eg GN ee De ee Ve Ge GR Ge dive E Ge 60 Plotnumbers EE DE GE De sele
23. RA AA AA AA AR ee ER AA Ge ee Re ee 52 Genetic Resources Center 5 47 49 52 ET le AE HA EE EE N N EE OE EA 7 83 GENOMIC DNA EE AR MT EN AE ER N OE OES 60 113 60 E UTC 39 Germplasm iese RA 5 8 10 11 13 17 31 35 39 46 47 49 52 53 56 83 MEER EE EE LA OAAR OR IE DE EE ER IR 53 AE WEE 56 El AE AE EE EE AR EE OE OE N 10 len EE EE AA EE OE EE N anes 46 O AA OR RE EE GSE R eos 47 TREE EE EE EE OR EE EE EE OE TEED 39 ME EE OE OR OR OE IE OE OE EE EE LONE N 39 Germplasm acoulsltton ee ee O 10 Germplasm characterization ee ee ee RR AA ER Re GR Re AR Re Re ee Ge nr ee ee ee Re ee ee 8 11 a9 tee UL e s EE EE ME EE OE ER ER 39 GERMPLASM EXCHANGE ee ee ee ee ee ee ee ee ee ee ee ee ee see ee ee ee ee ee dee ee de ee ee ee ee ee ke ee ee ee ee ee Re ee 10 76 Germplasm information ee ee ee AA ee RR AA AA ee GRA Ge ee Re ee Gee ee ee AA ee ee 52 e Vue EEN BE eli ae ean eg Re Ge Ge GR ee ib 52 Germplasm Nursery ege Reeg SE ee ae a a ee ge gee ge ee 39 Germplasm nursery screenhouseS ee ee ee Re AA Ge AA AR ee nan nn naar cn ee RR ee ee AA ee ee ee 35 ee EE N EO EE HA OE OE EO EN 7 83 GIBGOBR EE 64 Glaberrima esse ee ek Ak ee ee ee 7 8 14 15 17 23 25 27 28 30 31 35 39 46 47 83 Glaberrima germplasm ee ee ee AA AA AR Re AA ee AA ee ee ee AA ee Re RR ee AA de ee ee 13 Glaberrima satiVa is ege SE Aare AAA ea Pe Ge ee 39 Glabrous EE ert iy AA a A aa a eae 39 Glaszma er Ee EE
24. Re Re Ge AA Ge AR Re Ge ee ranma 79 C C 8 14 17 23 25 27 28 30 64 73 74 104 AA OOR EE OO EE EE AE AA A AS 74 ER EE EI wal Mirae E E AO EE 73 MB ME EE RE A OE ER EE OO Ad 14 C O Cl Tira A OR OR N NR OE RA RE 79 G OIRRIE EER RE DE ER EE A Ad Ee EE Gun Ee Ee Dee Ee 79 C o IRRI Cooperative Project A 79 MAST N AE N ORE OR OO N NR erence eran re 79 C o IRRI Liaison Scientist se ee ee ee RR RR ER RR ee Ge Re Ee ee Ee Ee ee ee ee ee ee ee ee ee ee ee ee ee eek ee ee Ee Ee ee ee 79 le EE EE 68 85 104 Ee D EE 104 Gana Postal as it tactica cers 79 Call aia dd ege 79 Cambodia ET 79 Cameroon cto E ts N RE EE iii 79 Capabilities 32 23 N rani dt tdci 53 CarbolUraN rats AE EE EE OE EO 15 35 O NEE RE MO OR EO NE 68 85 ete EE AE EER asa 56 77 ee aa 7 83 ee B D RAEE ME EES AR OE OR OE AE TE OE RE RE 7 83 CCE Business STER IE KO EE N EE EE 31 Ce NOP AN AMG a RE ole be Sees Scenes io ie 77 EARTH 68 CENARGEN ie eege ee a da a Oe Sida 79 GenitralAirican BEDE SR RS ER re ee n ie Dee Se RR RE a n Ee ER ED ER OE Gee EE N be iii 79 Central Post Entry Quarantine Station iese ee ese ee RR AA AA ee ee GR AA ee ee Re ee ee AA ee ee ee 79 107 CGIAR iii N tonto ER ONE EO tae ER ia 52 ACC Ad EE AE ME N 52 ee EE EE EO AE RE OE a 7 83 Characteristics A GR ER Sa Ke ae RE AR ER AE Ee De 11 GhatacterizatiON 2E EES Senden Ge ee OE IE TE Ad SNR a canada De ee 8 39 52 53 73 UE LE 52 MOFPNO AGKONOMUC ie sa AR OR atten ez OE N RE RO eal 5
25. Stir until CTAB dissolves Add BME just before using RNAse A Ribonuclease A 0 1g 10 mg ml Type II A Tris HCl 0 5 M 10 mM 200 ul NaCl 5M 0 5 mM 30 ul supH20 9 77 ml Heat in boiling water for 15 min Allow to cool slowly to room temperature Dispense into 1 ml aliquot and store at 20 o Working stock maybe stored at 4 C TE buffer pH Tris 500 mM 5 mM 1 ml 7 8 EDTA 50 mM 0 5 mM 1 ml SUPH O volume to 100 ml Autoclave Table 8 6 General stock solutions for DNA extraction Solution Chemical Composition Amount 88 Chloroform isoamyl alcohol 24 1 CTAB 10 EDTA 0 5 M EtOH 70 EtOH 95 EtOH 99 5 NaCl 1M Chloroform Isoamyl alcohol CTAB NaCl supH O volume to 480 ml 20 ml 100g 40 95 g 1000 ml Mix NaCl and HO Heat to 65 C and add CTAB Stir until CTAB dissolves EDTA NaOH pellet upH20 volume to 93 05 y 109 500 ml Stir vigorously on a magnetic stirrer The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to 8 0 by the addition of NaOH Autoclave EtOH absolute supH O EtOH absolute supH20 EtOH absolute supH20 NaCl UPH O volume to Autoclave 70 ml 30 ml 95 ml 5 ml 99 5 ml 0 5 ml 29 22 g 500 ml Table 8 6 con t Solution Chemical Composition Amount 89 NaCl 5M Phenol chloroform 1 1 Tris HCl 1M NaCl upH 0O volume to Autoclave Ph
26. ainia ee Re Se GR ee NE Re Ee EER SE 10 28 ads ER EO EE EE OE RE EE EN 31 la EE acd tye AE ED IE EE OE EE E EO OE thas 83 IRRI s policy on germplasm evchange AA 49 IRRI s Policy on Intellectual Property Rights A 49 IRRI s Seed Health Unnt ee ee ee AA ee RR Ge AA ee ee AR cnn Re ee AA ee ranma 49 IRAP S Upland FaiM seresa A pee bee ey che eee tea a EE 5 IRRIS BoOard AE IE EE OE EE OE baad OE RE dee AR 10 dIE RA EA Aa ad 10 ASO III eee AE N EE a RR EE N 56 85 Isocitrate dehydrogenase A 68 85 Isopropanoli EE Eg Ve A Ge ee ge ge Ge ge pg en 56 Ela EE EE EE RE ME EE EN 56 77 Isozyme Protocol ER ci Me a RE es Ve ein li 77 Ela N ME RE AE dE SE AE 8 11 56 85 EE EE AE ER N EA TE EE EE N 27 28 TONOWING RE EE EER RE N EE EE OE AA 28 ER RR EE EE die eae ee OE N 79 ME EE ME ME N N EE RE EE EE N N GN 54 J J 31 39 er EE N TR EE N ian RR OE EE al 85 JACKSON EA EE tee ee ee ete a E ER 17 Jalan Merdeka E EE 79 Japan BERE edie at aie re es a etre a a a 14 Japonicas A el i A 17 Valletta Geetha Ata ae ie ee ie ea ee Ae ee ae 56 BEE le EE EE eed AA A a ie 35 39 Joaquin Rodriguez Garcia 79 JUY AI ii aa retire arr eran 5 JUNE dd kee Ee egen RE EE heen Met Lis 11 13 EER AA EE 11 Novemba AR N MA OE et at at eed er 13 K K 17 31 85 KPSOA EED ET Gat erate EED AR ED sua ence 104 Kameswata Rao iii ii NE na 17 GIERE ll EE IE EE OR EE EE sande 79 Kb Re GER ES ER N N RC RT EL EE DE N N do ee 56 68 85 KEDNA DEE DE EE SE DER EE GE A
27. dee ge De Ge es 39 PUMPING gue N NE N ER OE EA RE ates 56 oi ARE See RE N ON OE RR N 56 Punctatas n ENEE AE AE A i n 7 8 83 MAAR AE N EE HO MO RE ON N EEN 83 re el EE N EE ED OR Ed 35 Purple Shade EE EE RE EE raa a A a aaa 39 PUSE CAMPUS iaia A ee eel ee rel ee eee 79 PVP EA ER RE OE tad 68 85 Pyridoxal 5 pnosphale s d eae es A a Be Ge Eg SE ge Ge 85 Pyridoxine HCl ra vic es ii eae geet A EE EEN Ve N vate 104 Q Qty 25imle2 dH2O ss skies Ea Ee EE oe a 85 Das See AM EE EE EE EE dia ene tend 85 Quality Controlar RE EE EE d N bad OE OE N 79 Q ara tine Office ni AE KEN NE OO EE EN 47 ed ER EE EE OE OE tees RE NE 47 R A RE oa daha td eh vk sda EE adapt DE AN OR NA 17 Random Amplified Polymorphic DNA 56 RAPD EE AE ee EE ED IE SE DE De eres 8 11 56 58 64 68 85 RAPD Protoeol 2 SI EDS ER its 58 Ralie as ES NO DR ALOM Ee RE GE MED Ee EE EE DA Ee sl 17 DE RO EE OE RE EE MA EE EE EE N EE 17 Ratooned EE A A sat De ED EE Ge aut ee MALE ah et oe 35 RATS THIS ME ED ED ED AE DE ER N EDEN DE le ee 17 Rattooned EE SE EE EE EER See GE Ee EE EG RE EE LS SU el Gh calla 35 Ready iS 10 germplas EEN 10 SE Ve BEE 17 RT ER EE OE N EE OE OE OE N 46 Germplasm RE EE N ias tae 46 126 R commendati ns EDE E sce ea bh wd cs Sa roe ee Se ak ees cad eva ced EE Leta aan 17 RECUPSOS GEMCIICOS eege Ee Kee ee ia 79 Recursos Maierales ees ee ee ee ee Re EE ee ee ee RR ER EE ee ee RR ER EE ee ee ee Re RR Ee ee ee ee ee Re ER EE ee ee Re Ge e
28. double checking and quality control Check the selected sample against the seed file and the pre labeled envelopes against the original container Mix the selected samples and divide using the pre labeled envelopes as follows a For Active and Base Collections gt 120 g O sativa and O glaberrima 2 x 100 grain samples for viability testing 2 x 200 grain samples for seed health evaluation 1 or 2 x 60 g sample for Base Collection 2 to 5x 10 g samples for pre packing 21 1 x 20 g sample for duplicate storage Bulk sample for active collection if amount is greater than 60 g otherwise prepare planting materials For the wild rices 1 x 50 species for base collection 2 5 x 20 seeds for prepack 1 x 50 seeds for duplicate storage bulk sample for Active Collection b For temporary storage insufficient seeds 5 to 120 g and samples with viability ranging from 50 to 85 for japonica and 50 to 89 for indica 1 60 g planting materials 1 or 2 x 100 grain sample for viability testing 1 to 5 x 10 g samples for paper pre packs c For planting low viable seeds lt 50 viability prepare 60 g planting material d For embryo rescue insufficient seeds lt 20 grains For wild rice hand threshing verification and cleaning are done at one time Ten grain sample is taken for viability testing and the cleaned samples are transferred to paper envelopes for final drying 14 Place the cleaned samples again in the dryi
29. ee ee Ge AA sees Ge AA AA ee RA ee AA Ge AA rra 77 Electrophoresis gee his ee Ree ee Dh ae ce Pa cals 77 GEE ol his ied ae A eda Ge vane EE N EE RM ed 52 P E N OR EO reteset 56 68 85 ei RE EE RE RE N 35 39 se ae RR N ER EN 39 ei EE EE EE EE EE tel 39 ei EE RE EE ER EN 7 A EE EE N N N AE 39 52 Cultivated Species EE UR 35 CUSO4 SH20 EE E E ai 104 Cylogenetical MEE ER EE EE AE EK N 8 ele ER EE EE EE IE EE HEN 74 D D 14 23 25 73 74 104 DE OE EO ahs EO o OR 79 DAPO Box 7777 Metro Manila eie se ee Ge AA Ge ee ee Re ee ee AA ee ee ee AR Re ee ee ee ee ee ee ee 49 Dark Green AE OE N OE EO indent ene an eed 39 RT e RETTEN 8 56 BERE ER RE ER vate ete ed ME EE Patan eee 15 35 39 DAT REGS RE tent eed hihi tee N ee ee oes ees Mental eatin ee eren tata 17 35 A AA EE ER N EE EE EE N OE 39 BIER Ge e MR ER ER OE EE OE EG 54 Data Integrity EE REKE sa bagi es N EE eed 54 Data management icc ER OE RE EE a 11 Data el LR OE RE EE EK AE EE EE 54 Datta sheets EE 53 DA TP EE EE EE Ey een DE ET cme rrr ea RR ee Ee De ED rere 68 85 DM EE EE RR ME EE N 35 AA EE NEE OD EE RE EA EE AE 77 Ben GE 68 85 Be MAER EE EE bone he ened cde nad EE EE beans cation AO N 39 109 De aeiale EE EE EE et ce en N EE EG RR EE 77 Biel AE EE OE OO ER EE EE EE ee 13 POD FUANY AE EE ME RE EE EE T ete 13 Dedicated EE 8 GRC databases applications ie ee AR AA Ge AA AA RR AA Ge ee ee ee GRA ee ee ee ee ke ee 8 DER O rota DE ER EG Oe De A
30. flowering of photosensitive materials Accessions planted late are likely to remain at the vegetative stage and never flower until the following year 2 Storage potential of japonica rice can be enhanced when grain filling coincides with the cooler environment that is prevalent in late December to early February Therefore early planting is also desirable 3 If possible areas to be used should be followed to minimize dropseed or volunteers On the IRRI Experimental Station ES a fallow is practiced every other season After space has been allocated and the schedule for the growing season decided then planning and specific procedures follow Selection of planting materials The type of materials included in the seed multiplication program depends upon priority and the available area The nurseries that are usually included are initial seed increase or the post entry quarantine planting seed increase for long term conservation regeneration and special seed increase for other purposes e Initial seed increase also known as post entry quarantine seed increase includes all new acquisitions that need seed multiplication for the first time and all materials planted previously and that have not assigned accession number due to insufficiency of seed produced low viability or seed infection e Seed increases for long term storage are the multiplication of materials that are not yet in the Base Collection e Regeneration is the seed increa
31. iodide solution 1 5 Destaining solution 5 5 1 Hydrogen peroxide 10 ml H202 30 Distilled water Magnesium chloride 6 hydrate MgCl 6H20 10 169 Distilled water Potassium odide KI 759 Distilled water Distilled water 250 ml Methanol 250 ml Acetic acid glacial 50 ml Table 8 5 Extraction and suspension buffers and RNAse A solution 86 Stock Concentration Final Concentration Amount Solution CTAB 1 5x CTAB 15 159 Tris HCl 1M pH8 75mM 75 ml EDTA 0 5M pH8 15mM 30 ml NaCl 1 05 M 614g supH20 volume to 1000 ml Mix Tris HCI NaCl and H2O Heat to 65 C and add CTAB Stir until CTAB dissolves CTAB CTAB 1 10g precipitate buffer Tris HCl 1M pH8 50mM 50 ml EDTA 05M pH8 10mM 20 ml supH O volume to 1000 ml Mix Tris HCI NaCl and H2O Heat to 65 C and add CTAB Stir until CTAB dissolves Extraction Tris HCl 1M pH8 100 mM 10 ml buffer for method 1 NaCl 5M 1 4M 28 ml EDTA 0 5M 20 mM 4 ml CTAB 4 4g BME 14 4 mM 0 1 ml SUPH O volume to 100 ml Mix Tris HCl NaCl and H20 Heat to 65 C and add CTAB Stir until CTAB dissolves Add BME just before using Table 8 5 con t Stock Concentration Final Concentration Amount 87 Solution Lysis buffer Tris HCl 1M pH8 10mM 1 ml NaCl 5M 1 4 M 28 ml EDTA 0 5M 20 mM 4 ml BME 350 mM 2 4 ml CTAB 1 1g PVP 5 59 SUPH O volume to 100 ml Mix Tris HCl NaCl and H20 Heat to 65 C and add CTAB
32. materials to smaller and finer cloth bags suited for drying Net bags are the best as it allows flow of air freely Immediately after blowing dry the materials using a slow process or by passive drying by placing them in a drying room with a temperature of approximately 15 C and 15 relative humidity to obtain higher seed storage potential When the harvests are already dried transfer the seeds to brown paper bags to facilitate handling and identification of the harvest Second seed blowing is needed to further remove half filled and light grains that were not removed during the first blowing After the harvesting period arrange the material by plot no and accession no to prepare for verification and authentication of harvest prior to processing Chapter 3 Seed Conservation Introduction to seed conservation This is a combination of processes that enables the upgrading of seed and seed lot quality with the ultimate goal of obtaining the maximum genetic composition with maximum viability potential This is handled by cropping season and involves the following processes e Harvest verification e Seed cleaning and selection e Viability testing e Seed health evaluation e Final drying e Packing e Storage e Duplicate storage e Maintenance Harvest verification This accounts for the success of the regeneration process lt determines whether the seed lot represents the composition of the original sample and the sufficiency o
33. provided by data management based on variety names species name donor code and previous name b Compare the donor code and the country of origin of the incoming materials and the probable duplicate c Compare the seeds taking note of the grain characters d Ifthe seed characters are different the sample will be accepted in the genebank e Ifthe seed characters donor code and country of origin are the same mark the sample as probable duplicate f If the seed characters are the same and the donor code and country of origin are different plant the seeds in the field side by side with the probable duplicate s and compare the plant characters If the plant characters are the same mark the incoming as probable duplicate Otherwise the sample will be accepted in the genebank 3 Note if the sample is a population or a mixed variety 4 Prepare seedfile a representative sample of seeds of the original genetic composition placed in pre labeled small packets for use in verification purposes 5 Process the seeds for storage and assign an accession number if the amount is sufficient and viability is acceptable otherwise separate seeds for planting 6 Take note of the amount of seeds left after taking the planting materials This information is needed in case replanting is required 7 Encode all pertinent information 8 Update the donor on the status of the materials received Protocol for seed distribution e All germplas
34. rere cre 85 BR RE AE EE EE MO OR RE EE EE IN 39 Eaboratory Manual inr a AG HOE ES EE ED Ee EED aia Ge ee EER ee te 104 Physiological Gtudes AA 104 AQUA RA EE ED EE EE EE o EE N 79 Lamda ol EE EE aa 85 Lamda DNA 250 nig TO EER RE Reg Re SEED ESE RED ER ED GE AD Ee AR ER ee tee ER teeta Pe GER ee ge ee Page 85 Lamda DNA TOOTO EE EE EE RE DE a petits 85 kamda DNA EE OE OR ER OE EE AE OE EE EN 85 kamda DNA SOUNO AE ER EE OE ER EE OG ONE OE OE OE ech 85 Bil TT tcs RR Pe ee ee Ee EE Di 8 46 BE RE EE ER RE OE OE ee 68 L arginyl b naphthylamide AAA 85 ATID OU Aerts ees ER cas deeg Es 7 8 83 ATION ER ET LE EE EE OAR AE OO 7 83 BAR AR AR N AE DE EN 39 Leaf Grinding Scarlatti 56 Leaf Sample Preparation ee ee ek AA Ge AA GR Ge AA Ge AR Re RA ee AA Ge AA RR ee ee ee ee ee 77 BEER OE OR OOR OR ER RE OE EE a dened aden dened S A 39 118 BEE EE EO ER ER DAE N OE EE 85 GO Ee EE ES EDE ER Ee e ee Ee Leg 85 Leaves straws ie ei DEE EE E GE anidan 35 BT WEE 7 35 83 Leersia hexandra teo ana aiii 8 83 Lecersla Derrlel conos EE EE ee AS S 8 83 Leersiatisserantis Ee EE ee aaa ER ERGE ee cocos 8 83 Leucine aminopeptidase ie ee ee ee ee Ge ee ee Ge AA ee Ge ee Ee ee ee ee ee AA ee ee 85 Rek ER KA NE EER EE EE da toa e 79 Life Technologies EE EE iced ENE DR SEE RE EER HOER EE GR Ee Pe Be Gee Ee Ee Dee GELE De ERA ees tines GER aie 64 Big ht Brow AAR EE EE OR RE ERK DO 39 Ute Lume Ce ER OE lo trancas 39 Lig lt lg ER EO RE EE N OE
35. support laboratory with facilities for cytogenetical study of conserved germplasm and tissue culture for embryo rescue and propagation of low viability accessions a molecular biology laboratory for the study of genetic diversity using isozymes RAPD AFLP microsatellites and DArT access to gt 10 ha of field space each cropping season on the IRRI Central Research Farm Upland Site a protected zone of the experimental station for the multiplication and rejuvenation of germplasm and also field characterization In 1983 the genebank was named the International Rice Germplasm Center IRGC In 1995 it changed its name to the International Rice Genebank The acronym IRGC now stands for International Rice Genebank Collection Table 1 3 The composition of the rice germplasm collection in the International Rice Genebank at IRRI Only samples with an accession number are included in the table Number of accessions Species name Wild O glaberrima O sativa O alta australiensis 36 barthii 216 brachyantha 19 eichingeri 29 glaberrima 1 543 glumaepatula 54 grandiglumis 10 granulata 24 latifolia 40 longiglumis 6 longistaminata 204 meridionalis 53 meyeriana 11 minuta 64 neocaledonica 1 nivara 1 251 officinalis 278 punctata 60 rhizomatis 19 ridleyi 15 rufipogon 1 022 schlechteri 1 sativa 90 348 OOOOOOOOOOOOOOOOOOOOOOO 6 Hybrids 9 Chikusichloa aquatica 1 Hygroryza aristata 4 Leersia hexandra 2
36. temporary identification number and variety names are important Incoming materials Withdraw the prepared planting material and sort by country of origin temporary ID and amount of seed Sort and assign plot numbers following the procedure for the registered accession Note All incoming materials are hot water treated For regeneration there is no need for hot water treatment since conserved accessions have already passed Seed Health inspection and at the same time low viable entries might be affected Materials which do not pass the previous inspection and that must be re multiplied should also be treated with hot water Hot water treatment is done by dipping the materials in hot water 52 57 C for no more than 10 15 min Materials are then re dried but not using high temperature to prevent caking It is better to air dry first before redrying at no more than 35 C Seeding and seedbed activities Seeding method For O sativa the type of seedbed used is either a dry bed or modified wet bed method In the dry bed the seedbed area is plowed and cultivated by several passes of a machine drawn rotovator Beds are raised approximately 15 cm above the field level and furrows are made on the bed using a furrower with 10 cm between rows On the IRRI ES a tractor draws a seedbed maker with a drum furrower at the back such that beds are constructed with just one pass In the case of a modified wet bed the field is prepared using a sta
37. the Director of Plant Protection AUTHORIZED CHANNEL As specified in the Import Permit Central Post Entry Quarantine Station Los Banos Laguna or Seed Health Unit IRRI As provided by consignee Director Directorate of Plant and Quality Control Private Bag X258 Pretoria 0001 South Africa or as provided in the Import Permit As provided by consignee As provided by consignee c o IRRI Cooperative Project of the Ministry of Agriculture amp Coperatives P O Box 9 74 Togo Import Permit Turkey Import Permit USA Import Permit green amp yellow tag for cultivated red amp white tag for wild species Vanuatu Import Permit Zaire Import Permit Zambia Import Permit Zimbabwe Import Permit 159 Bangkhen Bangkok 10900 Thailand As provided by consignee As provided by consignee Plant Germplasm Quarantine Center USDA Bldg 320 BARC E Beltsville MD 20705 USA or as specified in the permit tag As provided by the consignee As provided by consignee As specified in the Import Permit As provided by the consignee Table 1 1 Taxa in the genus Oryza the species and genome groups Species 2n Genome Sect Oryza Ser Sativae O barthii 24 AA O glaberrima 24 AA O glumaepatula 24 AA O longistaminata 24 AA O meridionalis 24 AA O nivara 24 AA O rufipogon 24 AA O sativa 24 AA Ser Latifoliae O alta 48 CCDD O eichingeri 24 CC O grandiglumis 48 CCDD O latifolia 48 CCDD O minuta
38. wild rices 1 Prepare to 2 to 5 pre pack samples containing 20 seeds in 8 x 5 5 cm foil bag 2 Prepare another 2 50 seed samples one for the base collection and the other one for duplicate storage 3 Weigh the remaining seeds for bulk samples in Active Collection This bulk sample may contain materials coming from one or more seasons separately packed in coin 26 envelopes before placing in aluminum foil bags Each season has a corresponding weight of seedstock and other related information 4 Encode all related information Duplicate storage Accessions held in a genebank are valuable and often represent plants which are no longer available or which are endangered in the natural environment All samples in store should have a safety duplicate sample stored elsewhere From IRRI seeds are shipped once a year to the National Seed Storage Laboratory Fort Collins Colorado USA based on the agreement between IRRI and the USDA ARS see Appendix 3 6 The materials are kept in sealed boxes in a room at 18 C Duplicate storage procedure 1 Pack 20 g sample in pre labeled small aluminum foil packets for O sativa and O glaberrima 50 seeds for wild rices 2 Accumulate the samples in the Active Collection room 3 Request for space and documents necessary for shipping to Fort Collins Colorado at the start of every year 4 Forward the accumulated samples together with the temporary list to the Seed Health Unit which
39. will handle the preparation and shipping 5 Provide the final list of the accessions to be sent 6 Include 10 to 12 control samples for viability testing prepared as follows a Separate 10 to 12 representative samples in each crop year b Take the initial viability percentage following the appropriate guidelines c Prepare 12 x 100 grain samples for each accession and seal in labeled aluminum packets 7 Prepare the packing list and the necessary documents 8 Request the phytosanitary certificate 9 Coordinate with the Shipping Section for the export permit and shipping 10 Update the data files Duplicate storage Accessions held in a genebank are valuable and often represent plants which are no longer available or which are endangered in the natural environment All samples in store should have a safety duplicate sample stored elsewhere From IRRI seeds are shipped once a year to the National Seed Storage Laboratory Fort Collins Colorado USA based on the agreement between IRRI and the USDA ARS see Appendix 3 6 The materials are kept in sealed boxes in a room at 18 C Duplicate storage procedure 1 Pack 20 g sample in pre labeled small aluminum foil packets for O sativa and O 27 7 8 9 glaberrima 50 seeds for wild rices Accumulate the samples in the Active Collection room Request for space and documents necessary for shipping to Fort Collins Colorado at the start of every year Forward the a
40. 2 039 Succeeding EES bars EE EE DR ES cu guna Ged DEEG ED DEERE EE Ee E Es Do Gee dees oder eae 39 Chemical Components sas EE OE EE cani 104 Chemical Ee eil eier Le EE 85 e dle De AE EE OE AR RO EA cere creer rere ee 31 Chikusichloa aouatica nono ed ee ee ed ee ee ee Re ee ed ee ee ee AA 8 83 Ghikusi6chloa EE sacs A aa Eege 7 83 E UI EE N ER A AA EA A E EEA WN Te Rade Alves do EE oo 14 dl EE A A N EN OE EE OE N 7 83 Chloride 6 hydrate 0022 AE i VEE a eee 68 does AE RE EO OR OE canas 85 Civil ae EE RE HO N A EER EE ER 31 Classifications ER ER A EA ee GE GE Ge Ge 39 Cleaningiselection geseet eg AE E dee 23 25 CAOS a Been N ER EE EE ME SE AE bees Ee 77 AVET S AE EE N ER EE OR OE N OE OR DEE N 77 CODES NA Deed A a aN das aed a AE A da laa ded ee lati EA ves vealed 39 Ee E EE A a eA A es 77 EE EI RE EA EE 77 Ee E E 104 CODE EE EE AR AE ER EE EE DR 39 GM EE EE EE OE EE EE RE OE 39 A EE EE EE EE EE RE 56 A AEE T A A E A 56 ed Te teddie ot ER RE OE 39 GOlOMDIA ER EE RR EE EE EE EE NTN 79 Color ER EE EE OE EN EE Eet AER 39 eeler geen 39 er eis OR ER EE EE RE RE Aral 10 30 ee el OE EE RE EE EE EE RE EE EN 39 Methuen Handbook AAA 39 Commercial EG ER AE OE OE EN OE RE N EE N HA N ER 60 Computer Services ARE AE EE EE NE EE OE EE N ON 52 Computer generated list ee ee ER AA ee RR Ge AA ee Re ee GR AA Ge ee Re ee GR ee rana 46 eo EE EE EE EO EE EE EE 85 Bois AA EE EE RE OO EE EE OE EE AE AN 79 COMSCORE AUN ON ati A E egene Ia A 7 10 ric
41. 2 45 ml M pH 3 3 Incubate for 15 min at 50 o C Table 8 2 con t Enzyme Stain Composition Amount Malate dehydrogenase NAD 25mgml 2ml Mdh Maleate buffer 1M pH 5 ml 6 0 Tris HCI buffer 0 5 M 10 ml pH 8 5 Distilled water 29 ml Just before use add MTT 10 mg ml 2m PMS 1mg ml 2ml Malic enzyme Mal Protect from light Incubate for 10 min at 50 C DL malic acid Tris HCI buffer 0 5 M pH 8 5 MgCl2 0 1 M NADP 5 mg ml Distilled water Just before use add MTT 10 mg mI 250 mg 20 ml 1 ml 2 5 ml 25 ml 1 ml 81 PMS 1 mg 1 ml mI Protect from light Incubate for 20 min at 50 C Table 8 2 con t Enzyme Stain Composition Amount Peroxidase Pox 3 amino ethyl carbazole 20 mg N N dimethyl formamide 2 5 ml Acetate buffer 1M pH 5 ml 4 65 CaCl solution 0 1 M 1 ml Distilled water 42 ml Just before use add 0 7 H20 solution 1 ml Leave for 60 minutes Phosphogluconate Phosphogluconic acid 10 mg mi 1 ml dehydrogenase Pgd Tris HCI buffer 0 5 M 10 ml pH 8 5 MgCl 0 1 M 2ml NADP 5mgm 1ml Distilled water 34 ml Just before use add MTT 10 mg ml 1ml PMS 1mg ml iml Protect from light Incubate for 25 min at 50 C Table 8 2 con t 82 Enzyme Phosphoglucose isomerase Pgi Shikimate dehydrogenase Sdh Stain Composition Amount Fructose 6 phosphate Tris HCI buffer 0 5 M pH 8 5 NADP 5 mg ml Glucose 6 pho
42. 3 3A2 3 30 Yellow 1B8 40 Gold 3 4AB8 41 Light Gold 3A6 7 50 Brown 5E7 8 6 7E6 7 51 Light Brown 5 6CD6 8 52 Brown Tawny 6 7DE7 8 56 Blackish Brown 6F5 8 60 Green 27 28CD 61 Light green 28 29ABC7 8 62 Yellowish Green 30AB7 8 63 Dark Green 28 EF 70 Red 9 10AB7 8 11 AB7 8 80 Purple 10 14 DEF7 8 81 Light Purple 10 12 BCD4 5 13 14ABC3 5 82 Reddish to Light 10 14 AB4 6 Purple 83 Purple Shade 10 14A2 3 100 Black F1 39 40 Chapter 6 Germplasm Exchange Receipt of germplasm Incoming materials originate from direct collection by genebank staff in collaboration with national counterparts or donation from other public and private institutions farmers and private individuals and processed following the guidelines below 1 2 10 Record date of receipt donor and origin of seed package Bring seed package to Seed Health Unit SHU to open the box and for post entry inspection Check the contents of the package for the accompanying papers such as seedlist passport data phytosanitary certificate and import permit Check the seeds and compare the names on seed packets with those in the accompanying seedlist GRC and SHU staff Note discrepancies and missing samples if there is any Send acknowledgment letter stating status of seeds as early as possible and or request for clarification for any discrepancy from donor Release seeds and documents to GRC with recommendation on appropriate health protoc
43. 35 39 ele e EE N OE EE ES 31 ell EE RE KON N aut a EE OE EE RE EN 39 ee e DE OE OE EE OE OE RE AE AR N 7 Indonesia Rd Se EE Ee EER RE Ee Ge SE das Gash gas GR EE Ge EE aia 7 14 79 83 riole TE AE N OE EE EE RO NE OE EE OE 47 information EE EES ER EE sec edad E Ee De S Ee TE Ee GE Ee EE SEE De ee DE 49 Information Technology Services ie ee RA ee ee Re ee GR AA ee cnn nc RR ee ee ke ee ee ee ee Re ee ee 54 NE EE N RE OR nip AE crt ere RE SN aaa 79 ele Ke dl EE ie EE EA OE ER N RE EE AG 17 ELE De GEE OR OR On aia 39 Insitute a 5 program EE EE OE ER OE EE RE EE EE adas 5 Institute s Local Area Network AA 5 Instituto Colombiano Agropecuario ie ee Ee Ge AA AA ee RA Ge ee ee ee ee RA ee Age ee ee ee 79 instructional Manual EE SR Ge A eg oe se ee 60 64 instrument Repalf EE aie EG Ve Ge Be SE ee ee ee 31 Delia RE OE AE OR ER N 79 Integrated Pest Management 17 Intellectual Property Protection on germplasm ee ee ee Ee AA Re ee ee ee ee ee 10 International Network es n a a Ee a ee ee ve ee 5 10 Ex Situ CONCCUONS ss ie Ee Se n Ee ee ee ee Ge A E ee gee Ee ge ed ee 5 10 International Rice Genebank ee ee ee ee ee ee ee ee ee 5 8 13 49 83 adopted WEE 13 NAME ii ds E A A E dee Gan vada A EA LEIA AAA A ee A 8 Bro edutes AE EE A aes A A N N RE 5 International Rice Genebank Collection ee ee ee ee ee ee ee 8 39 A N EE ET EE E EE A 8 International Rice Genebank Collection Information Gvstem ie ee e
44. 5 11 Replace the culture solution twice a week 12 Transplant the seedlings to pots in the nursery Cytology Pollen Mother Cells Fixation and Staining 1 Fix panicles in fresh fixative 6 3 1 ethanol chloroform acetic acid with 0 25g ferrous chloride per 100 ml fixative for 24 h at 4 C 2 Store in 70 ethanol at 4 C 3 Prior to staining wash spikelets in 70 ethanol at least three times and blot with paper towel Transfer to clean vials with sufficient amount of snow carmine to submerse the spikelets Incubate in an oven at 50 C for 24 h 4 Keep at room temperature for at least 3 d before squashing Slide preparation 1 Remove anthers with fine forceps and add one drop of 45 acetic acid Squash gently with a fine needle and remove debris Place cover slip and heat slide gently Apply enough pressure to flatten cells a Fw RN For temporary mounting add Hoeyer s solution to one side of coverslip and let dry for an hour at room temperature Do the same for other side Microscopic observation analysis and recording 1 Examine slides using binocular microscope first under low power objectives then shift to high power objective adding oil for oil immersion objectives 2 Perform meiotic analysis using appropriate form see Appendix 9 1 3 Take photomicrographs of noteworthy materials 66 Root tips Germination pre treatment and staining 1 Germinate seeds in sterilized Petri dishes lined with mois
45. 50 ml centrifuge tube 50 OND Add 2 5 volumes of 95 EtOH and mix well by inversion to precipitate the DNA Centrifuge sample at 4000 x g for 5 min and discard liquid Rinse the pellet with 1 ml of 70 ethanol and dry the pellet Re suspend the pellet in 50 ul TE buffer see Table 8 5 DNA Quantification 1 Prepare a 0 7 agarose gel by weighing 1 75 g agarose Transfer this to a 500 ml Erlenmeyer flask Add 250 ml 0 5x TBE see Table 8 7 and boil in a microwave oven for 6 min Allow to cool to 55 60 C Get a gel mold and seal both ends with 1 masking tape Place in a level platform and attach a comb Pour agarose unto the gel mold and allow to solidify Pour 0 5 x TBE buffer into the electrode tank Remove masking tape from the ends of the gel mold Mount the gel mold on to the electrode tank with the comb oriented towards the cathodal end Make sure there are no bubbles in between the tank surface and the gel mold Gently remove the comb Load 10 ul 1 Kb ladder see Table 8 7 on the first well and load 10 ul each of the 4 different concentrations of lambda DNA 500 250 100 50 ng 10ul see Table 8 7 on the next 4 wells The concentration of the lambda DNA can be varied depending upon the harvested DNA Apply a series of 3 ul drop of 10x loading buffer see Table 8 7 on to the surface along the width of the parafilm Follow this up with a series of 10 ul DNA samples exactly on the same spots of the loadin
46. 68 ENT OE EE ida tad 85 Poteoatgt ebe eeneg Eege MG ER OE EG NE N N DEE 15 17 RR EEN 17 RSD ES SE Ee 47 Ruaraidh Sackville Hamilton 49 PRUPIP OG Lo EE ER OE OT EO A OR OE 7 8 35 83 aile HR EE ER OE N 83 RUIE EE ER IR RE OO EE EE ER EA AN 77 BOMA EEN 77 SO KEE EE 60 68 S S 7 46 68 83 PIKE EE AK DEE HARE ER EE EA OR N ER 31 Ee Lu EE 60 127 Sample sis sissies AE EO IE EN AR ER IR ete 23 25 39 Active GOMES CU ONS is te Se Ee dit ais vel oe es 23 25 EER EE EE IE EN OE N RA ES EE EE nie 39 Sample CategOrV ir EE EE EEN ee Ee ER GER EERS Ee Re ee Re ED OE ee n ee ene eats 47 LU WEE 7 8 14 15 17 23 25 27 28 30 35 39 46 47 83 SAV He EE AE o EE NAN ON 39 la N EA EO OR EO N EE OE E 7 83 Schlecht se EER AE ev EE RE OR ee De Gee ee ee E E ee ee ER EE oe Ee 7 8 35 83 Schlechterianae ss RE ia Eege ee oe dee 7 83 eed BA AA AO EL OR EE N EE AE SEE nis ie eaters ceeds 64 PEEP Ed ER OE ME AE OE EE EO NG 64 Score RAPD EE ER EG SE ti 58 es EE EL ME LO EE ERG 77 SCreenhouUse titan 10 35 Screenhousetaciiti s iii ii GE ee NE De bee 35 Screenhous s Es N ge a A ee ea ee eu e 8 39 alter O N oe ee De Ee ee ee eae 68 85 Second Ed id ge a di de ede ee ges 39 El le ie EP SE ER ee a el ain ees E EV ke 39 Section EE ee E dane Ge veda hatha sda va vad dua Ee EE De ie ee 39 73 ad EA EE EE N OE N N EE 39 e EE 25 47 Seed conservation Se ees ee ee NENNEN nan ee VAN 23 ite e e RE 23 Seed distributi n a 47 Pro
47. 9 74 ER EE EE EE ER EE RE N 39 MUNN DOR EE RR EE EE EE ER EI EE RE EE 39 Ip BT 1 EA EE OE OE OE OE EE E EO 7 83 B B mercaptoethanol AA 68 S Ee A aa EE AE ee ees AE N EE N 39 039 RAIN SEE DE DE EE SE o EE 56 58 64 74 15 20 MIE EE RES AE e Ee De Ee ER ee 56 LOMO EE AE AE RE RE MO EG ER ts 58 64 takes aDeSe ER ee E e tak 17 ET EE 8 60 International Rice Genebank Collection cccocccccccccccococococononononononocononococononononononos 8 IPC Utica EE EE ME OO HE A ON OOI EE EE 60 State EE RD AS EE MOE DES EE 60 DN EE TA ES DE GE ME EE Ge GE O Dr ee bandas 60 Steriborer ss EE EE Ge ER N ah naa Ee RE NE EE EE DR ES se sia caste ED ER EO ea oe 52 Stemborers ESE do Ek oe 17 Stock CONC A alii 64 Stock CONCONUAU OME iii aa dis 58 60 85 129 ellen e TEE 35 SR EE MEE EE ET AL EA EER AE N 85 tell EE RE EE a 85 STR ale EE ESE EI EN ER ER ss ee NE E DE ee Ee estan 85 Sie AR ER iria 104 GEIB ee EE RE EE EE RE EE EE EEN 77 System a oran pad ch neers rere n Ge eee 77 dIE EE AE EN OO AE EE OR OE 77 EN EE EE ER EE AE OON AE HE OE OE 77 ELE TA GEE EE N OR EE rara 77 EER LE RE DR EO EE AE 17 TEE 73 GI EI EIER e WEE 7 83 SUDUN AE EO EE OE AE OE EE A E ON AE ONS 39 lee Tue DEE 39 Characterization EE EE A i de eau cade cena ee Ge Ge ee PD A gu 39 SUDH20 BEE 58 60 68 85 Supplementation sie Ae Cai A es Gee ge We dee as el eee 17 SUCIA date ea ddan dale ee ee ee 39 56 TDSC AIA EE GE A 39 ole Uil N N EE N N N ER
48. AA A DA ge ee ee ee 85 Ee ee EE 85 Glucose 6 phosphate dehydrogenase ee ee AA Ge AA ee AA ee ee ee ee ge ee 68 85 EIERE 7 8 83 Glumaepatula i e EE AE iia edn uel aa ee A Gein ee ene A es 83 GUMS EE AR EE EE EE EE N 35 Glutamate oxalodeetal Es a a EE ede De Ge oe ee Age ee 85 EI RE N RE OE EE ER EO ON OR RE N 104 Glyphosate AR EO OE OR N AR EE 35 Got MIB gies getest a 85 len AE EE EE EE EE OE OE ER EE 68 dC AE NE EE EE OE OR 7 8 83 eT geta ER OR RS ME ORE EG 7 8 35 83 Gral EE 83 clie ER AE EE EE ER EO EE RE EE EE N 46 47 52 ele ET SR NE EE bent ee EL EE EE OE AS 46 UR AR EE EE EE AR OE OR EE OE EER KIN 47 AU RE ER OR AE OE EE EE ER 47 GRC Data Management Room 54 GRC databases applications ie ee ee AA AR AA Ge AA Re GR AA AA Ge AA Re ee ee AA ee ee ee RA ee 8 COGIC AC RE EE ER AE EE EE E 8 GRC NUIS Pry cita il EE RE EE OE EE OD 35 Elle TEE 35 ACTOS OE ea Lact eed ddd ME NE EE ON 35 Grind KR EE 56 Guildiord St iris DE Ee ER 31 GUY AN ate ER ERA EE N OR EE ORE EE 79 H H 27 39 56 58 60 64 68 73 74 104 A SA EE RE AE AE EE Gloag sansa ER EE needs 27 114 e ie AE OE ME EE OE EN 58 60 64 68 85 A evi ORE ORE EE RR DR Ek 68 85 TAS OA REEL a EE aioe iets 104 HSBOS RE ME OE acacia 104 nt E EE ati ene rere AE RO N ee Dee 17 Hande i AAR OR ER LE EE RE EE ER N 47 Harvest VerificatiOn escea a ER N ia 23 Harvest de ETA AE EO ia 39 HEL ES EER RE DE RR Ee GR a EE AE de 68 74 85 Cy 0 lene eae MEE AO
49. APA eege eene la ete Ee A ae 79 Empresa Brasileira MAR i EE RE EE OE RE NE 79 dle RR EE OM LE IE OER OE EE EE OE EA 28 ol ei SAO EE OE EE OE OE EE ORE OE ER OE EE 28 Endopeptidase AE N EN OE RE EE OE OE 68 85 AE AT AE N RA OE OE EE EE 68 85 Enrique delos Reyes eis EER REEKSE EE SEG KERR EE SEER EE GR OER Re SE ROES ERNS SR RR aerate 31 ell ER ME EE AR RO EE OO N EE EE 85 LONG EE EE EE MO LE EE EE 85 Erlenmeyet snr EE RR EE EN N EA 56 73 104 SE EE EE NA ER EE ER N N N 13 do EO EE OE EE OE EE EE NE N 68 85 Estesterase ie SG A Ee Ge Ee DE GN ee BA ge dee ONE EAR 68 Dl ER N EE OE OR N ea 56 58 64 68 85 56 LS le ER EE ER ee ei lo a ieee 68 85 Ethylenediaminetetraacetic AA 68 EtOH ER EN N ae EE OE ea a 56 68 85 Europe i GR EA NE KA EO EE N EE EE op 7 83 EVALUATION RE OE EE E A EE EA EE Ne 11 39 52 Ex Situ Collections RE EE ER ta te tins 5 10 InternationaliNetworkK EE DEEG GESE anadir E NEE 5 10 DEAN EE EE ME OE N EE 39 DE Ee EE EER 52 Germplasm information EERS EE Teba GEE GREG AE EE ee 52 DEd AM EE EE 39 ETA AR OE ER EA KA RR EE EE OE N 17 39 F F 14 104 Ede eatin in eee lie e E Es 39 56 DE EER EE OE OE N ER ED EE RO EE aoe 56 FAOn MT ER EE EO NE OE EN a 5 10 47 49 53 FAO designated oermplaem esetet nttt restr tstn retn nsttntttnntnsttnsntnsttntunntnnsrnssnnn nts 47 Fast Blue BB vies A EA EE A EE EE 85 sine Ne de OE ant aea EN A E E E 85 TA IE EE atte eee ee eee areas hee ee as 31 GUUR NEE TE NE EE RE N
50. BCC Sub Saharan Africa O rhizomatis 24 CC Sri Lanka Ser Australienses O australiensis 24 EE Tropical Australia Sect Brachyantha Ser Brachyanthae O brachyantha 24 FF Sub Saharan Africa Sect Padia Ser Meyerianae O granulata 24 GG South Southeast Asia O meyeriana 24 GG Southeast Asia O neocaledonica 24 2 New Caledonia Ser Ridleyanae O longiglumis 48 HHJJ Indonesia Irian Jaya Papua New Guinea O ridleyi 48 HHJJ Southeast Asia Ser Schlechterianae O schlechteri 48 HHKK Indonesia Irian Jaya Papua New Guinea Table 1 2 Genera number of species distribution chromosome number and spikelet structure in the subfamily Oryzeae adapted from Chang and Vaughan 1991 Genera No of Distribution Tropical species T temperate t Oryza 22 Pan tropical T Leersia 17 Worldwide t T Chikusiochloa 3 China Japan t Hygroryza 1 Asia t T Porteresia 1 South Asia T Zizania 3 Europe Asia N t T America Luziola 11 N and S t T America Zizaniopsis 5 N and S t T America Rhynchoryza 1 S America t Maltebrunia 5 TropicalandS T Africa Prosphytochloa 1 S Africa t Potamophila 1 Australia t T The need for genetic conservation Traditional varieties and the wild species of rice are being lost through genetic erosion Farmers adopt new varieties and cease growing the varieties that they have nurtured for generations and eventually lose these varieties The wild species are threatened with extinction as
51. Consequently they need to be grown in 30 cm wide mouthed pots without holes Wild species are also known to have stronger dormancy than the cultivated species 1 The germinated seeds are planted 1 2 cm apart in a seed box containing moist fine clean preferably sterilized soil mixed well with appropriate amount of ammonium sulfate 2 Granular insecticide e g Furadan is applied 3 4 days after planting to protect the seedlings from ants and other insects 31 11 12 13 14 15 16 17 The seedlings are watered carefully with a fine spray and grown under partial shade until a week before transplanting The seedlings are transplanted at 30 DAS to water tight pots with good quality soil mixed with about 5 g of complete fertilizer Water level is maintained to at least 1 cm depth For species of the O meyeriana complex the seedlings are transplanted in pots with light soil and good internal drainage to prevent water logging as they thrive best in mesophytic conditions For the highly stoloniferous species such as O schlechteri and some related genera like Luziola Leersia and Hygroryza a modified flat bed is constructed and used for growing and maintaining a single accession The pots are laid out at least 100 cm apart to provide sufficient ventilation between plants and enough space for plant management All species of the genus Oryza grow well under full sunlight except members of the O meyeriana complex and O
52. D N EE GEE EL ED DR ee 58 64 KO DELE ER MR LE eN SDA N EE ER n 60 68 85 Keep SERE ED DEE EE CEC ER EE ED EE ee N ee Di 31 O AR EE RE EE EE EE EN 31 Kenya EE ME DI EE RE GE OE EE GR OE SG DE GO De tk 79 KOM EE 79 Kenya Agricultural Research Institute AA 79 der EER EO DE EE N RA EE 68 85 104 UE 85 117 EER AE EE AE EE EO LE N este 39 ele N EE EE ta 68 Kimwipe EE ME EE OR EO OE EE EE EA N 60 lu ER EE EE OE OR N 56 le N EE OE OE EE N N OE EE OE EK 56 Klaus Lampe Laboratory ee ee ee ee ee ee ee ee ee AA EINE E E ee ee AA ee ee 56 Adele RE A ME EE lee ke anes 104 giele del ER NE OR RA ME NT 85 Kadak GB GIAN ON OE N ORE EE EE 85 Kodak GBX Developer 60 K dak GBXFIXe AE N AT OE RE EE OE EE 60 KORG ER EK ER EE OE ME EE OR RE OE iia 14 ed NE OE NE N EL EE KO EE lage 39 L L 56 58 60 64 68 73 85 104 ly EA EE EE EE AO ER eae EE 58 RE EE RR EE EO EE eee NE 60 MO EE OE EE RE OE EE ER EE AE N 60 RE EE NE EE RE EE OE IE EE IE 60 di RE TE ME OE OR RE EE AE NS 56 genomic DNA EE Ee ee ee E 60 RE ME OE EE OR RE ae A EE 60 Mi ME EE RE OE EE OR EE EE OR 64 LATE EE 60 LE SXSTR EE EE EA DE a dd te at 60 d ER EE EL EO IE EA EE 56 ERA EA AA EE N err 56 L BME susi is de EE GE RE ei Gta at eae Ee dead 56 L IDNA SE A ER oe NEE ee eg 56 64 lr CG 56 ESPALDA ee ad 56 ag hia Ee ie ee Ee e Aad sedate tas a iid 56 Mg dd HA le EE N nn sane EE AO ieee 60 BEN OE OE AR OE LR EE EE Ee 39 AR EE EL ELE EE EE EE EE 39 EE AE RA EO EO N AE
53. Director EL EE RE RE N N RE RR N N EO EE 79 BITE EE EE RE dc OE DER ces ee ee N Ra GO NN 60 77 ROS dle ER RE EE OE EE EO ET EE EE a 77 NO MAREE AE HO ON AE RE EE N OE ER EE 60 DisodIUM ER EE EE EG Ee De Re AEE asd EE EER N ee E se ee 68 85 104 Pie RR EE EE EE AE EE OE Ee 74 KN ME EE OE A EE OE EO EO EE OE EN 74 DL alanyl b naphthylamide A 85 PIE ese EE ME OE RR ON EE OE EE OR ER NE 85 DESTA EE DE Eeer 85 NIE e 85 BEE RE hal hort ME OE OE LE N 56 58 60 64 68 85 collect ES E ee E TE DS be nn 56 COMANO WT 56 110 EE EE EE AE RE EE EE OR OT a eaten 56 Loaddimg AR RE N ce EE RE EO EE 60 Stalon AI AO EE ER LE EE EO EE 60 VUE aa a a Ee 56 DNA EXtr cti h EER a a a rice a e ea EE a R ere 56 DNA Quantification iss EEN is Se GR seeding OR Ke VR A A AS 56 DNA Sample Preparation ee ee ee ek AA Re GR Ge AA Ge AR Re ee Re Ak AA Ge AR Re ee ee AA ee ee 56 Protocol eege EER EE eebe Re RE EE ness 56 DNTP eebe Re EE IR EE Ee eebe ER EE Ee ee 60 68 85 RV HR SE 58 RV Be SE MA TE ON EE OO AE EO 58 Documentagreement Ae 53 DocumentatiOn EE EE AE ER she Sues ee esau eats aca bene Re ca detente noun A 52 DOCUM INES Forse EE EE ER OE aerate 46 GREG ER tree reece beeen 46 BR LE EE ER HE EN ER OO MR ON N 49 lee BEE 49 Drop WEE 74 77 ER N RR ie aan ieee Wl leat EL EA ee ee 77 LE ONE EE N EO eevee EE DE 74 Moie EE ae A yaaa eed ue eee 13 DropsSeedS eege ie E AAEE EA ke dio 17 BAAR RE OE ER EE ER OR OR IE EE EE N 56 60 le EE es E
54. E EE EE AA EE ER E 60 n La EE EE EE EE EE ON ER KG 56 Dryer S ER AR RE N ck OE OR N N EE 77 el 77 BERE EE AE ete eater EA EE RE marta pout 13 DTT AE EE EE EE wee eater ee ae 68 85 Duplicate Storage AR AE EE OE N N 28 30 Re EE RE EE OE EE RE tbn 10 11 13 60 77 electropho eS S EE RE des EES DASS 60 77 ET EE todas 11 o EE EE RE N N EE 10 DAT MR ER EE OE OE Rd 13 Dye Line Characteristics After 4 hour Run 77 MY ei ER EE E E EE N OE ER 77 E E 14 104 EG oe aes Mee Kee eet pa est Ek EED EE DE ea ee at tet 68 East Africa TEE 7 79 83 e E e ICT UE 7 deel AE EER EE EER is 14 drol sa EE ER ae es N EA OR ER EE EE OE EO 64 deed sa MSC ira ER EE AL OE RE EE EE Fr 64 AR EE ER RE N OE EE OE EO EE OE OE 60 PERE DE Ee EE EE OE ee Ee ED EE GE eech 60 Sharktooth Et RE EES n eae e eebe EE EE Geer 60 Editideselect O ee n ute ee ER De n RE op RE OR Ke ae ee eae ee 47 EDTA Si re oa DE OE rere ree renee rece eee a 68 85 104 RRE 104 EE ie eebe ee eebe ees 7 83 da EE EE LE AE HI ER OE EE 39 A it EL EE RE EK ee ee EA se GR E Ge ER EE ge ed 7 8 83 Electrical Eng RE OR EE ER EO EE eee 31 111 Electrophoresis ocio N eee EE ET ee 56 58 60 64 77 85 Crude Extract Preparation sees ee ee ee ee RA Ge AG AA ee Ge AA Ge Ge ee Re ee Ge AA ee ee ee 77 Co LUT ne ER AE EA EE RE EA AE DE OE 60 77 la AE RA RE LE OE EO EE tines 64 FI6CtrOphOretG cc NE EE RE R N RR RE eee 85 ad EE OE DEE EE 47 dien EE OE a N ec OE EE terrae 47 EMBR
55. E EG de ee OR AE OG 39 Deche Zieser ee ee eege eege eg 39 79 Dehydrogenase OE OE EE EE EE EE OE 68 85 Denat EE OE EE EE EO EE OR EE 60 AECH DNA ie OE EE EE RE OE EE OR 60 BEd LE OE RR EE RE LR RE OE EE AE ai eee 31 Deoxyadenosine 5 triphosphate AA 68 Deoxycytidine 5 triphosphate A 68 Deoxyguanosine 5 triphosphate ee ee Re AA ER Ee AR Ee AA ee ee ee ee ee ee ee ee ed ee ee ee 68 Deoxynucleoside 5 triphosphate AAA 68 Deoxyribose EE RE EE GN dee N se ee De VG DE EA de EHS ay 68 Deoxythymidine 5 triphosphate ee ed Ee Re AA Re AR Ee AA ee ee de ee ee ee ee ee ee ee ee 68 Department EE ai iia wa EE DE de ee ENEE ge EA de n se Ee De En 47 Agriculture Aina oie en ia GO eee 47 AT e e WEE 35 nature IMPOMAaNnCe iio ee LG ae ce Ee aie ge 35 AT TE ul e EE 39 56 gul EE EE N ieee eh OO N N N AI 56 NET e EE rae ee ieee teen aaa Rada ee EE 28 56 De Staining OR N ER OO ved N a vee eee 77 85 DISCO ER N eee A es ER a 77 Determine mixtures Off ty Pes AAA 23 BL pg AO AE N EE RE IN 39 56 EE to ias 56 DOTP ocios ia RA 68 85 DA Oi A eel eee NE ER IE RE 85 DH2O VE 56 58 60 64 68 85 104 Dhaka ARE EA RR EE N N 79 Dle EER OE ME RR EE EE N 79 Belge de RE OE RE ege 64 Dle red de EL EE AE OR EE ER OE EE EE 68 85 Diphenyltetrazollum ss Ee REG DEA et 88 liniers 68 DE ER ME EE OE EE EE OE EE N 79 IIe te 79 Blare EE EE OE went thane ARE N 79 ARE EE RE OE EE 79 Plant Prote Con ER AE OR OM EE EE OR EE a a naadi aa Senda 79
56. EE ED EE E EE 15 Several proofreadngs ie ee AA Ge AA Ge AR ee Ge AA ee Ge ee ee ee RR ee AA ee ee Re ee ee ed ee Re ee 14 128 Shape ess ER TE ee cle ee eet ae ee et 31 Sharktooth E E A ek tt o ee geed See EE bi 60 ARE EE EE N EE OR EE OE OE EE 60 Shikimate dehydrogenase ee ee ee RA AG AA AR ee Ge AA AA ee ee rra 68 85 do Tu oe TE AA N AE DEE HE APE 85 led EE EE EE 30 ora EA ER HE EE N ER EE AE OE Se 30 Shipping Section EE EA N OE EO EE ER 30 al MEE ON EA RE OS HE EE EE 46 79 eu eo EO EE ER EO EN RE OE EE OO OE 60 see 210 11 EE EE AE ER EA OO EE EE OR 28 SINGER ar N DER OE ER N OE RE A N 11 49 52 ol AE ES ENE EE EE OR EA EE EN eee 39 AE AREA EE EE EE EN EE EE O O 13 lu le EA EE EE OE AE EE a RE rrr reer 13 labon Ee e Ge ee Ge Ge ER 77 Snails This EE ED tse dad a NA 17 Sodium molybdate 2 hydrate ee esse ee ee ee AA ee Ee RR rr 104 Sodium phoshate 2 hydrate ie ee ee RA AA ee AR Re GR AA ee ee ee Re ee ee AA ee ee ee eek ee 104 Solution EE EE ER EO N EN EE N N EE EN N 104 ole EE AE a OR a dz 104 Somali NN ee oe 79 SOUT OX EE EE N N EE OE EE EN N EE ON 53 South Central Americal DERE N OE AG GE ED ED ee E 7 83 SoutheastASsla ii ek ee Ge Nee dE NEE 7 83 el AN de AE RE RE EE HE EA AEA 79 SOUL ASIA OR ll EE N OE N 7 83 SoutheastAsia EE EE SE ME ER Ee SE EE Get GP Ee OS Ge be Gee Ge Ee eb ee el DO ee 7 83 SR OE OT EE EE ON EE EEN 17 lS EE ES SE OE EE 7 35 39 83 lS ER EE EE SE OR EE ED OE 3
57. EE NE 39 Bld EE EE EE N OR EE EE rere 60 ell e TEE 60 Lig ule EE a a ne ei en ee eee 39 LiguleleSs ege EE a i ER EEE E VOE ENA AAEE ee 39 LIPIDS es AE HE EER AL IE MO OR ER OE EE OE EE 79 Line Characteristics EE A ee EN Ge Ge GV E E E A Ge ee N ee 77 BI EE 39 L leucyl b naphthylamide ooomcoccconnncccconoccccnnnncnccnnnnnncnnnnnecc cnn rn 85 Load TOUR oai da ORE A E EA DAD Ai id ai 56 58 64 LOAadING ici AA Ge aoe ee eee 60 77 Grude Extract de ee ANE dE ee ee ee ve 77 DNA A ata ee adh ae ee daca GE ee Ge E Ge E Es 60 London WOTN QLD ER EE N EE EE OO 31 ede Te TES EE EE EE 7 8 35 83 LONgIStaminala EEN 7 8 35 83 LONGIStAMINata s a a ea A neds ieee 83 LOS BANOS ARE RE EE EE EE aaa 13 39 79 Los ROyOS EE iiini N EES ege 85 En EA N AE EE EE EE ER EE OR EA AR 39 IUGR SR OE OR E EE EE OE EE N EE PL 60 Hire ER EE OE OE ON MR N EE EE ER 7 35 83 SA EE EE EE EE N 8 83 BEESTE EE OE OE OE EE 56 85 M M13 15 17 35 58 60 68 74 85 KL BEE OGE SG ENS Ge Mee ES RE EG ee erent coe er N 85 MEE N OR OE MA OR RE NN 85 M DRG O O AR ER EA 85 Mp cho EG E anta ido DE RD ee Re Ee bg De RE reer ER ES GE mercer ee De cree 85 OR EER MO cda 85 OK e EE ER RR OR EE RE ata 85 deet ee SE ON EE TR ER ii 74 M NaCl Re ee Ee oe 56 Get Hu Ce LAE OE EE SG EE ER GE de tbc 85 d Re EE 56 EA NE AE EE OE EO ED EE 8 17 M 2 2 aa ia 73 MA e 68 Ee le ET 79 Malta cia aaa 73 PE costaria ira dit 73 119 Mall AA a ER Sh Ac OR NAN HA EIE 85 Mal
58. EEN EE EE EE IG 39 Note ES ER DE EE EE NE OE EE OE OE EE OR OR OE EE EN 35 Abbreviations ACFONYMS AA 68 e e TEE 8 11 39 A bromo napthalene EE EE EER AeA NEE ER ee ER ad aa eM eG ee ee EARS 74 ABS eRe AE ED De ERE EERE EE N GR SEER Care Cece Er ae 17 PICCOSS SE EE 35 52 54 CGIAR ESE DR sa e El cen tae 52 Genebank Seide HOL N BEE 54 Ee lte 35 e El E E EE 49 Accessionsiplot ES EE GE en rane EE Ae ren nei bent ole etre heen ee 17 ACCOMPANYING scenic ila ean et eee eat 46 seedlist EDS DEE N GE EE Gal beni A E SR doo 46 Ae tee ee EER OE EK HE 85 Te eli RR OE EE dee AEN 74 Acidphosphala ER AE OE OR RO RE RE OR de 85 ER AK AE OE RE N EE EE OE N 68 85 Acrylamid bis acrylamide ese ee ese ee ee AE A ET AA ee ek Ka EA Ke ee ee ee Re ee ee ee 85 Acrylamide ER EE MR OE OE EE EE N N 60 85 Acrylamide bis acrylamide ee ee ee ee AR AR Ee AA Re AA ee AR AA ee ee AA ee ee ee ee ee 85 te ER OR RE RE a N EO ells 10 23 25 28 RE AE N N EE DE EO AE ee 28 Activ Collection EE SE DE ata 8 10 13 23 25 28 30 31 103 PACU aah REKE EE RE EE N OE ER N EE EE tiene 39 Add 147 UTE as EE ER OE OE OE ORE OE AE OE HEN 64 Add 20 ul si coa EE ee ES EE DE Oe ee EE Ge ee Ee RR ER Ge SE oe oe 60 ORE ME ME AR N EE AE ER AS 64 Add 25 RE AE AE EO AE EE OE EE IS 74 Add 300 RT e EE DE RE RR EE ea Cee een erection reece ered ie ree EE ee EO es ei Pe 56 OE SERE RR EA EE AE IE OO N 56 Add RU ET NEE 56 ORE NEAR EE EE OR EE EE EE OE KEN 64 Add 500 REK
59. ERAL DEPARTMENT OF AGRICULTURE essens essens KREE KREE EE RANGE RR RAN ENEE REK Ka KAR 74 IMPORT PERMIT sesse es sake kak eke R REKEN GER EKEN GR ERA KERKE RAK GE NEE ER RARR KERE REK GER RE ER RAK EE RE RAK KEEN 74 Table 8 6 General stock solutions for DNA extraction ee ee ee RA RA RA RA Ge ee ee 88 Table 8 8 Solutions for RAPD E 92 Welcome The staff of the Genetic Resources Center assigned to the conservation of rice germplasm are pleased to present this revised Manual of Operations and Procedures of the International Rice Genebank lts preparation has been an excellent example of mutual support that the staff provide When joined IRRI in July 1991 it took me several months not only to learn the basics about rice the genetic resources of this important crop and their conservation but also how the genebank operated Since then the various operations and procedures of the genebank have rationalized and upgraded Many aspects of these operations have changed significantly The genebank was fortunate to be included in the refurbishment program of the Institute s facilities Our ageing storage room cooling equipment was replaced a seed drying room installed and laboratories remodelled New screenhouse facilities at IRRI s Upland Farm were added for the cultivation of the wild rices Our data management capability was enhanced through connection of powerful personal computers to the Institute s Local Area Network In 1995 we also receiv
60. GCIS User s Manual IRCGIS User s Manual A separate user s manual for the operation of IRGCIS is being developed The manual aims to Provide the user a step by step procedure on how to log on and use the system Assist the genebank staff in carrying out each genebank operation Aid the germplasm users on how to request the desired germplasm and related information Serve as a guide to refer back at a later stage especially when some clarifications are 47 needed or when problems are encountered during the operation For a beginner it is essential that the User s Guide be read before using the system to have a better understanding of the task to perform Data integrity The IRGCIS incorporates the validation rules for all traits described for each sample in the collection Through this mechanism encoding errors are automatically intercepted and the user is warned that an invalid value has been entered A prooflist is also provided by the system to verify the encoded data against the original data Data security The system takes into account the list of authorized users specifically genebank staff who can access the different options Access to Genebank Activities options where updating of data is being performed is restricted to person in charge of the operation Hence data is secured from unauthorized modifications and deletions The daily scheduling program automatically creates a dump file of the database Informati
61. International Rice Genebank Operations Manual Table Of Contents TABLE OF CONTENTS sinies ee sin ese eie dee ee be see ee ee ie ee ei 1 Vd Keel 5 CHAPTER 1 CONSERVATION OF RICE GENETIC RESOURCES AT IRRI 6 The conservation of rice genetic resources at IRRI ee 6 The need tor genetic Conservation aa 7 The Ge SDA AO RA ee 7 Germplasm acquisition and CONSSIVAON omssici ic citas 9 Germplasm exchange AA EE EE EE 9 Germplasm characterization and evaluation kee 10 Data management 10 CHAPTER 2 SEED MULTIPLICATION OF ORYZA SATIVA AND O GLABERRIMA GERMPLASM oe oe N ere NEEM ee een ee Ne uie eie ee ed 11 Seed multiplication of Oryza sativa and O Glaberrima germplasm A 11 Selection of planting ELE EE EE EE EE ER 11 Seeding and seedbed activities ee RA AA Re Re ee 13 e E a 15 CHAPTER 3 SEED CONSERVATION vicncninencitnsncnccsccncntncninscensnsnecsssninencntecieenens 20 Introduction to seed Conservation ENNEN 20 gels Ee ie AA N DE EE NG 20 Seed cleaning and selection 22 o EE 24 sesd health Sua OM ase inva EE ARE IE RS ES NR EER meer Ee NS Ee ea Ee ese ie 25 Els OE EE TT AE ER EE 25 PACKIN EE ER RE E TNA 25 BUC Ae Storage ARE AE RA EA AO EE 27 Ree EE 27 MAL ee he AP e 28 CHAPTER 4 GERMPLASM NURSERY SCREENHOUSES esse sesse sesse 31 The germplasm nursery SCreenhousesS ke 31 Cultivated es RO EE AE EE NE Ee 31 WU MON EGS AE N OE N ER EE AE EE 31 CHAPTER 5 GERMPLASM CHARACTERIZATION AND EVALUATION
62. LE EE OE EEN 8 Stee 68 85 doele es el MA adhe OE EE EE DE 31 dd ERROR EE OE EE EE EA EE HER EE 68 Pesquisa Agropecuaria RER ee E aaa ee ee AE ee ee A Re ee ee ee ee ee AE Ee ee 79 Pest Control ER EE RS ED EE ee ee onto Eeer DE ee cease ege De od 17 KUR EE IE AE EE EO peer DE reer 14 73 74 77 ARE ER EA EE ME EO OE ares 68 85 ad AE EE EE ER EE EO OE ER 68 85 PHE ae Ed EER AE EE EA 60 73 85 104 tei OR OE EE EE OE EE ONE OE RO OE EA EO DE os 104 MEER RE RR EE N EE EER EE a ee 73 sak a OE ME LE ON 85 dakke EE EE ER EE EL OE IE ERG 73 MEME MEE HA NA OR N ENE N EEN EE ii 85 PHA 8 N RR EE MA EE RE EE OE EATA 85 da ER EE A ees EE RE KEN N N 56 Ma AE BL HOE EE ER 85 ad ET N EE DEd RR EE EE N N 17 Phenazine MethosSullate ES EG TA Ne ee ee ge ee ee 68 85 Philip LE RE EE N EE N 46 Philippine phytosanitary A 10 Philippine Phytosanitary Certificate 49 Philippine Plant Ouarantine ee ee se AR Ge AA AA Ge AR rca rn rca 49 Philippine Plant Quarantine Service ie ee ee ee ee AR Re RR Ge AA ee cnn nn ee Re ee ee ee ee ee 10 e UU EE 7 10 13 39 47 49 79 83 eds LEER OR EE title 68 le Eegiel E 85 NV eieiei ele 85 PhosphoglucoSe Leomerase AAA 68 85 PhHOtoperlod SOnsitive ER ER til 35 Physical Plant Sefvic s Unit ts its ese EE aida 31 SUE EE EE 52 Physiological te RA AE EE EE N 104 Laboratory VELLE N RE N N tias 104 de UIE TA OE AR EE 30 46 47 ele ITS ER N N EE RE EE MEE 30 Phytosanitary Certificate 47 dd et RE EE
63. N EE EA EE AD 56 Ou RE AE EE Ee OR EE AD 56 Ada 700 RE EA EE EEN EL OE N ere 56 Le KAN TEE 64 TE EER oe RE a EE De EN reer EE EE cerca cree 64 ede EE EE EE EE ae Gee a N 85 ee ER heavens aie te leh EA OE Oe OE 39 60 EN EE EN EE OE MA OE IA ceed 60 REM ie A OE EO OE IE RE N OE ER 39 Addressed aere e a ava ale Ge T EG Ee 49 79 Australian Plant Quarantine Office iese ee RA AA Ee AR GR AA AA ee ee ee Re Ge ee ee ee ee 79 BE EE NE ME AO N N N EE N N EEN 49 ENE EE leah NE EE HE A IAE 68 85 ae Ee sea A RE NE OE OE OE AN EK 104 ARE MA MA EE ENE EERS EE EE 104 ae dos EE ON ER TA T 104 Adapted ARE ER EE OER AE ER diia 13 International Rice Genebank iranier eneterreaga aaa a ee ee RR Ge ee ee RR ee 13 NAAK ER ER OE RA OE OR EE RE 79 EE RE EE E E TEE eege 8 11 56 64 68 Prepare RR EO EE EE AE OE EE 64 EE EE EE EE EE 64 dd das OE EE RE EE N 64 AFLPTM Analysis System IVAFLP Small Genome Primer kt 64 jle AE EE EE 7 83 Ndl TEE 7 Akter 13 id EE EE RE N ER N RD AE AS 73 After electrophoresis ii Ere REEDE ER EER DEE BESEER EN GEES planea EP ee ee se s 58 60 64 After pre amplification AAA 64 Gee AR ER ER AE OE N ER see eeas 56 58 64 85 Ge ok EE Bie aa see EE EE OE N EN ta dee 68 85 Ned LE OE RR EE RE OE EE EE EE OE 5 Agricultural Research Service ee ee ee GR AA AA ee Ge AA ee ee RA ee ee ee Re ee nt 10 ede dd RARR EO RE EE EE OE EE AE N 10 47 79 D partrent 7 EE RE EE AO OR EE 47 Federal Department esse esse ee ee ee Ee
64. O AT 85 Board ME RE EG EE GR aac EE 49 TITUS RE SE IE ER ER ab ee into 49 Bagor AE RE ER EE ER ER OE EE 79 Box 9 159 A OM N OE RR OO N EER 79 RR EE RE EE T EE ida OE EG 64 106 BPIEorm 10 ERGE EE RE OER EE 47 Oe ORV GE in Ga N N de A ee ee ee ee EE 47 CT EDGE EA OE ME HE OE EE EE ME RE 7 8 83 ad EER OE EE AR OR NEE AE OR EE EE OR 83 A RE He ee ER PA EERS EE ER EER ar agg been Ee ER ee 7 83 PIEEO DEE EEN 85 British Solomon Association ccccccccccccccccceccececececececeeacacaueceeseueneneueueaeacaeseseeesesecececeeeaeaeaneeseseaeeeess 79 British SOlOMON ISIANGS iii a ERG Ge ee AD ARE Re a GN Ee ge Ee Ge Gee Ge Eed 79 Et el ET EE 74 Bromphenol EE EE EE DE EE ee Re EE sees EE ER EE GE ES Ge ee 60 77 85 ie MI iaa 39 Brownlow MEWS EES OE ti Es Ge eed OE GE EE ee De ie E sna ee GE Bee Deep Ee ER OER Gee ek Seier 31 BRR E EE EE EO LA OE AE 79 SES AR N MA RE NE II DEK LE 79 Brunel Datussalaf Ee EE EE GREG Ge ee aii 79 B tler teen EE ON GE DE GE Ee ee as a gid Ge ee 60 ae EE AR AE N NR N a aaa 60 Buffer System ES ee de a oh i Me ge E 77 Buffer Systemilh 0053 EE ESE a Ae islet ee Ee ee DE Ee ee Eg 77 Buffer System ege hie oe eel de Ee OG Ree 77 Bunds EE GE A A Ge ee 17 del Ti AR MR ME ME OE OE OR 17 Bureau GE A AN Ehe ge a EEN 10 47 Plant Industty RR OR RE ed ee ae a ae OR De de EE NET 10 Quarantine Oe use gereent od ew es Rd PO ea es se ee 47 A ER EE 79 Bvumbwe Agricultural Research Station iss ee ke ee AA Ge AA
65. OE N 58 60 ING UO ER RE EE EE EE OE EE N AAN 56 ee EA BAKE EE OR EE OE OE Gus 49 ERROR AE RR EE ED IE OE N OE EE 17 68 104 Ede OR ER ER AE EE eta te OE 58 NE IN ok RE ia EE EE OE 104 IV Dee lu ET e TEE 68 85 Nigel Ruaraidh Sackville Hamilton iese esse ee ee ee AA Ge AAR Re Ge AA ee ee ee ee ke ee ee de ee ee ee 49 NE AR EE AE OE ORE OE ESE KS 79 Ile E NM N OE MO VOER EE EE 79 IV IC BEE 7 8 83 November MERE SS EG EG EE GE ED SE o ee EE 11 13 Julie EDE EE EE ee ER DE GE EG RD ED dei Ve ge koe 13 MEME EE EE EE OE AE EE RE OE Ee 13 NP EE a Ht essere on ERT AL ES ED A esche 17 9 RS oe 17 122 NSSE ee ee Oe aed cad outa sa wea De GE bd 10 N tetramethylethylenediamine AA 68 IK UE e ME 56 Number ERROR EE EE DO GE bed Gr a Da ee ee eebe 39 LEER EE ER RE Ei 39 O O7 8 13 14 15 17 23 25 27 28 30 35 39 46 47 79 83 characterization 23 EE DEE EE GR ac EED as 39 STAGE i oss cass EE RE EE EE 39 Observed EE 39 ole EE OE N ET EO ER RT EN 56 58 60 64 68 77 85 PIE ENG N AE EE ER EO ER EE EE EE 56 KT BEE 56 ER N EE OR GER EER EE EE OE EE EE RE ETE N 56 SEE RA EE EE OE RT NE a EE ea 77 Oe ER GO dia EO OE ER E 10 13 GER OE N na EE ER eed 10 October 1994 ES GE GE ceded ES ii ee ee ee Ee 5 10 49 etd EER OE NE EO OE OR ae aad OE EE OT ES 39 OfficinallS 000 OE RE EE N OER OO dua eaten ea EO a a 7 8 83 Se EE 28 Operations tachi A ee De aie lee eee tae 5 Manual SEE EE EE Ee daca ssa RE EG EG GE Ee e
66. Stock solution for culture solution Solution Composition A Amonium nitrate NH4NOs dHO volume to Sodium phoshate 2 hydrate NaHsPO 2H20 dHO volume to Potassium sulfate K SO dH O volume to Calcium chloride CaCls HO volume to Magnesium sulfate 7 hydrate MgSO ZH O dHO volume to Manganous chloride 4 hydrate MnCls 4HO Ammonium molybdate 4 hydrate NH4 gMo70244H20 Boric acid H3BO3 Zinc sulfate 7 hydrate ZnSO 4 7H20 Cupric sulfate 5 hydrate CuSO 5H20 Iron chloride 6 hydrate FeCl3 6H20 Citric acid monohydrate Dissolve each separately in 50 ml dHzO in beakers then combine Add concentrated sulfuric acid H2SO dHO volume to Amount 91 4g 1000 ml 40 39 1000 ml 71 49 1000 ml 88 60 g 1000 ml 324g 1000 ml 1 500 g 0 074 y 0 934 g 0 035 g 0 031 g 7 700 y 11 900 g 50 ml 1000 ml Yoshida et al 1976 Laboratory Manual for Physiological Studies of Rice IRRI 98 Table 9 4 Amount of stock solution to take per preparation Stock Solution Amount ml per 4 liter solution per 20 liter per 60 liter solution solution A 5 25 75 B 5 25 75 C 5 25 75 D 5 25 75 E 5 25 75 F 5 25 75 Adjust pH to 5 99 0 0 01 NODS RE RE EE RE OR EE E 77 OUT ER N ER A a OO EA 85 LG AR EE E EE EN 74 D Me cco EE e E gege deet gece 85 OS I EE NE uelutaydnubdnentes 85 SK cate RE EE ED N EN 85 Mo dl EE N EE oO N N 56 58 0 7 tege EE ee ees ee 56 OS ZDNe
67. Tris EDTA Na Boric acid up CO volume to 300 ul 200 ul 40g 75 ml 200 mg 84 8 ml 108 g 939 55 Y 1000 ml 91 TBE buffer 1x TBE buffer 0 5x TBE buffer 10x up volume to TBE buffer 1x upH20 200 ml 1800 ml 1000 ml 1000 ml Table 8 8 Solutions for RAPD Solution Agarose 1 4 dNTP mix 100 mM EtBr staining solution Loading buffer 10x TBE buffer 10x Chemical Composition Agarose TBE 0 5x dATP 100 mM dTTP 100 mM dCTP 100 mM dGTP 100 mM Amount 3 5g 250 ml 100 ul 100 ul 100 ul 100 u Mix together and dispense into aliquot Ethidium bromide upH20 250 mg 50 ml Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved Transfer the solution to a dark bottle and store at room temperature Ficoll EDTA Na 0 2 M Bromphenol blue SUDH O Tris EDTA Na 40g 75 ml 200 mg 84 8 ml 108 g 939 92 Boric acid upH O volume to 55g 1000 ml Table 8 8 con t Solution TBE buffer 1x TBE buffer 0 5x Chemical Composition TBE buffer 10x upH O volume to TBE buffer 1x upH20 Amount 200 ml 1800 ml 1000 ml 1000 ml Table 8 9 Solutions for microsatellite Solution Acrylamide gel solution 6 Acrylamide bis acrylamide 40 APS 10 Chemical Composition Acrylamid bis acrylamide 40 Urea 5x TBE upH 0 volume to
68. Trustees in September 1994 the institute will not seek intellectual property protection on any of the germplasm it holds in trust and provides germplasm on the understanding that a recipient of germplasm from the International Rice Genebank will not take any steps to apply intellectual property rights to these materials Protocol for requesting information 1 Send a request either thru a letter addressed to Dr Nigel Ruaraidh Sackville Hamilton Head Genetic Resources Center IRRI DAPO Box 7777 Metro Manila Philippines 44 b electronic mail to Dr N Ruaraidh Sackville Hamilton r hamilton cgiar org or 2 Access germplasm information directly thru the System wide Information Network for Genetic Resources SINGER website http www cgiar org singer An MTA must also accompany all germplasm given to staff at IRRI 45 Chapter 7 Documentation and Exchange of Germplasm Information Documentation and exchange of germplasm information The data of all the rice germplasm conserved at IRRI are efficiently managed and maintained by an information system known as The International Rice Genebank Collection Information System IRGCIS Background IRGCIS is a comprehensive information system developed jointly by the staff of the Genetic Resources Center GRC and Computer Services CS at IRRI The data are managed in Oracle8 and its application was developed in Oracle Developer2000 System accessibility The system is availa
69. a It has two ecogeographic races indica and japonica including temperate and tropical japonica e Oryza glaberrima African rice that originated in West Africa e Twenty two wild species of rice that are found in Asia Africa Australia and the Americas Only a few are closely related to the cultigens Eleven other genera in the tribe Oryzeae All are distantly related to rice but some species such as Porteresia coarctata which has tolerance to saline conditions are being investigated for their value in rice breeding through the application of biotechnology Further details about the rice species and related genera are given in Tables 1 1 and 1 2 Table 1 1 Taxa in the genus Oryza the species and genome groups Species 2n Genome Distribution Sect Oryza Ser Sativae O barthii 24 AA Sub Saharan Africa O glaberrima 24 AA West Africa O glumaepatula 24 AA South Central America O longistaminata 24 AA Sub Saharan Africa O meridionalis 24 AA Tropical Australia O nivara 24 AA Tropical Subtropical Asia O rufipogon 24 AA Tropical Subtropical Asia Tropical Australia O sativa 24 AA Worldwide Ser Latifoliae O alta 48 CCDD South Central America O eichingeri 24 CC South Asia East Africa O grandiglumis 48 CCDD South Central America O latifolia 48 CCDD South Central America O minuta 48 BBCC Philippines Papua New Guinea O officinalis 24 48 CC BBCC Tropical Subtropical Asia O punctata 24 48 BB B
70. a SE cea Ee cae GE os is 39 METERS EE EE ed at ER EE EE AE OR ER EE An 79 Wetter 7 83 Verified DE SEG EE EDE AL SEER EE EE EG baal meet N DE Actes re eae ts Fok ee Ge ee 39 Viabuityai a a EE EE EE De e E 27 Vitro Germination ss EE EI EE Ee ED EE e De EE alt 73 tee En EE 17 VOlA0 ED RE RE ED IG Tee eee nearer rl 64 Me GEGEE EE ME RE N EE N EO NE EN 58 Me MT RE EE N N RE EE OK EE 60 WwW W31 60 68 LO AA ER a ee atin ved Bea 56 Wanscher 2a Sete Sec Nee ie Ne NE EE EE EE N OE N 39 MEE EE EE ME N RE OON 56 DNA estate reien EE ER RE laa 56 WAT RE EE OE EE RE OR AE EE ETD 74 EE Sear a dni EEN 74 Web page ER OR N OE EA ED OE 52 ME es RE N OE OE RE DE N EE EE ts 49 TR EER SE EE EE RE EE EE OE ED 17 132 VO PUIG i cs fe EE RT A RE OE eee 56 58 MEDEDELING 56 DONADOS iii EE OR N RE EE IE 58 Welcome ER EG RE RR GO EE bois 5 UV rt CR EE 31 West Africa EE ER EE 7 83 Whatman E EE EE EE ote 77 Whatman NO EE EG RO Ge EE a a e De ED ads 77 ae OE AE EE ON EO OE LE RE ET AE ON 77 UU UR MERK OAR RO EE AE MOE AS 39 lies N BE EE N N EE HE EE OE 35 46 Winadmlll Lane SE eege eege Ee oe dias ee 31 lee BAAT ER EE 52 JC EE SE EE GE N EE EE case 52 RR ee EE OE OR ON EE ME OE EE NA 13 WWW EE whe vs ah ec A A td 52 X Xylene cynole EF ige es GE i en ede Se Ee ke Ge ie vee i ge Ee De en Ee ME De ee eden 85 Y Yellowish Greeff seks tra eties eb Ee be Eie ee Dee gee Vee ee Gee eboek ralla dador 39 Yokohama Plant Protection Gtation nonononnononono
71. al N N OE NEE EA RE AL 85 Malate dehydrogenase AAA 68 85 EE EE sete EE ad aed ae oie ke Rootes te cet sheet cee St ta 79 EES N EE AE AE AA AE EE AA rect 14 79 Malatesta EN ML IE EE ETE 85 Malel s eie DE ot RE eege dE EE 85 MEER EO N TR EE OR EE nae es 79 Ela IE EE ME NE N IE AO EE EN AE Ed 68 85 Maltebrunia casa GR EE EE aa basa a RE Chat SO EE Re ee Ge DE Ge Ge ee bras 7 83 MERE RE EE OE OO EO OE A OR EE EE N 52 characterizatiOn ES arias 52 e DIE SERE EE OE OR OE OE OR EE ER EE rere 52 Manchester M34 de EE RE OR EA EE EG 31 MANQANOUS vise RE EE a 104 CET Ee Ee ee dela ais US Ge Ge Ge ee Se N ee de Ge ee ee 5 ed ME ARE AO OE ER a oe EE N EN 5 Manual VE WO e T TEE 47 Material Transfer Agreement 47 49 dii EE AE NE N ER oa Paco ua gh N A N NR ENE 47 Materials ES N De A A EE 13 39 Sele tiOn Ee ee GE Ge a de ee 39 EC EE 79 May November EE vis cae cae sai ata ene Ee a oe e Ge sad de en ee ee Se ee 13 oe EN EE OR EE EE EE EE EE IE N 28 MIB 20 205 ES ES a E Ge ee 79 de AA EE da ER N OE AN ER ees 68 85 ME A AEEA TOTE A a e ibas 68 EET RE N ER N EE NE OE OE N EE 39 Measure TR RR EE dde dela 39 Mechanical Engg RE EE N ME EE EG 31 Mercaptoethannol ER ER e RE N EE EE NE EE Dieter 77 UE e ei OO EO EE EO EE rea 7 8 83 Ee ale AE RE N N EO A EK ENE N 83 MeSoOphytlC EE OR EE ER N OER 35 ET ER AE N OE N OR OE OE EE EE 39 Methuen Handbook seni SE DE da ica Ee Dee Ee ER EE Ga A GER ra BA ee be ee sd EE 39 GOMQUES EE ER GE de ee
72. appear and rinse 1 5 KI solution Pour on to the gel Bands appear as white on a blue background Score quickly because they disappear after a few minutes N a benzoyl DL arginine 5 mg ml B naphthylamide Fast black K salt Tris maleate buffer 0 2 M pH 3 3 Incubate 20 min at 50 C a naphthyl acetate B naphthyl acetate in acetone Phosphate buffer 0 1 M 5 ml 15 mg 45 ml 50 ml 50 ml 5 ml 15 mg 45 ml 1 mil 50 ml 79 pH 6 5 Fast garnet GBC salt Incubate at 50 C until first band appears Remove from oven and wait for the other bands to appear 15 mg Table 8 2 con t Enzyme Glutamate oxaloacetate Transaminase Got Isocitrate dehydrogenase Icd Leucine Stain Composition Amount DL aspartic acid a ketoglutaric acid Tris HCI buffer 0 5 M pH 8 5 Pyridoxal 5 phosphate 1 mg mI Distilled water Just before use add Fast Blue BB salt 100 mg 50 mg 20 ml 1 ml 36 ml 40 mg Protect from light Incubate 15 min at 50 C DL isocitric acid Tris HCI buffer 0 5 M pH 8 5 NADP 5 mg ml Distilled water Just before use add MTT 10 mg mI PMS 1 mg ml 100 mgml 1 ml 10 ml 1 ml 36 ml 1 mi 1 mi Protect from light Incubate for 10 min at 50 C L leucyl B naphthylamide 5 mg ml 5 ml 80 aminopeptidase Lap Fast black K salt 15 mg Tris maleate buffer 0
73. binet in the culture room until the shoot and root emergence Inspect the tubes occasionally to find out if the seeds have germinated Transfer the tubes with seedlings into the lighted shelves in the culture room The 65 shelves should have a 12 12 h light and dark cycle 3 Let the seedlings grow until they are about 8 cm long These are now ready for transferring to the culture solution 4 Label each hole of a 9 x 12 styropor with the accession number of the seedlings 5 Put the styropor in an 11 x 14 tray filled with distilled water 6 Remove each seedling by scooping out the agar media with a spatula 7 Wash the roots of the seedling with distilled water in a tray to remove all the agar media adhering to it Do this very carefully to avoid damaging the roots 8 Sandwich the lower portion of the stem 1 cm from the base of each seedling with a 0 5 x 2 foam Insert the foam with the seedling into the corresponding hole of the styropor 9 Transport the seedlings to the Phytotron Replace the distilled water in the tray with culture solution see Table 9 4 for culture solution preparation and Table 9 3 for the stock solution Allow the seedling to grow inside the glass chamber 21 29 C 70 RH or an indoor growth cabinet 21 29 C 70 RH 12 h light at 900 m 2s 2 for 2 3 weeks or until the seedlings are at the 2 3 tiller stage 10 Maintain the pH of the culture solution daily It should remain at pH 5
74. ble in a client server environment Oracle client software is installed in the workstation The system is accessible to genebank staff for their daily activities Major data i e Passport morpho agronomic and evaluation data of the entire collection are accessible to germplasm users thru IRRI Intranet using either of the following web page address http genebank irri cgiar org 81 irrinome irgcishome html http grcsvr4 irgc main htm A data subset of all the accessions in the collection is also accessible through The System wide Information Network for Genetic Resources SINGER a system that provides access to CGIAR center germplasm databases through the Internet on the World Wide Web WWW The webpage address is http www cgiar org singer System objectives IRGCIS is designed to manage the genebank operations more efficiently It aims to e Assist the genebank staff in day to day activities e Facilitate data recording storage and maintenance of germplasm data e Allows to request the desired seeds and provide direct access to information pertaining to accessions in the genebank such as 1 Passport data refer to accession ID and the data recorded when the sample was originally collected i e accession number scientific name cultivar name collecting number collecting date name of collector and place and date of collection etc 2 Morpho agronomic data refer to the morphological and agronomic description of an a
75. bt ae 56 2 6 MIN SE EE eae caine A a GE os EE ED Aen titel tnt ini ait adele ol teal te 60 UPH 20 EE ion EE 60 ER EE EE EER DEE EE IE ti 74 C 74 dissolve ES e a Ee ie De EE ee Ee Re aR oe 74 EL RE RR OO NA N OE ege eg N a aA 74 27 28CD EE ee ate A 39 28 29ABC7 O EE ii ae et a ete ae ata tari 39 AOS aii ME ER ET NE 39 Ee TE EE cet OR EE IE RE OE N EE 39 ele 104 ER ED EE N MERE EE AR RE RE EE A HE MR 35 QM Aare a EE LE OE EO ER N EE EN 7 83 Mol EIE ER OE EE ME AA 17 3 39 E lt EE EE ah DE Ee e N e tual ee 58 RE ER EE RE OO OR EE EO OE 58 30 O EE N EE 39 OG AE E EE des acts EE Eeer 56 ee EE EA EE RE OE LE EE EER EE nad 56 DEE 56 30 0 0 ale EE 17 EE 39 BOME AA Ve e Maen tas SE ME Ee ER EG EE EE eta 17 RR N ae EE EE EE DE OE EE OE 17 ERA RE EE EO ORR EE EL OE OR 39 101 3 5 C 73 ER AR N EI en See ea ele eee 39 A EE EE EE OR OE EE RE Id EE OE AD 39 3 amino ethyl carbazole ie ee ieee iaaa AR Re AA ee ee AA ee Re ee Re AA ee ee AA ee ee ee ee ee ge ee ee 85 ee RARR EO RE OR EE EE EL EL OE REEN 56 ea EE OE EE EL EE EE EO EA OE EED HEN 85 4 4 5 dimethylthiazol 2 yl ee ee ee ee Ee ee ee ee Ee Re ee ee ee Re ee ee ee ee Ee Re ee ee ee ee ee Re ee ee 68 85 ASC is EE EN EE A OR EO ER EE EE 85 ER OE RE LO RE ER ER IE 77 45 oe EE EE EE ER RE ER EO AE 74 46 60 48 C14 El elie EE EE OR EE ee a EE OR ER 77 ie RA AE OE A A AA 104 5 50 ee N RR ER EE N N OE erch 77 EE dit 77 ie NN 77 DO MM EE ER EE
76. cceptable limit Generate data sheets see Appendix 3 2 Pre clean the seeds by blowing in a ventilated column to separate unfilled grains and light density materials Verify again using the seedfile Determine the selection to be done based on the recommendation during the verification process and the current storage status Examine the seeds and hand sieve with graded mesh sizes if mixtures off types vary in size to separate slender and bold grains Remove discolored deformed infected soiled immature damaged seeds and off types Determine the actual action to be taken based on the quantity of clean seed This will determine the packing system to use Prepare and label all the necessary envelopes for use in seed testing viability testing temporary storage and final drying to minimize labeling errors Submit the selected samples together with the seed file pre labeled envelopes and the original seed container for double checking and quality control Check the selected sample against the seed file and the pre labeled envelopes against the original container Mix the selected samples and divide using the pre labeled envelopes as follows a For Active and Base Collections gt 120 g O sativa and O glaberrima 2 x 100 grain samples for viability testing 2 x 200 grain samples for seed health evaluation 1 or 2 x 60 g sample for Base Collection 2 to 5x 10 g samples for pre packing 1 x 20 g sample for duplicate sto
77. ccession Examples panicle length seedcoat color maturity awn color leaf blade color etc 3 Evaluation data information on the reaction of an accession to different insect pests e g stemborer leafhopper and diseases e g Blast bacterial blight and physio chemical stresses e g drought flood etc It also includes data on grain quality e g amylose and protein contents etc 46 System menu structure IRGCIS has 5 main menu options see Appendix 7 1 1 Acquisition handles the activities related to the acquisition of samples and manages the passport data It covers the registration of samples assigning of temporary Ids until samples are assigned IRGC accession number for inclusion to world collection Multiplication handles the activities related to seed multiplication rejuvenation Characterization handles the activities related to the characterization of the morpho agronomic traits and manages the characterization data Seed management handles the activities related to the management of seeds in the storage and distribution of seeds to the users It manages the seed data pertaining to seed stocks and viability duplicate storage in USA and seed request and dispatch System administration maintains the common files used by different modules It provides system tools such as granting access to selected menu options and monitoring of user s log in and data processes Features and capabilities
78. ccessions this trait can be measured at post harvest Distance from panicle base to 1st spikelet insertion Actual measurements in mm from 5 samples taken when panicle has fully exserted Awning The awning character is recorded after full flowering as 0 absent 1 short and partly awned 5 short and fully awned 7 long and partly awned 9 long and fully awned and 999 mixture Awn color The color of awns is recorded at flowering as 000 awnless 020 straw 040 gold 052 brown tawny 070 red 080 purple 100 black and 999 mixture Awn length Exact measurements in mm from 10 spikelet samples Apiculus color Apiculus color is classified at flowering and at maturity into 8 classes 010 white 020 straw 052 brown or tawny 070 red 071 red apex 080 purple 087 purple apex 100 black and 999 mixture Sterile lemma color When the terminal spikelets are approaching maturity the color of sterile lemmas is classified into five classes 020 straw 040 gold 070 red 080 purple and 999 mixture Lemma and palea color For the wild species data are taken at early reproductive stage because it turns black at maturity Eleven colors are recognized 012 green striped white 044 green striped gold 053 brown spots 054 brown furrows 056 blackish brown 060 green 062 yellowish green 083 purple shade 090 purple spots 100 black and 999 mixture For the O sati
79. ccumulated samples together with the temporary list to the Seed Health Unit which will handle the preparation and shipping Provide the final list of the accessions to be sent Include 10 to 12 control samples for viability testing prepared as follows a Separate 10 to 12 representative samples in each crop year b Take the initial viability percentage following the appropriate guidelines c Prepare 12 x 100 grain samples for each accession and seal in labeled aluminum packets Prepare the packing list and the necessary documents Request the phytosanitary certificate Coordinate with the Shipping Section for the export permit and shipping 10 Update the data files Maintenance Maintenance is necessary to keep everything in shape at any one time This should be done through frequent monitoring and coordination Germplasm Unless monitoring of seed viability and amount is done the genetic stability of a certain sample cannot be ascertained Monitoring is necessary to determine the amount of seeds in store at any given time and if the stored seeds will germinate to produce new plants This is necessary to decide whether or not to regenerate the sample Seeds should be monitored at the start of the storage period and at regular intervals during storage Viability monitoring 1 2 3 4 5 Determine which accessions require monitoring based on the last germination date performed initial germination variety g
80. cia ER EE AE RE EE OR N 14 39 Lu EE 76 Appendices EE 39 O SE EE EN EE EE EE EE N 28 49 52 74 Le ue DER RE KO ER AENA IE ieee 23 25 Lee ue ir EE EE EO EE AE EO rt e ER leer 23 25 Append TN 27 APRON GIN RE AE OE EE EE AE EO RE EK 27 Lee DEL EE snare EO AE EO RO EE 30 O MERE EE OE AE OE OE OE NE GR AE ORE NR N 47 Apply rell Ueleg ER N EE RE OE OE OE EE EE EE EO ee 17 AR ER EE LE EE ON OE EE EED 60 68 85 ole EE RE EE EE ME OE N AE OE EE AE 74 PRA RE AE A EE EE EE EER 68 85 Areas Isoladas Norte Pargue Rural ees se ee ee Re ee ee Re ee Re ee Ge Re ee ee ee ee Re ee ee ke ee Ge ee ee 79 Arginine aminopeptidase AA 85 elk iS ER OE EE N EE EE EE RA N 54 SE RR AA E 7 83 E AA RE EE HE EE GEE EE RE EE OE EE EE 7 LR ORE HE EE OE EE OE OE OE EE N 39 ASSON WEE 47 53 ELC Peete OE EE HE EG ORE AE ee 53 RECI RE EER GE Ie e T a 47 Ee Il LEE 14 elle TV DEE 14 Assisi SEA o a aa ua nee Tea eth EE BEDE ed 52 53 GSM SANK a 52 53 105 PTL cscs ees ER ER ER eco N uae ted IE ENS OE HD EN N 79 Auricleless EE Meed oe WEE GO Re ee sa nani sae data sa Ee oe ee ke E 39 Auricles ia SE EE E Ee ee EE ee 39 AUS ME ER EE rata 7 79 83 Australian Plant Quarantine Office uie ese ee see ee ee RR RE ER EE EE Ee Re ee ee ee ee ee ee ee ee ee Ee ee ee ee ee ee ee ee ee 79 le lo AEI EE 79 TE d ENEE 7 83 id Ee AS N OR saa gra eege OE ais 7 8 83 Atithorized Ehannel ii N Ee EN EG AE ee tad 79 B B 14 17 23 25 30 64 104 BES a 85 Ee te Ire UU
81. creer errr errr 58 85 kele RE RE EE belt ds 60 104 OO dada 104 heated ER EE AE EE OR AE AE AE Ota fod Sede Ed 60 500 Mi ET AE ass til dada la atados 85 A ET EE OE eaves 85 A EE 10 5 10 SERE RE EE EO OE OE ER la dal 77 KERE EE AE EE EE EE EE EE 39 BOOG ese EA N EE E EE EN GE OE ER Ed 56 EE AE ER EE EO EER EO EE 56 AE ARE MA HE RE EE EE ORE e EN ss 39 DOr EER AE HE OR NE EE OE He OE EE EE 39 ee ele AE EE AE OE ER EE OR OE EE Ed 104 TR RE EE EE EO DA 39 AE RE EA EE EE EE EE EE OR HE EE DR 85 URE EE EER EER EE RR EE AA ME EE Ehe 60 De EE AT AE EER Ee dee 64 BX E EE EE EE LR EA ER EE T 85 6 60 IR EE N EE EE EO N ER AE 85 600C AR EE N ON 58 nos AE EE EE OE EE OE OE en EE Ee 58 COO Cites eases cate neon RD EE 85 el ER HA OE MO RE EE NE 85 102 AE ER as ates ces EE OE N OR AE Lees ag cir 39 AA RR EE EER EE EE OR OR N 39 BEE geed EE ME EIE EE EE OE OE 39 eel GE AO EE EE ER EE Eege Ee dE AE EE 85 104 BMO7O244H2O EE AE RE EE AR EE EE EE 104 7 70 Add o 56 72 60 TO KERE EA EE AO OE EE AE OE MED tec aut 85 Ee LR MO N EE ER RO OE EE N 68 104 8 BL Oger RE eege a a a Eed eg 77 CONTRO cect at singe EE EE OR ER ethic ER N dat 77 S0GB DL Etape Ee geess so Ee IE A eG ee n Aer Ag Ee 54 ele EE EE N EE EE bat cht 56 85 St let selle UR EE EE EE 74 9 9 73 GADABT O RR deed E OE N EE AE EE EE EE 39 A EE RE EA EE OE EK OE EE OE RD 39 AR RE EO RR EE OR IE HE ER ER N 39 ARE EE EA EE EE WAN EE EE OE A 7 83 EIS EE EE ON E 39 EER RA ME EO ON
82. d gt 2 cm For the wild species actual measurements in cm 37 are taken from 5 leaf samples Ligule length Actual measurements in mm from 5 samples Ligule pubescence Based on ocular inspection using hand lens and classified as 1 glabrous 2 hirsute in specific places 3 generally hirsute and 999 mixture Variety group Classification of accessions into variety groups is based mainly on the morphological features of the adult plant and to a certain extent on grain appearance Four major groups are recognized 1 indica 2 sinica japonica 3 javanica and 4 intermediates hybrids Since the variation in morphological features among varieties is continuous it is sometimes difficult to make an accurate classification Population Six types are observed 1 homogeneous 2 mixed with other glaberrima plants 3 mixed with sativa plants 4 mixed with other glaberrima sativa plants 5 mixed with barthii plants 6 mixed with other glaberrima sativa and barthii plants Harvest Postharvest stage Rhizome and stolon formation Observed when the plants are ready for harvest Six classes are recognized 1 vegetative crown 2 vegetative crown and stolon 3 vegetative crown and weak rhizomes 4 vegetative crown stolon and weak rhizomes 5 strong rhizomes and no tubers 6 strong rhizomes with tubers and 999 mixture Leaf senescence 1 9 999 The leaves below the flagleaf are observed at the time of
83. d This line is the tracking dye that moved because of the electric current and the table below describes these respective lines for each buffer system Tracking Dye Line Characteristics After 4 hour Run Buffer System Line Characteristics 3 Diffused about 2 cm thick Solid about 1 mm thin Diffused about 2 cm thick Make a second cut across the gel slab s width 9 cm from the origin The area between this cut and the origin is the gel slice where the isozyme bands can be 70 visualized after staining Trim off the upper right corner of the gel slice to orient it Carefully detach this slice from the gel mold tray by running water between it and the tray s bottom The rest of the slab may be discarded Do this for the 2 other gels 4 Place this slice on a slicing bed Draw a thin steel wire through the gel s thickness to come up with 1 mm thin slices for scoring Transfer these scoring slices to their appropriate plastic staining trays making sure their trimmed corners are oriented in the upper right sides Do this for the 2 other gels Each enzyme will have a distinct staining procedure Refer to the recipes see Table 8 2 for each one 5 Incubate the scoring slices in an oven Incubating temperatures will depend upon the enzyme Scoring 1 When the isozyme bands are visible in each scoring slice discard the stain in the staining tray by pouring it into a plastic bag for hazardous waste Carefully wash the
84. delible ink e Distribute and check the tags at least one day before pulling Keep the tags of non viable accessions for counter checking later e Flood the seedbed the higher the water level the better but only to 3 5 cm above the bed This will facilitate pulling and washing off of soil from the roots Pull the seedlings carefully and tie them with the wire of the tags One bundle represents an accession Note plant stand to decide what further action is needed on a particular accession After pulling arrange the seedlings in ascending plot numbers in seedling trays Separate accessions with insufficient seedlings from the batch For insufficient seedlings that can be accommodated in 1 2 row field plots transplant on the best part of the field Entries with less than 25 seedlings will be planted in concrete beds in the nursery area Number the seedling trays This will aid in the distribution of seedlings in the field Field activities Land preparation Standard land preparation procedures for lowland rice farming O sativa and upland culture O glaberrima are followed Land preparation consists of one plowing and 2 3 passes of harrow or rotovator The final stage of land preparation is leveling for lowland culture and furrowing for upland culture Field layout Plant spacing depends on the nursery being planted The number of accessions to cover a given area can be computed based on the desired spacing and the number of ro
85. dle like leaf samples from 40 day old seedlings can also be used Put these into their corresponding depressions in the spot plate Cut the leaves into small pieces and add 2 drops of ice cold distilled water or 2 drops of 0 01 mercaptoethanol Mercaptoethanol can prevent oxidation of samples Macerate each leaf sample with a glass rod to produce a crude extract After each maceration be sure to rinse the glass rod tip with distilled water and dry it 69 Loading the Crude Extract Te Put 6 small strips of Whatman No 3 filter paper 2 pieces 15 x 5 mm per strip for System 2 pieces 12 x 5 mm per strip for System Il and 2 pieces 10 x 5 mm per strip for System III in each depression of the spot plate and allow the crude extract to be absorbed Prepare the gel insertion points in each gel mold by first pulling back the plastic wrapping film from the cathodal end Then using a sharp scalpel make a slit across the gel s width 5 0 cm from the cathodal end This slit is called the origin Be sure to cut gently to avoid damaging the gel and the mold s bottom Also do this to the 2 other gels With a Pasteur pipette line the origin with 1 bromphenol blue to serve as tracking dye To load the crude extract samples use a pair of forceps to take 2 strips of filter paper from a depression in the spot plate Blot these on a paper towel to remove excess crude extract This will prevent contamination of neighboring lanes in the gel mold d
86. e 79 een AR AR EE OE EE AE EE AR ees 14 A RE ME OE EE N AE EE AE OE EN 39 47 64 GE gel el 64 Head EER EE EE De vue De DE ER ER ISL Ge EG DR iio 47 SO CUO O NO 39 Reierence si EER RE occa se tanta Seas ee Pegaso oad cade EE De asl T gv Shea E ed Es 39 BEUR OE EE OO OE OE EE RA EK 31 Refrigeration 8 Airconditioning AA 31 Registered OF 2 2335 a AR OR OE EE AE IE ON RR RE EK 46 SIE Eu e Le EE 14 17 ad LULL Beep acer OE OE EE EE ENE OER OER eer erect 64 ER Ad AO AE EE OD EE EER N 64 Remove E AE AE NE Wee Hc A GE EG Ge ee N EG 58 60 64 73 Se 60 POR EE 58 64 tube f Re A dee EE ee 73 RE UE cain hii ete OE RR eee le ee ee ae 30 47 germplasm RE ER EE GE EES Ee eee ee 47 A euer Ee Valea aa ada ath alsa ENEE Eer 47 phytosanitary EE 30 AT UTC 47 Import Permit RA ieee ede A ee N 47 Research Genetics ii A ER Re a i ND 60 Elia el die EE OE EE alterne 64 la AE EE IE EE OE EE EE T RE IG 47 GIG aes N RE N IE EE OR ER N N N ET N RE E 47 PRM RE OE RR HE EE OE RE EE ES EN 8 23 25 28 31 73 also El EO OR EE OE AE ER EE RR EE EA 7 8 83 ld led ii AS RE ER N 7 83 RhynchoryZa Subullata RE OE EN 8 83 RIDONUCGIGAS AE EE AE OE ER N OE RE NE OE N NG 85 lei 5 8 23 25 28 30 35 39 47 104 Rice genetic El EE 7 CONMSERVAUIOND ios RE EE RE EE N er Eed eg 7 Rice ds IERE OE RE RE EE RE ER EN 60 sil EENDE ET RE EE OE NE O ER Dieaai 7 83 Hudder eebe ade ced tect ed Ge ci sass ee ee EE A Re De ee ee 7 8 35 83 EO NE N AE EO EE EE EO EE N ee 56
87. e airlock to ensure door closing in this system the outer door cannot be opened unless the inner door is closed and vice versa and to control the flow of warm air into the cold stores 6 Installation of plastic curtains to minimize flow of warm air 7 Inthe drying room an alarm signal is connected to the RH and temperature sensors Beyond the limit the machine will shut off 8 Inthe Base Collection a red light signals that the door is open 9 Provision of a standby generator specifically for the genebank aside from the generator serving the whole IRRI A strong coordination with the Physical Plant Services Unit is necessary especially the Refrigeration and Air Conditioning Section Contact Physical Plant Services Unit Person in charge IRRI Tel Refrigeration amp Mr Alfredo Mazaredo 6804 Airconditioning Mechanical Eng g amp Maintenance Electrical Eng g amp Maintenance Instrument Repair Civil Eng g amp Maintenance Mr Enrique delos 6803 Reyes Table 3 1 Seed viability monitoring schedule for the cultivated species Number of years interval Indica O glaberrima Japonica Active Base Active Base Viability Collection Collection Collection Collection 85 89 3 5 90 95 5 7 4 6 96 100 7 10 5 7 29 1 Mew T W and J K Misra 1994 Manual on Seed Health Testing IRRI 113p 2 Wall 50 50 19 Continental Can International Corp 1200 West 76th St Chicago Illinois 60620 Cable Contican Chicag
88. e ee Gee ee Ge De de ee 5 ORACLE ES fact er oe A A ee AT GE Ge ve toe ee ee ee da ed 11 Oracle Developer2000 ir EE etn te agin Ge EED ee Ee Ke ER Pe tte age os Re ede 52 OPAC OS is ee EG EE EE Gee ee De Ee A ES Ee ve ee NEG ee Ee ee 52 els AR OE EE OR EE N EE OE DO EE N 74 Orya AE ER EO RE hance 7 35 73 83 Oryza Re LEE OU EE H Oryza SATIVA E EEER 7 13 73 Seed multiplication EE Ed San ei a Ae at et eds 13 dl RE 85 OZ de EE EL EE OR N hace lanes etario 7 8 83 OutCrOSSINd EE ER EE ES A EH 17 35 Oxaloacetate transaminase eie ee ee ee ee ee Re Re Re Re ee ee ee ee Re ee Re Re ee ee ee ee ee Re ee Re ee ee ee 68 P P 17 79 POS Ee DAD ED EE ED EE RE DE ER DE de DA pana eie SA og 79 Pack AE EE EE EN EE OE EE EK 30 Peay SOM AA ER RE OR EER AE OO ER et ate 17 dl TE EE 7 83 PAKISTAN i ae ee ee EE 79 Palea EE E R TAATA DE oul ER o e 14 39 Panicle exserti0n ed e Ge ee ade 39 Paniclethreshabllif y is EERS EER ERROR EE DER acted GEREG Re Me ge ke dee EE Res ERG EG ke sydd ee ee ee ee 39 Papua New Guinea 7 79 83 Parafilm ss ES EG EE Ge EO ED REG Ge coi cas es ee dE dn DE GR Ee de 56 le le EE EE OD MR EE EE EE ED EE EE GE ED ER DE GE ie 56 Passport morpho agronomic eie ee ee ee Ee ER ee ee ee ee Ee Re Re ee ee Ee ee ee ee ee ee ee ee ee ee ee ee Ee ee ee ee 52 Pasteur EE RE sac a E Aa 56 77 ER TR EE ER ad 56 58 60 64 68 123 Cl ul lt RE pretest dav TE ia thew set waa OE IE EE N 58 64 Pts ER AO ER RE
89. e ee ee ee 11 International Rice Germplasm Center 8 EI OT KA EE HE EA OE AL RE OE N 8 International Traffic OfiGe ie EE FEE eg ee se ad ee Ee ee GE Be dee ee Ge ee ee 79 INTERNET AE EE EE N AE METE OE OE OR E ON N 11 52 World Wide EE OE ER LE RE N N 52 AITO e o p RE ada DE EE EE EE N 23 39 Germplasm characterization AAA 39 Seed conservati M EE 23 ae ER OE E A ET 60 68 BURN EE 60 AREA RE EE 60 dle MERE EE HE OE OE RE GEE N 60 EA NN 60 delta EA dia NE OE 60 CC ue ER ER EE EE OO EE NEE EE N AE AI 60 ld RR EN AE EE EE EE EE RE EE N 17 IRCGIS User s Manual ee ee ee RA EA ee Ge AR AA ee ee ee AA ee RA ee ee 53 It esse ase AE AE EE RE DU N RE EE AN 53 EES ell ER OE N EE EE EE OO OE AE OE EER 53 stele RE DE ER NE EE RR N ER VEG 8 39 47 52 ASSIGN sss EE eck ED EE DER N GE Ee aes RR RE ee Ee EE DE ee DE nes EE ee DEEG Be le 47 IRGC Information System see ee ee AA Re GR Ge AA Ge Re ee RA ek AA Ge RR ee ee AA ee ee 47 Jee 11 14 47 52 53 54 IRGCIS Users Manual ee ee RA Ge EA ee AA ee AA ee ee ee ee AA ee ee AA ee ee AA ee ee 53 ANAM ED OE ER AL OE LE AE RE EL OE DE ets 7 83 A ED EE ER N RE ee RE 5 7 8 10 11 13 17 30 31 47 49 52 79 104 A A ER EE ER EE RE EE ER OE OE OE ESE iEa 47 116 LISE RE RE ER ER DE eer KIE N 15 17 35 IRRI Experimental Station ee ee ee ee ee Ge Ge AA Ge AR Re ee ee ee AA Ge de RR ee ee AA ee ee 13 IRR genebank AE EE EE DE EK EE OE OE 10 Il EE ER EN N RE EE EDE 85 IRRI Seed Health Unit Ee
90. e ee ee ee ee ee RR RR RR RR Re 65 Conservation de va anes cactus EE ER EE RA N ORE ER 65 o AP o e e 65 a ele o o o PO 66 DEN a a AAA A A eae dea 67 APPENDIGES i n 68 ISOZYME PROTOCOL ci iaa 69 IMPORT PERMIT sesse ass sake ken ea RRE NEE EKEN GR EE RER KERE RAK GE RENE Ran EK ER RAN AK GER RE ER RAK EE RR RAK KEEN 72 THE DIRECTOR GENERAL C O IRRI esse ek EE RENE GR ERG R RR RARR RR EE EE RENE R ER GER ARENA ER 72 DIRECTOR OF AGRICULTURE eens kk REENEN GRANE REKEN ER GEK AR AK GRANE RE RAK AK GEK AR EK ER RAK AAR KEN 72 IMPORT PERMIT sesse es es sa seke kaak noc EER RR ER GR GEKEER ERKEN AR GR NEE GRA EK EKEN EK GER RE ER RAK EE RR RAK KEEN 72 BRITISH SOLOMON ASSOCIATION seent KEREN ERKENNEN RAK KA RARR AR RAK EK AR RAK EK BEK AR AK AR Ra 72 THE DIRECTOR TTT 73 JALAN MERDEKA 147 ess KREE nana R REKE RARR REK ER RARR 73 BOGOR 16111 sesse REK ER KERE ER REK AR ARE R AR RAK ARE KERR ER AR RAK ER AR RAK ERA RAK KAR RAK ER AR RARR BEK AR EK AR RAK RA AR Ra 73 THE DIRECTOR TTT 73 PLANT QUARANTINE STATION sesse sakke KERR ERKEN KEREN KEREN EKEN ER HEER AK GR KEN ENKER KEREN EN 73 Usel IR 73 Ge Re Re Ge Ge KEE KREE KENE NK ER 74 INTERNATIONAL TRAFFIC OFFICE osse ea ana sakke Ra R KERR N KERR ANS nen RAN SKEER ARE KERR AR EK GER RENE KEN 74 DIREC RECURSOS MATERIALES ees sak aka KEREN RR ERGE RARR GEK RAN AR KEREN GR RARR GEK AR ER AR RAKA 74 PLANT QUARANTINE SERVICE os sesse sakke k Raka RER EE cnn nen RARR ER RANGE KERE RAK GR KEN ENKER KEREN EN 74 FED
91. e from 1500 6000 seeds The need to preserve the genetic structure of an original sample should be considered in determining the frequency of regeneration It is ideal to store original seeds or produce them with the least number of multiplication cycles because this prevents or minimizes the loss of less adapted or susceptible germplasm samples The following guidelines have been adopted for the International Rice Genebank 1 At IRRI Los Ba os Philippines 14 N 121 E alt 21 m there are two distinct seasons the wet season WS lasting from June to November and the dry season DS lasting from November to May Seed multiplication for long term conservation is undertaken only in the DS During this season fewer pest and diseases problems are observed relative humidity at harvest time is lower and pre harvest germination of weak or non dormant accessions is zero to minimum 2 Growing diverse germplasm in a single environment presents some problems with regard to some cultural practices Therefore it is advisable to choose fields with a good irrigation and drainage system during site area selection 3 Time of planting plays an important role in seed multiplication Factors we have considered are 1 Photosensitive materials are sown earlier preferably during the first or second week of October so that the late vegetative stage coincides with the short days of the year around December to February in Los Bafos Short days induce
92. e genetic resources AA 7 Conservation support 73 Conservation Support Laboratory iese ee ee ee RR ek AA Ge ee ee ee Re ee ee AA Re ee ee ke ee 73 ere EA EE AA ME EO HERE EE 17 seedille EE DE BEE N cet AE EE RE EE N EE ate EE ED Ge ei GE not 17 BRA ER ME EE EE ART ER OE EE aa 104 Gontaining EE 28 56 A ME DA ED EE ES RE RE DE ED GE ES ED Coke ioe tea hoes ee 28 BL WEE 56 Gontican Chicago EE EE 31 Continental Can International ie ee ee RR AA AA AA ee RA AA AA ee AR Re ee RA ee ee ee ee Re ee 31 108 BO OC i bat NR EO OE EE NE A A eee nee NG 77 Convention on Biological Diversity ie ee RA Ge ee Ge AA ee ee RR narrar 5 GOOI EE EE EE Ee laa ca son OE RE NICO 58 104 ORE ER EE RE OE OE EE EE 104 Me EA EE RE AA N EE N IE oe 58 Cooperative DEPAGRI IRRI Program 79 Seed AE RE EE ET N ee eee estes 79 COPY AR ERA EE EA HO EE EE EE N 46 E EER ER ER AE OE EE OR OE RE EE ER ER DE N 46 A AR AA EE OE E 39 ed ME EE EE ON EE OR AA OE sas 31 COSA RICT AR AE OE KO EE ER OE EE EED 79 COST CMICIOM CY RR AE EA EO OE DA 13 CONWY AE EE EE DR EO OE EE AE ee 39 Ouere EE N OR RE EN 73 oe EE ER ME KM EO N NE ees 74 ARE OR EA AR REEN ER IE EE 79 Crop Year hte ene EE EE N Sel Ae a eee 47 ode as hele a EE KO dle Ave vane ee N N 53 Cross pollination 2 Ee ER Ma ele aa a ies ee ee 17 od ME AAR EO NEE ee eae 77 Loading eege Ale EE de a ee vee Ee VER ge ee ge vee SEENEN 77 Crude Extract Preparation ee ee ee ee ee
93. easily onto the assembly Secure the clamps to the IPC outer glass plate sandwich by moving the levers towards the IPC panel Place the assembly in a flat surface with the IPC panel facing up Check if the glass plates and the spacers are flush Place the precision caster base on the lab table with the open cavity facing up Place the gray precision gasket into the base The cam pegs in the precision caster must be pulled out to accommodate the apparatus Place bottom edge of the IPC assembly into the precision caster base with the bottom edge of the assembly resting against the gray gasket of the precision caster base Once resting on the gasket use the cam pegs to connect the base to the clamps Push each cam peg into the corresponding hole on the clamp with the lever in the up position Slight downward pressure applied to the top of the IPC assemble may be required to engage each cam peg Lay the IPC assembly flat on the lab table with the drain port facing up and the precision caster base facing towards you Make sure that the assembly is level to prevent gel leakage Gel preparation 1 2 Prepare the gel by mixing 100 ml 6 acrylamide solution 50 ul TEMED and 600 ul 10 APS Mix gently by swirling Filla 100 ml syringe slowly with the solution and attach the tubing assembly Remove the bubbles from the syringe by forcing some of the gel out When all the bubbles are removed from the tubing place the luer taper into the in
94. ed a new name the International Rice Genebank This Manual describes how and why we carry out the various operations and procedures of rice genetic conservation at IRRI Since its original compilation in 1995 several modifications have been introduced in our work activities These are incorporated in this revised edition of the Manual It shows how we are meeting our obligations under the Agreement signed with FAO in October 1994 to place the collection at IRRI in an International Network of Ex Situ Collections under the auspices of FAO as well as the Convention on Biological Diversity We hope readers of this Manual will find it informative and useful in their own conservation programs am grateful to the staff of the Genetic Resources Center for their hard work in drafting and producing this Manual Our mention of tradenames is not an endorsement of any product but only information on those we currently use in our activities M T Jackson Head Genetic Resources Center IRRI April 10 2000 Chapter 1 Conservation of Rice Genetic Resources at IRRI The conservation of rice genetic resources at IRRI Rice is the most important cereal crop and the staple food of more than half the world s population The genetic resources of rice have been used effectively to increase the productivity of the rice crop They comprise the following materials e Oryza sativa Asian rice that probably had its origin between the Himalayas and Indochin
95. ed water Acp Adh Il Trizma base 46 05 Sodium 1 2 hydroxide Got ME Pgd Pgi Citric acid 5 85 Boric acid 9 28 Pox Distilled water Distilled water Mdh HI Trizma base 2 6 Trizma base 16 25 Histidine HCI 9 6 Citric acid 11 08 Distilled water Distilled water Gel buffer system and III concentration 20x Gel buffer system Il concentration 10x 77 Table 8 2 Stain recipes Enzyme Stain Composition Amount Acid phosphatase a naphthyl acid 50 mg Acp phosphate Acetate buffer 1M pH 10 ml 4 65 MgCl 0 1 M 1 ml Fast Garnet GBC salt 25 mg Distilled water 39 ml Incubate at 40 C Some bands appear quickly some will need overnight incubation Alanine DL alanyl B 10 mg I 5 ml aminopeptidase naphthylamide Alap Fast black K salt 15 mg Tris maleate buffer 0 2 45 ml M pH 3 3 Incubate for 20 min at 50 o C Alcohol dehydrogenase Ethanol absolute 1 ml Adh Tris HCI buffer 0 5 M 5 ml pH 8 5 NAD 10mgmi 1 ml Distilled water 41 ml Just before use add MTT 10 mg ml 1 ml PMS 1 mg ml im Protect from light Incubate for 10 min at 50 C 78 Table 8 2 con t Enzyme Arginine aminopeptidase Arap Catalase Cat Endopeptidase Enp Esterase Est Stain Composition Amount L arginyl B naphthylamide 5 mg ml Fast black K salt Tris maleate buffer 0 2 M pH 3 3 Incubate for 15 min at 50 C 0 7 H20 solution Pour on to the gel and wait for bubbles to
96. ediate most plants moderately lodged 7 weak most plants nearly flat and 9 very weak all plants flat 999 mixture Culm diameter Measured in millimeters and measurement is done from the outer diameter at the mid portion of the culm during flowering or at late reproductive stage and classified as 1 thin lt 5 mm 2 thick gt 5 mm Internode color The outer surface of the internodes on the culm is recorded as 041 light gold 060 green 084 purple lines 080 purple and 999 mixture Taken after flowering the best time is at ripening stage Node color The solid portion of the culm is classified as 041 light gold 060 green 080 purple 081 light purple and 999 mixture Taken after flowering or at ripening stage Flag leaf angle Leaf angle is measured near the collar as the angle of attachment between the flag leaf blade and the main panicle axis after flowering or after the pollen dehiscence Five classes are recognized 1 erect 3 intermediate 5 horizontal 7 descending and 999 mixture Panicle type 1 9 999 Panicles at near maturity stage are classified according to their mode of branching angle of primary branches and spikelet density at near maturity 1 compact 5 intermediate 9 open and 999 mixture Secondary branching Secondary branches bearing the spikelets may be 0 absent 1 light 2 heavy 3 clustering and 999 mixture This can be scored anytime aft
97. eeds were multiplied Documents needed for sending seeds abroad e Seedlist e Phytosanitary Certificate issued by the Quarantine Office of the Bureau of Plant Industry Department of Agriculture Philippines BPI Q Form 11 upon application BPI Form 10 e Import Permit some countries require an Import Permit to be attached to the seed package Table 6 1 e RSHT results for seed lot above 100 g and to be sent to India e MTA IRRI s policy on germplasm exchange Germplasm is freely available on request to bona fide researchers in both public and private sector institutions to NGOs and farmers Since 1973 we have distributed over 786 000 10 g packets of seeds for wild species just 20 seeds per packet gt 20 to collaborators outside IRRI free of charge We require an import permit from the requesting country see Table 6 1 All seeds are checked by IRRI s Seed Health Unit before shipment to ensure safe exchange of rice germplasm world wide A Philippine Phytosanitary Certificate accompanies each shipment Fumigation hot water and other treatments as prescribed by the Philippine Plant Quarantine and recipient authorities are undertaken IRRI uses a Material Transfer Agreement MTA for all germplasm designated to FAO under the terms of the agreement signed in October 1994 IRRI s Policy on Intellectual Property Rights Under IRRI s Policy on Intellectual Property Rights see Appendix 1 1 approved by the Board of
98. electrophoresis Attach the top and bottom safety covers Attach electrode wires to the power supply and pre run the gel at 120 W to achieve a gel surface temperature of approximately 45 50 C Pre electrophoresis prior to sample loading will create a uniform gel temperature and bring the gel temperature to the recommended run temperature This will help eliminate any smile pattern from developing early in the run Loading the DNA samples and gel electrophoresis 1 2 3 4 5 Denature DNA samples by heating in a thermal cycler at 95 C for 5 min and immediate chilling on ice After the pre run turn off the power supply and remove the top safety cover Clean the well area again Carefully insert the teeth of the sharktooth comb into the gel 5 1 mm deep Load 6 ul for 46 wells or 4 ul for 72 wells of each sample into the wells Loading of samples should not exceed 20 min to prevent cooling of the gel and to maintain the denatured state of DNA Attach top safety cover Turn on power supply and run the gel at 120 W maintaining the tempearture at 50 C for 1 h Running time would vary depending upon the primer used Generally stop the run after the bromphenol blue leading dye reaches the bottom of the gel Disassembly 1 2 Staining After electrophoresis turn off the power supply and remove both safety covers The upper buffer chamber can be partially emptied by inserting the drain port connector into the dra
99. enol Chloroform 146 19 500 ml 50 ml 50 ml Equilibrate the mixture by extracting several times with 0 1 M Tris Cl pH 7 6 Store the eguili brated mixture under an equal volume of 0 01 M Tris Cl pH 7 6 at 4 C in dark glass bottles Tris HCl upH20 volume to Autoclave 60 55 g 21 mi 500 ml Table 8 7 Solutions for DNA quantification Solution Agarose 0 7 DNA ladder 1Kb EtBr staining solution Chemical Composition Agarose TBE 0 5x DNA ladder 1 Kb 1000 ug Loading buffer 10x supH20 Ethidium bromide upH20 Amount 1 759 250 ml 50 ul 150 ul 800 yl 250 mg 50 ml Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved Transfer the 90 Lamda DNA 500 ng 10 ul Lamda DNA 250 ng 10 ul Lamda DNA 100 ng 10 ul solution to a dark bottle and store at room temperature Lamda DNA stock of 0 25 ug pl TE buffer Loading buffer 10x Lamda DNA 500 ng 10 ul TE buffer Loading buffer 10x Lamda DNA stock of 0 25 ug pl TE buffer Loading buffer 10x 200 ul 480 ul 320 ul 500 ul 300 ul 200 ul 40 ul 576 ul 384 ul Table 8 7 con t Solution Lamda DNA 50 ng 10 ul Loading buffer 10x TBE buffer 10x Chemical Composition Amount Lamda DNA 100 ng 10 500 ul ul TE buffer Loading buffer 10x Ficoll EDTA Na 0 2 M Bromphenol blue supH O
100. ent the water requirement of late maturing entries in the batch it is advisable to have flush flooding and drain system This can be done right after the harvesting of matured entries in the batch Use overhead rainbird or sprinkler type irrigation for O glaberrima Note When O glaberrima materials start to mature droplets of water may shatter the grains thus furrow irrigation is advisable Pest Control a Snails This is considered as the most destructive pest in lowland rice cultivation during the early stages of crop growth It can totally wipe out the plots overnight To minimize the damage e f seeds are available raise more seedlings for replanting e Apply molluscide at least a week before final soil leveling or do snail picking specially in the early morning or late afternoon when the snails are exposed e Monitor the presence of snails two days after molluscide application and if needed re apply molluscide or pick up gather the live snails It will also important to destroy egg masses to control the growth of the population e After transplanting and there are still snails in the area drain the field or re apply molluscide e Construction of small canals in the paddy soil will help as the snails will stay in these low lying areas This will also facilitate picking since these creatures will stay in these areas e If the materials are not affected by age of seedlings at transplanting as in late maturing and pho
101. er flowering Panicle exsertion 1 9 999 The exsertion of the panicle above the flag leaf sheath after anthesis is classified as 1 well exserted the panicle base appears way above the collar of 36 the flag leaf blade 3 moderately well exserted the panicle base is above the collar of the flag leaf 5 just exserted the panicle base coincides with the collar of the flag leaf 7 partly exserted the panicle base is slightly beneath the collar of the flag leaf blade 9 enclosed the panicle is partly or entirely enclosed within the leaf sheath of the flag leaf Rating is based on the majority of plants in the plot and 999 mixture Panicle axis The panicle axis can be 1 straight or 2 droopy at maturity and 999 mixture This can be recorded anytime from grain filling to maturity Texture of panicle axis Data are taken when panicles have fully exserted 1 increasingly hispid scabrous towards tip pubescence of the axis covered with hairs stiff rough or minute 2 not increasingly hispid scabrous towards tip 999 mixture Panicle length Five panicle lengths are measured in centimeters from the base to the tip of the panicle using a coded measuring instrument at near maturity as follows 1 very short lt 11 cm 2 short 11 20 cm 3 medium 21 30 cm 4 long 31 40 cm 5 extra long gt 40 cm For the wild species actual measurements are taken at early reproductive stage while for cultivated a
102. es with shorter panicles glassine bags are a good substitute The net bag is pinned to the bamboo pole Prior to bagging labels are prepared using shipping tags written with plot number and date of bagging with indelible ink The labels are attached inside the net bags The panicles are harvested 30 days after bagging or when most of the seeds have shattered If sufficient seeds are obtained the plants are discarded and disposed through burning However for species with low seed set like O rufipogon and O longistaminata the plants are ratooned by cutting about 20 25 cm from the culm base a little amount of ammonium sulfate is applied and maintained until next flowering to maximize seed production After harvesting the panicles are dried and kept inside the drying room for 2 weeks before carefully hand threshing and cleaning the seeds To ensure plants do not spread by seeds or rhizomes specific measures are followed a Seed multiplication of all wild rices is done inside the screenhouse in pots b Adisposal area a pit about 3 4 meters deep from the surface ground is designated for burying discarded and burnt samples c A modified incinerator or burning facility is provided to accommodate burning activities especially during the rainy season d All drainage canals inside the screenhouse are covered with fine mesh screens to further control dissemination of seeds through water Waste materials from the canals are regularly hauled b
103. etic diversity studies of rice This chapter includes the different protocols for isozyme DNA sample preparation RAPD microsatellites and AFLP DarT protocol will be added in this chapter in the 3rd edition Protocol for DNA Sample Preparation Leaf Grinding 1 Small scale Grind to a fine powder 20 mg leaf samples clean and uninfected with a pellet pestle into a 1 5 ml microcentrifuge tube while suspended in liquid nitrogen Store in 80 C until further use 2 Large scale Grind 5 8 g leaf samples clean and uninfected using a mortal and pestle with enough liquid nitrogen Transfer ground samples in a 50 ml centrifuge tube Store at 80 C until further use DNA Extraction Method 1 based on Gawel and Jarret Plant Mol Biol Rep 9 262 266 1 Ina 14 ml tube aliquot 10 ml extraction buffer see Table 8 5 and add 10 ul BME this is good for 12 extractions 2 Warm extraction buffer in a 65 C water bath for 30 min 3 Add 700 ul extraction buffer in 20 mg of homogenized leaf sample in a 1 5 ml centrifuge tube 4 Incubate for at least 1 h at 65 C in a water bath Mix by inversion once in a while 5 Remove tubes from water bath and allow to cool down for 4 5 min Add 600 ul chloroform isoamyl alcohol 24 1 Mix by inversion for 5 min 6 Centrifuge at 13 000 rpm for 5 min 7 Pipette out 500 ul supernatant and transfer to a new tube 8 Add 500 ul ice cold isopropanol can leave overnight at 20 C Mix b
104. eves Eege ageet 53 Alda ARE EE OE ORE OO N 13 Bel AE EE OR OE OE EE OE NE EE ER 13 FEGISOH2O n MS oe Re be tated ES ett beer ht deed vaste trad e 104 Federal Department sesse aa aaae ee ee ee e ee ee ee ee ee ee ee ee ee 79 elle il ER OE EER DE ER EE EE OR OR EE OER ER 79 FOSO ate GR terest ac ee OO Re De on ES OR tact ee sat Ge ee rs 104 EE Ee er ER Ee ee E ee GE Re tener DE ie dira EE EER DE 7 83 Bes EE AR DEE AE AE EE TE EE HE ER N 85 ARE ed AR RE EE EE RE EE OE N OE N EN 17 dk de SERE ER ET OE OR EE EE EE N 17 PIM SLAMS RE N RA OO OE OR EE N 79 112 EE EA N OE EET DNE AT E N EE EI 60 ant ed MT EE ML TEE N EE NE TE eege 28 dl Re TEE 64 Final GONCENt ATION sis si RR EE tea tins te ee Ge EE Ve Ge Ee Ga Ee ee Ee Ge o tana ied 58 60 85 EISHEreS ii Gee Ee oe Ee GER ER EE ER Ee ee Geng 79 dd Ce ARR a a a e ER abit 60 85 discard EE nde concen ea Sasa EG EE 60 Flag healt ARE IE OE OE EE RE OE EE and ee noes 39 elle EE OE EE RE N OE RE rad 28 EAR ME N OE OR EA N ER EN 28 ele EE RR OO EO EE EE 77 eat une 85 Seil e TT ae ee ee a Pe ee 10 30 LB EE EE DE EE OO OR RE OE ER EE OE OE 30 Fructose 6 phosphate ER eiiis se ge eevee dee Ee ee Eg Ee gee de Ee Ee eg ED de es 85 Full Strength oe RE ays EE anniek Cae yee adh SE OS ls ee eles 104 FUL ACAD Es Tei a DR dg bias dual seca ath ae ee Ge AA EA LAA a DE GE Ge a 35 FUFTGWEE ES N N sity eda dio dans E ee ee a De ee ENN 15 G G 10 13 14 23 25 28 31 35 47 49 56 68 74 79 85
105. ew seeds lt 5 g one row 5 to 10 g three rows 11 to 15 g five rows 16 to 24 g eight rows gt 25 g in estimating the number of rows consider also the viability of the materials For wild species there is no need to specify the amount since the materials are planted in pots and these are pre germinated in petri dishes before transferring to seedboxes g Update the database with this information e Group the planting materials using the IRGCIS based on variety group maturity and country of origin 1 Japonica or early maturing accessions lt 100 days maturity or accessions without records on maturity but are coming from Korea Japan or China 2 Other variety group or maturity between 101 125 days 3 Late maturing or photosensitive materials with maturity greater or less than 126 days or accessions without maturity data coming from Indonesia Malaysia Thailand or countries that usually have late maturing varieties e Sort the materials further according to the estimated number of rows Other sorting can be done further based on tillering this will help determine spacing and number of entries per unit area for low tillering accessions to obtain sufficient harvest e Generate the seedlist for each group e Manually sort the prepared planting materials based on the computer generated list e Proofread the list against each envelope Make sure the seeds are in correct order and grouping e Generate the plot numbers us
106. f harvest It also provides a guide to seed cleaning and selection Harvest verification procedure 1 Retrieve the accession number of all the harvested materials 2 Determine and compare the composition of the harvest with the seedfile 3 Determine mixtures off types if present This will serve as guide during the seed cleaning process 4 Trace back possible errors and locate other seed sources from another season if the harvest does not represent the original sample 5 Discard the materials when the seed lot does not totally represent the original sample 6 Update the database with information on the harvest status Seed cleaning and selection This is done to improve the seed lot by separating weed seeds and inert matter and eliminating poor quality seeds and off types In cleaning care should be taken to minimize damage to the seed and to avoid loss of good seeds Manual cleaning limits contamination and damage especially when the seeds are very dry Control measures should also be implemented to check the degree of selection and to minimize errors This is done in the seed processing room maintained at 40 50 RH and 22 C Seed cleaning and selection procedure for O sativa and O glaberrima 20 1 Identify the crop year for cleaning selection 2 Generate a list of accessions in the specified crop year the corresponding current storage status and the recommended course of action see Appendix 3 1 as follows a Initia
107. g buffer Mix by pumping the pipettor s plunger a few times without lifting the pipette tip from the surface of the parafilm Load the mixtures in the succeeding wells Close the tank and attach the electrodes to the power supply Run at 70 V for 1 5 h After the 1 5 h run turn off the electric current and remove the gel mold from the tank Transfer the gel in a staining tray with 100 ul of EtBr solution see Table 8 7 in a 1000 ml dH O Stain for 15 20 min Photograph the gel under UV light and estimate each DNA sample by comparing with the lambda DNA RAPD Protocol Preparation of reaction mixture 1 Prepare core buffer in a 1 5 ml microtube enough for 100 reactions 20 ul dNTP s 100 mM 250 ul 125 ul 355 ul 750 ul 2 NH rxn buffer 10x MgCl 50 mM SupH20 total volume Mix by inversion and spin to collect solution Prepare the cocktail in a 1 5 ml microtube enough for 10 reactions adjust amount according to need Cocktail should be prepared just before use 51 2 ul Taq polymerase 5 u ul 10 ul Primer 10 uM 138 ul SupH O 150 ul total volume Mix by inversion and spin to collect solution 3 Prepare the reaction mixture by mixing the following components in a PCR tube 7 5 ul Core buffer 15 0 ul Cocktail 2 5 ul DNA sample 25 0 ul total volume Flick the bottom of PCR tubes and spin to collect the mixture Overlay the mixture with 1 drop of mineral oil Stock and final concentrations per 25
108. ge AE N RA ER NE 39 METHUEN HANDBOOK OF COLOUR ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee 39 MEWS Ad TE EE EE EE ME EE RE OE EE EE 28 MT le ua EO EE a 79 EET RE RO ER LR AE EE ER OR NE dad 7 8 35 83 MOY SP lei OE N OR EO EE ER Wleael 83 EE ET ER EE EN OE HE ER N OE Ne 7 83 EER OE RR EE HEEN OE EE EE OE OG 64 Med EE ER EE OE RE EE ER OE ER OE d 85 ler EE ORE EE 58 60 68 85 ele EI AE RE RA OE ORE EE DO OR OE 85 MOCI GRA E 85 deen Ke EEN 68 MGS O45 he EE RE ME EE EE EE NE 104 Microcentrifude EE EE Re SERE EE EER ER ER ES ee ke ae GE Ee aisha inhibins 56 d Te ol VT EE 68 d Te o ue ET EE 68 120 Microsatellite gt 20 ee e a teas wat avs eet sett EE GE Ses eat Sone N ee coe Ee 60 64 85 Microsatellite DNA ER ER e N RR ORE OE EE OR aS 56 Microsatellite Protocol AA 60 Microsatellites Ee DE ae io Rea 8 11 56 lte a e EE 58 60 64 ll e dle AR EE va boa EE RE RE OE RE EE OE N N 64 lU e EE OE OR HE EE EE ER RI N 68 MERE KIE ORE EER ED 14 56 58 60 64 68 73 74 85 MENG red RE EE ON EE EE OR EO AE EN 35 lente HE ER OR N ORE OE OE EE EE DER 79 C o IRRI Cooperative Project 79 Minuta et EE OE EE GE ED AR Ed dua as a a 7 8 83 dle AE EO EE AE N ME OE EN 83 MEER EN EE EE OE AE DE 31 vase ME AD AR NM OR OE RA RE N 85 var Sk LE HER RE N EA N 85 MIXING EE N EE EO EE eh 60 EE N AR RE OK N EE ven tes 60 Mixtures offAyPE S Ee se Se see Ee seein e dee Ata ee ee Ke Se 23 25 MERE ER N N OO OR OE OE EE 85 MERE
109. gel with tap water Avoid tearing it 2 Pour about 50 ml of destaining solution see Table 8 4 into the staining tray Stand overnight 3 Discard the destaining solution by pouring it into a plastic bag for hazardous waste Again carefully wash the scoring slice with tap water Then add enough distilled water to cover the scoring slice within the staining tray Score the bands according to their mobility Gel Drying 1 Inthe staining trays trim the scoring slices some more with a scalpel cutting approximately 1 cm above and below their isozyme band lines These trimmed gel strips will be dried 2 Open the cover of the gel dryer Remove the thick plastic sheet Line the dryer with a sheet of 7 x 12 No 3 Whatman filter paper Moisten the filter paper with distilled water Align the gel strips on top of the filter paper at about 7 per sheet Cover the gel strips with cellopahane and replace the thick plastic sheet 3 Close the dryer s cover and turn it on by setting its control to 80 C for 40 minutes The gel dryer automatically shuts off at the end of the drying period 4 Switch on the tap aspirator that is connected to the dryer 5 After the drying first remove the filter paper with the dried gel strips Then turn off the aspirator to avoid back flow which can wet the strips 6 Store these as part of the data 3 Seed conservation This is a combination of processes that enables the upgrading of seed and seed lot
110. haracterization and evaluation Germplasm characterization is an important operation for a genebank The value of the germplasm collection depends upon the availability of information relative to the accessions Morphological and agronomic traits as well as reaction to biotic and abiotic stresses that are known to be in the individual accessions increase the importance of the germplasm Moreover systematic description leads to a more efficient use of germplasm in the collection This chapter focuses on the morphological and agronomic characterization of the collection Selection of Materials for Characterization The basic requirement for an accession to be included in the characterization planting is the absence of information about this collection thus newly acquired samples almost always make it on to the list of materials for characterization Another basis for selection of materials for characterization is the completeness of information about the accession Materials from previous characterization plantings with incomplete morphological and agronomic data are retrieved and are included in succeeding characterization plantings Selected materials for characterization are sorted based on the following available database country of origin cultural type photo period sensitivity The number of entries for wet season characterization schedule under Los Ba os Philippines condition is dependent on the number of evaluators The type of material
111. hoto enlarger is used for measuring grain dimensions Grain thickness Ten grain thickness is measured in mm using calipers Seed coat bran color Brown rice dehulled grains is classified into 010 white 050 brown 051 light brown 055 speckled brown 070 red 080 purple 088 variable purple and 999 mixture Endosperm type The starchy endosperm is classified as 1 non glutinous or common non waxy or 2 glutinous waxy 3 indeterminate and 999 mixture Classification is based on the staining reaction of the cut surface of endosperm n 5 to weak KI I solution Waxy starch stains brown non waxy blue black Maturity date This is recorded as the date mm dd when more than 80 of the grains on the panicles are fully ripened The duration can also be estimated by adding 30 days to the duration from seeding to full heading Life cycle The completeness of the plant growth in a growing season is observed 1 annual 2 perennial 3 intermediate 999 mixture Numbers in the parenthesis preceeding the character trait are the character code in use Note Numbers and letters in the parenthesis after the traits corresponds to the color based on the Methuen Handbook of Colours see Reference Reference Kornerup and J H Wanscher Methuen Handbook of Colours Second Ed 1967 Methuen and Co Ltd METHUEN HANDBOOK OF COLOUR Were cores COLOR CODES 10 White Al 11 Whitish 1 3 A2 20 Straw 2A2
112. in Centrifuge at 2 800 rpm for 30 min Collect the upper layer and transfer to a new 50 ml centrifuge tube Add 20 ml CTAB precipitation buffer see Table 8 5 and shake the tube until DNA precipitate forms Centrifuge at 2 800 rpm for 20 min Discard the supernatant Add 5 ml 1 M NaCl Add 5 ul RNAse A pre boiled at 100 C for 5 min see Table 8 5 and incubate in water bath at 56 C for 2 3 h or until the DNA dissolves Add 10 ml 99 5 ice cold EtOH Using a sterile pasteur pipette collect the DNA by swirling the pipette until the DNA adheres to the tip Wash the DNA three times as follows immerse DNA in 500 ul 70 EtOH for 7 min transfer DNA to another tube with 500 pl 70 EtOH for another 7 min and transfer DNA to another tube with 500 ul 99 5 EtOH for 5 min Pour off EtOH and air dry DNA pellet for about 1 2 min Add 300 ul TE buffer pH 8 0 and store at 4 C Method 3 based on Murray M G and Thompson W F 1980 Nucleic Acid Res 8 4321 1 2 3 Add 5 ml lysis buffer see Table 8 5 to 3 to 5 g homogenized leaf sample in a 50 ml polypropylene tube Add 5 ml of phenol chloroform 1 1 and mix well by inversion Centrifuge at 4000 x g for 5 min Remove all aqueous layer top layer containing DNA and transfer in a new 50 ml polypropylene tube Add 5 ml of chloroform and mix well by inversion Centrifuge at 4000 x g for 5 min Remove the aqueous layer top layer containing DNA and transfer to a new
113. in a tray with upH O for 10 sec 5 Transfer the gel to a tray with developer solution pre cooled to 4 10 C see Table 8 9 for 2 5 min or as soon as the bands appear with shaking 6 Return the gel to the fix stop solution for 5 6 min 7 Rinse the gel in a tray with upHsO for 2 3 min and allow the gel to dry at room temperature or at 50 C Documentation 1 Ina dark room with a red safelight place the gel on a light box The light box should have a white bulb 2 Position the Promega APC film with the emulsion side down over the gel 3 Turn on the lightbox and expose the film for 10 sec Film exposure may vary with different lightboxes or with different batches of APC Film Make test exposures first by exposing small strips of film at varying times 4 Develop the film in the following solutions see Table 8 9 for developer and fixer solutions Time Solution 1 3 min Kodak GBX Developer 1 min distilled water 3 min Kodak GBX Fixer 1 min distilled water will vary depending on exposure conditions 5 Air dry the APC film This is now ready for scoring AFLP Protocol Adapted from AFLP Analysis System IVAFLP Small Genome Primer Kit Instructional Manual GIBCOBRL Life Technologies Restriction digestion of DNA 1 Add the following in a 1 5 ml microtube enough for 10 reactions adjust amount according to need 248 ul AFLP grade H2O 80 ul reaction buffer 32 ul enzyme EcoR1 Mse1 360 ul total volume
114. in port on the IPC Buffer should drain immediately from the IPC After the upper chamber is emptied to the level of the drain port pull out the stabilizer bar and remove the IPC assembly Blot the bottom edge of the IPC assembly onto absorbent paper Carefully pour the remaining upper buffer out of the IPC assembly into a container Also carefully drain the lower buffer from the universal base into a container Never store buffers in an IPC Never add buffer to an IPC unless the clamps are in place Remove the clamps from the IPC assembly by first pulling the levers away from the IPC and then sliding the clamps off the IPC assembly Lay the IPC assembly flat on a lab table with the outer glass plate facing up Carefully separate the glass plate by pulling up gently near the top of the outer plate The gel should come apart from the IPC and become strongly affixed to the outer glass plate Remove the comb and side spacers Adapted from Promega s Silver Sequence DNA Sequencing System Technical Manual Rev 8 96 1 Place outer glass plate with the gel in a plexiglass tray with the fix stop solution see Table 8 9 for 20 min with continuous shaking Do not discard the fix stop solution Do this step inside a fume hood 56 2 Wash the gel thrice for 2 min each in a tray with upH20 with continuous shaking 3 Stain the gel with silver stain solution see Table 8 9 for 30 min in a tray with continuous shaking 4 Rinse the gel
115. ined rat baiting is better e Flame throw rat holes inside and outside the field regularly It should be at least twice a week especially during the flowering stage e Birds Birds are a major problem at the start of the grain filling stage especially when the materials flower ahead of neighboring fields Bird preference can be easily observed among accessions and therefore protection against birds should be implemented e Ideally bird nets are the best solution but are impractical to install throughout several hectares Use bird nets only on small area plantings lt 2 500 m2 e Employ bird watchers At IRRI bird watching starts from 0600 and ends at about 1800 hours e Use devices to scare off the birds e g scare crow sound creating devices striking colored materials etc Re identification roguing and purification Before pulling and after replanting in the field rogue rice plants growing off the row These are assumed to be dropseeds or volunteer seeds At the late vegetative stage obvious off types should be removed unless otherwise stated in the fieldbook that the accession or variety is cultivated as mixed Re identify the plants by comparing the seeds with the retrieved information in the fieldbook see section on fieldbook preparation Remove all obvious mixtures Note that this should be done several times as the flowering period varies among accessions In cases where a mixture is almost 50 or there are se
116. ing the IRGCIS The first group to be sown usually gets the first plot number and these are continuous across all plantings including seed source and nursery area plantings This will prevent mixing up of harvest and confusion in locating and identifying the harvest e Print seedlist with accession number and assigned plot number sorted by plot number e Number the seed envelope with the corresponding plot number e Proofread each envelope against the seedlist Several proofreadings are needed to check errors because in the field each entry will carry plot numbers only e Using rubber band bundle the seed envelopes in fives or tens depending on the number of rows and nurseries to which they belong This will facilitate in distributing the seed envelopes during seeding e Place the materials in ovens set at 50 C for 48 to 72 hours to break dormancy e Equilibrate at ambient room for at least 24 hours before seeding e Prepare seedbed labels Eight inch wooden pot labels can be used To save time effort and materials numbering can also be done by intervals of five or ten e Generate fieldbook using IRGCIS The field books should include plot numbers accession and some selected basic information e g variety group or eco geographic race origin awn presence awn color apiculus color lemma and palea color grain length and grain width which will help in re identification and purification later For initial seed increase the origin
117. itial germination results also reflect the storage potential of the seed in a certain environment Although all samples are tested for viability only seeds of high viability are processed for long term storage Viability testing procedure 1 Place the pre counted samples prepared during the seed cleaning and selection in the oven set at 50 C for 5 days to break the dormancy for O sativa and O glaberrima Equilibrate at room temperature 28 30 C for 2 to 3 days prior to germination Sow seeds in moist paper towels and place in germination chamber set with the following conditions a 30 20 C alternating temperature on a 12 12 h duration a 12 12 h light dark condition 99 relative humidity Count the number of normal and abnormal seedlings Evaluation will be based on ISTA rules 7 days after germination and a second reading at 14 days when necessary Prepare a third set if the difference between the two tests exceeds the maximum tolerable limits at a probability of 2 5 see Appendix 3 3 Encode related information 24 For wild rice only twenty seeds are germinated after breaking dormancy see recommendation Appendix 3 4 Although we have a set of germination protocols for wild species of rice we also observe some considerable variation between accessions Strong dormancy in some seeds has been observed and requires combination of dormancy treatments Seed health evaluation Only seeds of the highest qualit
118. jection port of the precision caster base Slowly inject the gel solution on to the glass plate sandwich Do not remove the tubing until the gel has polymerized Insert the flat edge of a sharktooth comb 5 mm past the edge of the outer glass plate Clamp it with a large metal binder to hold it in place Let the gel polymerize for 30 60 min Remove the tubing and the precision caster base from the assembly Clean the caster base and gasket of polymerized gel solution with tap H O followed by a dH20 rinse Pre electrophoresis 1 2 3 Fill the lower buffer chamber with 350 500 ml 1x TBE buffer see Table 8 9 Gently lower the gel assembly to the universal base Insert the stabilizer bar Fill the upper buffer chamber with 1400 ml 1x TBE buffer The level of the buffer should be about 1 cm from the top of the fill spout at all times during the run Gel 55 4 5 6 electrophoresis buffer can be heated to 50 C in a microwave oven before adding buffer into the upper buffer chamber This will reduced the time needed to bring the gel to the appropriate run temperature before sample loading and will greatly reduced pre electrophoresis run time Remove the comb from between the glass plates Clean the well area using a syringe Make sure to remove air bubbles and unpolymerized acrylamide Adhere a gel temperature indicator onto the outside of the outer glass plate somewhere near the center to monitor the gel temperature during
119. ket to properly labeled 4 x 1 x d tubes Pour enough 70 ethanol to immerse the seeds in each tube and wash the seeds by shaking the tube several times Pour off ethanol in a container 3 Pour 20 sodium hypochlorite NaOCl into each tube Leave this for 15 min to sterilize the seeds Shake the tubes occasionally After 15 min pour off the sodium hypochlorite in a container Pour sterile distilled water into each tube Wash the seeds by shaking the tubes several times Pour off the water into a container Do this step 3 times Transfer the seeds from each tube to petri dishes lined with sterile filter paper to blot dry them Plant the seeds in 5 x 1 or 8 x 1 I x d test tubes with agar media see Table 9 2 for media preparation see Table 9 1 for stock solution of culture media First remove the tube s cover and flame the rim of the tube For accessions with more than 20 seeds use a 125 ml Erlenmeyer flask The tube or the flask should be properly labeled with the accession number With a sterile spatula take a seed from the petri dish Put this seed on to the surface of the agar media Do this for the rest of the seeds For accessions with more than 20 seeds transfer 10 15 seeds per flask Flame the rim of the tube and the cover s underside before sealing it again Do steps 6 8 for all accessions Plant establishment 1 2 Stand the tubes with the seeds in a test tube rack and put them inside a dark ca
120. l canning first time the seed is processed for long term storage b Replenishment the accession is already in the Base and Active Collections with acceptable viability but seed stocks are depleting 10 c Replacement the accession is already in store but the viability has fallen below the acceptable limit regardless of the amount left d Temporary storage when the amount of selected samples is insufficient for long term preservation or the viability or the seed health test falls below the acceptable limit Generate data sheets see Appendix 3 2 Pre clean the seeds by blowing in a ventilated column to separate unfilled grains and light density materials Verify again using the seedfile Determine the selection to be done based on the recommendation during the verification process and the current storage status Examine the seeds and hand sieve with graded mesh sizes if mixtures off types vary in size to separate slender and bold grains Remove discolored deformed infected soiled immature damaged seeds and off types Determine the actual action to be taken based on the quantity of clean seed This will determine the packing system to use Prepare and label all the necessary envelopes for use in seed testing viability testing temporary storage and final drying to minimize labeling errors 11 Submit the selected samples together with the seed file pre labeled envelopes and the original seed container for
121. lo ER KEER EN AE EE EE Ed EE EL E RE IE 64 Prepare Mi dodo Se GE Ee es 64 Prepare L4 El RE EER AE RR OK OE AR OE OE 58 de RS ii RA RE OE OE RR OE bas ee EE EE OE EE 46 A EE ed DS Oe EE Ee A sea SE RE AE EG deus EG ALTE 79 Ao O O NON 79 Primer OU 58 125 Material Transfer Agreement 47 Print Phytosanitary Certificate Application Form iese ee ee ee ee ee ee Re ee Re Ge ee ee ee 47 PrintssecdliSt RR AA RE EE EE Ser OE RR OE 14 Private Bag AE MM OE AR EE RA ET EE NE 79 dele de AE NE OE EE EE OE EE EE OE 5 International Rice Genebank ee se ee ER AA Ge AA Re GR AA AA ee AR Re ee ee ee Re ee ke ee 5 dele SEA RE EE N RE OE EE N edness 46 newly received germplasm ees ee ee ee AR ee AR nc ee AR ee AA ee ee AR ee ee Re ee ee ee ke ee ee ee ee 46 rele li ER EO OR KO OE LE EE OE N 5 MSHE AR EE O AE OM ER OO EE N EE 5 Promega NI EE EO a Ad SAR 60 85 Position eebe Ee 60 Promega s Silver Sequence DNA Sequencing System Technical Manual Hey 60 Rromega AA AE EE EE OE N AE EE cece ere 85 Prooflist EE A ese ge ues Hea degen 54 Prosphytochloa i se GE tees A Wee einen Dee Ee Vee Ge sd 7 83 Protocols Ee Seege ides ee dee AE ee ee ee ate 47 49 56 DNA Sample Preparation ie ee se AA AA Ge AA AR AR RA Ke AA AA ee GR Re AA ee ee ee ee ee 56 seed distribution EE ET ER DE A ee ee i Ge ae ee GR ei se 47 AE ME EE va EE ORE EE ER N EE ania as 28 Pubes cencCce ES ee EE DE ge Dee eN Ge dvs nee a eg de vs
122. ls making sure no oil is pipetted out with the mixture Close the tank and attach electrode wires to the power supply Run for 1 h at 100 V After electrophoresis switch off the power supply and remove the tank cover Remove the gel from the molder and transfer in a tray with EtBr staining solution Stain for 20 min After staining rinse the gel with dH O Photograph the gel under UV light If the DNA samples were properly digested proceed to ligation of adapters Digested DNA appears as a smear with a fragment size of 100 to 500 bp Ligation of adapters 1 240 ul 10 ul 250 ul on a gt D Add the following in a 1 5 ml microtube enough for 10 reactions adjust amount according to need adapter ligation solution T4 DNA ligase total volume Add 25 ul of the mixture to each of the tubes with 25 ul of digested DNA Mix gently by flicking the bottom of the each tube and spin to collect the solution Overlay with a drop of mineral oil Incubate at 20 C for 2 h in a thermal cycler This mixture is now the ligated DNA After incubation transfer 10 ul of each of the ligated DNA to a properly labeled 0 5 ml new tubes Store the remaining 40 ul ligated DNA at 20 C Add 90 ul of TE buffer to each of the 10 ul of the ligated DNA to make a 1 10 dilution This is now diluted ligated DNA Mix gently by flicking the bottom of each tube and spin to collect the solution Pre amplification reactions 58 1 Transfer
123. lture Department of Agriculture Bandar Seri Begawan 2059 Brunei Darussalam British Solomon Association G P O Box 5 Honiara or as provided by the consignee As provided by the consignee As provided by the consignee As provided by the consignee As provided by the consignee AUTHORIZED CHANNEL Instituto Colombiano Agropecuario c o CIAT Apdo Aereo 6713 Zona Aduanera Cali Colombia As provided by the consignee As provided by the consignee As specified in the Import Permit As provided by the consignee 72 India Indonesia Italy Japan Kenya Table 6 1 con t COUNTRY Liberia Madagascar Malawi Import Permit Import Permit Import Permit Import Permit yellow tag for rough rice No Import Permit if dehulled Import Permit REQUIREMENTS Import Permit Import Permit Import Permit The Director National Bureau of Plant Genetic Resources NBPGR Indian Agricultural Res Institute Pusa Campus New Delhi 110012 India c o IRRI Liaison Scientist Cooperative DEPAGRI IRRI Program Jalan Merdeka 147 Bogor 16111 Indonesia As provided by the consignee Through Quarantine Station printed in the Import Permit label tag Yokohama Plant Protection Station Ministry of Agriculture Forestry and Fisheries Japan Direct to recipient if seeds are dehulled The Director Plant Quarantine Station Kenya Agricultural Research Institute KARI P O Box 30148 Nairobi Kenya
124. m beneath the mold 7 Gently remove the comb 8 Load 10 ul of 1 Ko DNA ladder see Table 8 7 on the first well and 10 ul of each reaction mixture in the succeeding wells making sure no oil is pipetted out with the mixture A gel can accommodate 54 samples in 2 comb positions 9 Close tank and attach electrode wires to the power supply Run for 3 h at 150 V Staining and documentation 1 After electrophoresis switch off the power supply and remove the tank cover 2 Remove the gel from the molder and transfer in a tray with EtBr staining solution see Table 8 8 in a 1000 ml H O Stain for 20 min EtBr staining solution can be reused but staining time should be for an hour 3 After staining rinse with dH2O 4 Photograph the gel under UV light Scoring and analysis 1 Designate a name or a number for each RAPD marker based on the molecular size and primer used 2 Score RAPD bands using a binary system of 0 in the absence of the band and 1 if the band is present 3 Data is now ready for analysis Microsatellite Protocol Preparation of reaction mixture 1 Dilute primers to 10 uM by adding 200 ul 1x TE buffer see Table 8 9 Each primer in the Rice Map Pairs set from Research Genetics is supplied at 200 ul of 20 uM in 1x TE buffer pH 8 2 Aliquot 5 ul of the genomic DNA 1 25 ng ul in each of the 40 properly labeled 0 5 ml PCR tubes or plate wells 3 Prepare the cocktail for 40 reactions in 1 5 ml microtube
125. m for distribution is pre packed in aluminum foil envelopes 10 g sample for O sativa and O glaberrima 20 seeds for wild rices e Processing of seed requests is on a first come first served basis e Requests for germplasm can be made electronically using the IRGC Information System or by e mail to GRC e Requests for germplasm to be sent to collaborators abroad should be referred to the Head of the Genetic Resources Center Receipt of request e Record seed requests received from IRRI staff and scientists from different universities institutions and National Programs and assign an IRGC request number e For general requests search the appropriate germplasm from IRGCIS databases references or request scientist concerned for nomination e For specific requests verify accession number e Generate appropriate list of germplasm accessions e Refer request to the appropriate scientist and or Division if the requested germplasm is not available in the genebank 42 Process initial verification of seed status whether seeds are available or not Inform requestor about the Material Transfer Agreement MTA that will go with the seeds and get feedback before processing the request see Appendix 6 1 Submit the request by using the IRGCIS Input all accessions requested and information about the requester Process seed requests using the IRGCIS from selection of source for distribution printing of verified list labels to be stuck
126. n 94 30 sec 1 57 30 sec 72 1 min 94 30 sec 1 56 30 sec 72 1 min 94 30 sec 20 55 30 sec 72 1 min 94 30 sec 10 72 1 min Hold temperature 4 C 2 After amplification remove the PCR tubes from the thermal cycler Gel analysis 1 Follow the procedure in the microsatellite protocol beginning from the step assembling the glass plate sandwich to staining 2 Score the AFLP bands Abbreviations Acronyms Acp acid phosphatase E C 3 1 3 2 AFLP amplified fragment length polymorphism Adh alcohol dehydrogenase E C 1 1 1 1 AgNO silver nitrate ALAP aminopeptidase alanine substrate Amp aminopeptidase E C 3 4 11 1 APS ammonium persulfate ARAP aminopeptidase arginine substrate BME B mercaptoethanol Rei degree Celsius 61 CaCl Cat CTAB cm dATP dCTP dH O DNA dNTP dGTP dTTP EDTA Na Enp Est EtBr EtOH GOT G 6 PD HO HCl HCO H20 Icd IPC KCl Kb calcium chloride catalase E C 1 11 1 6 hexadecyltrimethyl ammonium bromide centimeter s deoxyadenosine 5 triphosphate deoxycytidine 5 triphosphate distilled water deoxyribose nucleic acid deoxynucleoside 5 triphosphate dNTP mix deoxyguanosine 5 triphosphate deoxythymidine 5 triphosphate ethylenediaminetetraacetic acid disodium salt endopeptidase E C 3 4 21 24 estesterase E C 3 1 1 ethidium bromide ethanol gram s glutamate oxaloacetate transaminase E C 2 6 1 1 glucose 6 phosphate dehydrogenase hour s wa
127. na ca Vode dee Bae EG ie aen 14 assignment RE EE EE EO NE is 14 old se AA EE EE O EE ED 85 PIVIB 5612 RE N EE EN IE MM ER ER EE N IE EO 79 PM iii A A ee EA N E AA AL oe Ge Pe 68 85 Policy on Intellectual Property Piohts AAA 10 Pollen Mother Gells EE ER aie ee a N Ge es 74 Polyacrylamide ER LE OR ER ee OR OR ee 85 Polyvinyl pyrrolidone ee ieai ee Ge ee ee ee ee Ee AA ee Ge AA ee ee AA ee ee 68 Population SiXi EE Ie Gh i ee ee th ais ees ee a ed 39 Porteresia ORE EE AE EE 7 83 Porteresia coarctata iis ie EER Ee DERE GE EA EE GESE BEA EKEN GEE AE EO EK Ee ke ee ek Ee ti Seed eer 7 8 83 PO ER EE ER N N EE OE RE RE EE arca 39 Lei TTT EER N EE LI EE AE EE ER RE EE anh 39 ofdie ES EE OER il AE N EE OE IE 60 di TER ae N EE E EE IG 60 Posphogluconate debwvdrogenaee AAA 68 Post harvest Activities ie Ese ee EE EA EE GE Ek ESE gee ee EAR n gee De EE Gee sae bee de ee ees 17 Post Narvest characteristics iii iii 39 el Te ol RE EE ER RE EE RE ER idad 7 83 Potamophila par vilo amen deue hat dee een geb a ee ee egg De egg gese ers de eg 8 83 se ER N EE eege N OR AE EO OF Hee 85 AN EE AE ER IE EE EE OE dain is 56 58 se EE EE EDE EE DE N EO RE EE EE NEE A 68 85 Pre electrophoresis A 60 Ted Lie UR N EE RE AAEE EE OE OE N NG 64 VER EE ME ME OE ER OE ER TE AK 64 dinee EE AE AE EE EN OO EO N abe 39 Pre electrophoresisS AAA 60 Ed RR EE dd OE EE HE OE EE OR 56 64 ee NE MO EE REEN ENE TEE ODE OE EE N 56 BA RE EE AE DE OE EE RE AE EE EE EN 64
128. national collections Release seed and seed list to requesting scientist in IRRI Submit seeds seedlist accompanying letters Import Permit and phytosanitary certification application together with the Material Transfer Agreement to Seed Health Unit for seed certification if seeds are to be sent abroad Include procedures for growing wild taxa if there are wild rices in the batch Dispatch seeds by airmail air parcel or airfreight or handcarry Send covering letters and copy furnish requester if request was channeled through another scientist at IRRI Generate weekly summary of seed dispatched for information and review of GRC head File letters documents and acknowledgment Seed requests of IRRI staff for sending abroad GRC processes requests of IRRI collaborators in accordance with the arrangement between IRRI and FAO For safe germplasm transfer seed requests for sending abroad must pass through the Seed health Unit for quarantine certification Processing of seeds seed health inspection and quarantine certification may take from two to three weeks depending upon the volume of samples requested 43 Basic information accompanying the seed e IRGC request number e Name and address of recipient e Total number of accessions supplied e IRGC number e Crop Year when sample was planted harvested e Viability e Scientific name e Variety name for cultivated rice e Source country e Sample Category e Location where the s
129. nce herbicide is sprayed and a granular insecticide is applied a day after transplanting e Top dressing is done 30 DAT and 45 DAT with 350 g and 300 g of ammonium sulfate respectively for the indica and javanica types For the japonica types top dressing is done earlier at 10 DAT and 20 DAT because spikelet fertility is highly affected e Water level is maintained until panicle initiation For the upland types water is withheld 30 DAT to provide drier soil conditions e Regular monitoring of pest and disease incidence is coordinated with the IRRI ES and specific control measures are applied e For insufficient seedlings 1 3 seedlings transplanting is done in pots to ensure the continuous growth of the plant Additional care is extended in plant maintenance such that daily monitoring of plant health is a necessity e Panicle harvesting is observed for easy handling and verification Harvested panicles are dried and kept in the drying room for 2 weeks before finally hand threshing Wild rices Most accessions of wild rices require different cultural management practices for seed increase compared to those of the cultivated rices Most of them are strongly photoperiod sensitive so that the best time to grow them is during a season with short daylength to induce panicle initiation several species such as O meyeriana O granulata O ridleyi and O longiglumis grow better under partial shade while others grow well under full sunlight
130. ncn nono Ee Ee ee ee ee ee ede 79 Yoshida ie AA A ATA N De de ETA ad Ee A ee 104 AE N N EE N EA AE RE N E S 79 Z LAMA E shales eu IE RE N OR OR ada 79 alle lo RAD ER EE RE EE ON EE OE ME EE EE E 79 LIZA ER ER EE OE EER EA ME AE ET AT ame 7 83 ZIZANIOPSIS oi RE EE AE EE EO a N 7 83 Zizaniopsis villanensis se ee ee ee Ge Ee Re AE ee Ge AA ee ee ee ee ee de ee ee ee ee ee ede ee ee ee ee ede ee ee 8 83 LMS OA KEE EE 104 ZASOATEEOE TEE 104 elle EIER EE EE IE EN N EE OD OE OE EA A EE hae 85 A EE EN ER EEEE ee DS aus du De Ge ee EE 79 133
131. ndard lowland preparation Soil is allowed to settle for 2 3 days before the beds are constructed Beds are usually lower at 10 cm Furrows are also made using a wooden furrower at 10 cm between rows The seedbed width is 0 8 m Length is determined by the size of the field e Direct seeding O glaberrima is usually directly seeded The seeds are drilled to designated furrows and covered with soil Irrigate and then apply pre emergence herbicide However O glaberrima entries in cases where amount of seeds for regeneration is insufficient or have relatively low viability and it will be risky to direct seed these can also be seeded in beds or boxes and can be transplanted as in O sativa However water should be drained after the seedling has been established and subsequent irrigation will depend on the need of the plants Labeling Place pot labels at the start of the first bed leave several rows for a border and a vacant row before the first plot number Three row field plots require 1 2 seedbed row while 6 8 row plots needs 4 5 seedbed rows Count the number of rows depending on the desired number per accession with one extra row to be left vacant to distinguish one accession or plot from another e Recheck labeling by counter checking the number of rows with the desired entry requirement and label Seeding and seedbed management Distribute seed envelopes by bundle as described earlier remove the rubber band and then distribute each en
132. ng room while waiting for the viability and seed health test results for the final drying 15 Encode all related information Seed cleaning and selection This is done to improve the seed lot by separating weed seeds and inert matter and eliminating poor quality seeds and off types In cleaning care should be taken to minimize damage to the seed and to avoid loss of good seeds Manual cleaning limits contamination and damage especially when the seeds are very dry Control measures should also be implemented to check the degree of selection and to minimize errors This is done in the seed processing room maintained at 40 50 RH and 22 Seed cleaning and selection procedure for O sativa and O glaberrima 1 Identify the crop year for cleaning selection 2 Generate a list of accessions in the specified crop year the corresponding current storage status and the recommended course of action see Appendix 3 1 as follows a Initial canning first time the seed is processed for long term storage b Replenishment the accession is already in the Base and Active Collections with acceptable viability but seed stocks are depleting c Replacement the accession is already in store but the viability has fallen below the 22 acceptable limit regardless of the amount left d Temporary storage when the amount of selected samples is insufficient for long term preservation or the viability or the seed health test falls below the a
133. ntries originating from joint expeditions between national program personnel and IRRI staff or received for duplicate storage from national programs All incoming samples are examined by the Seed Health Unit at IRRI under the supervision of the Philippine Plant Quarantine Service Bureau of Plant Industry e Germplasm is rejuvenated and multiplied for long term conservation during October May for the production of the highest quality seeds under the lowest disease and pest pressure Seeds are threshed dried then hand cleaned In the seed drying room seeds equilibrate to e 6 moisture content before being packed in large aluminum foil packets containing or 500g for storage in the Active Collection Ten gram samples are also stored in aluminum foil packets ready for germplasm distribution The Base Collection comprises samples with two aluminum cans or 120 g e The viability of all accessions in the Active and Base Collections has been determined Actual viability determines the schedule and frequency of future monitoring Seed viability of all accessions is determined prior to long term storage at 20 C e Wild species are grown in pots in a quarantine screenhouse Perennial species are maintained as living plants when seeds are difficult to produce Germplasm exchange e Since 1973 more than 786 000 10 g packets of seeds only 20 seeds per packet for wild species have been distributed to rice researchers free of charge with m
134. number of days to first flushing of flowers are observed Culm angle 1 9 999 Culm angle readings are based on plants grown in the entire plot and taken after flowering Six broad classes are recognized 1 erect the angle is less than 30 from the perpendicular 3 intermediate the angle is about 45 5 open the angle is about 60 7 spreading the angle is more than 60 but the culms do not rest on the ground 9 procumbent the culm or its lower part rests on ground surface and 999 mixture Culm number Culm number is recorded after full heading as the total number of grain bearing and non bearing tillers after flowering 1 sparse lt 10 culms 2 medium 10 20 culms 3 prolific gt 20 culms Culm length Culm length is measured in centimeters from ground level to the base of the panicle using a coded measuring instrument after flowering 1 lt 51 cm 2 51 70 cm 3 71 90 cm 4 91 110 cm 5 111 130cm 6 131 150 cm 7 gt 150 cm For the wild species actual measurements are taken from 5 samples Culm strength lodging resistance Culm strength is first rated after panicle emergence by gently pushing the tillers back and forth a few times This test gives some indication of culm stiffness and resilience Final observation at maturity is made to record standing position of plants Plants in the plots are classified as 1 strong no lodging 3 moderately strong most plants leaning 5 interm
135. o 3 S225 321 aluminum foil packs Barrier Foil Products Co CCE Business Park Windmill Lane Denton Manchester M34 3QS UK Tel 44 161 337 8341 Fax 44 161 335 9101 4JHM 305 CTE Constant Twin Element laminate crimp sealer supplied by Hulme Martin Ltd 6 Brownlow Mews Guildford St London WC1N 2LD Tel 44 171 242 5448 Fax 44 171 242 2044 30 Chapter 4 Germplasm Nursery Screenhouses The germplasm nursery screenhouses The GRC Nursery has basically one main purpose to provide and maintain an environment that will result in optimum seed production both for the hard to grow O sativa and O glaberrima accessions as well as wild species of rice This chapter covers e cultivated species e wild rices Cultivated species For the cultivated species seeds are germinated in flat seed boxes and transplanted to concrete beds after 21 days or when seedlings are vigorous enough and can withstand stress upon transplanting The concrete beds are small field like lowland plots constructed inside the screenhouse about 2 5 m wide and 11 m long e Soil preparation is done by a mechanical mini hydrotiller after re shoveling manually e 500 g of complete fertilizer is mixed well with the soil as basal fertilizer application before final soil leveling and lay out e 2 3 seedlings are transplanted per hill using 25 cm spacing between hills and between rows leaving one row vacant between entries in the lay out e Apre emerge
136. of the actual layout Missing plot numbers should be included either in the list of non viable or insufficient seedlings Take note also of accessions plot that were transferred to the nursery area If at any instance there was an interchange in the distribution this should be noted Transplanting replanting e Transplant the seedlings using the same guide wire or planting guides during the layout with 2 3 seedlings to reduce mortality and ensure fast recovery e Collect excess seedlings for each entry and place them near the labels These will be used for replanting More healthy re planting materials can be obtained if they are divided in smaller bundles and properly placed in the paddy e Replant after about a week or two by passing through plots one by one Look for vacant hills and use the extra seedlings to replace missing hills This can be done two or three times Fertilization e Soil analysis is important in deciding the rate and type of fertilizer to use It is important to follow recommendations In the case of the IRRI ES the recommended fertilizer rate for dry season cropping is 90 30 30 kg N P K ha 1 The Spad meter can also be used to determine fertilizer requirement e The first step in nutrient supplementation is by root dipping of seedlings in 4 zinc oxide prior to distribution and transplanting e During the final soil leveling the first dose of nitrogen and all phosphorus and potassium are applied and incorporated to the
137. ol by SHU Pass on seed list and passport data to staff of data management section for documentation and assignment of batch ID for the incoming batch of seeds File a copy of the seedlist and other accompanying documents by the seed exchange staff Turn over to staff in charge of seed conservation seed samples and a copy of the seedlist Test the initial viability The number of seeds to be used depends on the quantity of seeds received If viability is very low advance the planting to the best possible time Note All foreign incoming seeds should be accompanied by a phytosanitary certificate and a Philippine import permit Processing newly received germplasm 1 Classify the incoming germplasm based on accompanying paper This will guide the curator on what steps lie ahead The newly received materials are classified by species as O sativa O glaberrima or wild species or if the sample is a landrace or a breeding line This information is usually included in the passport data Wild rices are handled differently from the cultivated materials Only promising and advanced breeding lines are accepted in the genebank Check probable duplications This is done to check whether the received sample s already exist in the genebank Comparison is done against existing accessions previous batches not yet registered or assigned accession number or within the batch 41 Procedure a A computer generated list of probable duplicates is
138. on Technology Services ITS is performing a daily backup copy of the file in 80GB DLTtapelV cartridge tape including the system application This protects the system against potential disasters Data exchange The output generated by the system is mostly in ASCII file format which facilitates reading and conversion by most commercial software packages System documentation The system is documented to serve as a reference source for any future modifications A copy of the documentation is readily available in the GRC Data Management Room 48 Chapter 8 Molecular Marker Laboratory Molecular marker laboratory Located in the second floor of the Klaus Lampe Laboratory the Molecular Marker Laboratory MML analyzes plant isozymes and molecular markers Isozymes are multiple forms of an enzyme This polymorphism revealed through enzyme electrophoresis is 1 useful in genetic diversity studies 2 helpful in determining F1 hybrids as it is controlled by co dominant alleles and 3 important in designating germplasm to the various isozyme classification groups Molecular markers are inherited DNA sequences that can be monitored Some of these markers are Random Amplified Polymorphic DNA RAPD Microsatellite DNA and Amplified Fragment Length Polymorphism AFLP All three are polymerase chain reaction PCR based Their reaction products are separated by electrophoresis visualized by different staining procedures and are used in gen
139. or As provided by the consignee AUTHORIZED CHANNEL As provided by the consignee As provided by the consignee The Head Plant Protection Services Ministry of Agriculture Bvumbwe Agricultural Research Station P O Box 5748 Limbe 73 Malaysia Mali Mauritania Mexico Mozambique Namibia Niger Nigeria Pakistan Table 6 1 con t COUNTRY Papua New Guinea Philippines Somalia South Africa Pretoria Switzerland Tanzania Thailand Import Permit Import Permit Import Permit Import Permit Import Permit Import Permit Import Permit Green and white address label seed Malawi East Africa or as specified in the Import Permit As specified in the Import Permit As provided by the consignee As provided by the consignee INIFAP International Traffic Office Direc Recursos Materiales Attn Mr Joaquin Rodriguez Garcia Insurgentes Sor No 694 80 Piso Mexico D F CP 03100 or as specified in the Import Permit As provided by the consignee As provided by the consignee As provided by the consignee Plant Quarantine Service shipment has to be sent Federal Department of via DHL Import Permit REQUIREMENTS Import Permit Import Permit with SHU valid for 6 months from date of issue Import Permit Import Permit Import Permit Import Permit Import Permit Agriculture Moor Plantation PMB 5672 Ibadan Nigeria or as specified in the Import Permit tag As specified by
140. or distribution Old seed stocks packed earlier are conserved in aluminum cans similar to the Base Collection Packing procedure for O sativa and O glaberrima For Base Collection 1 2 3 4 Prepare and label the aluminum cans and the lid using permanent marking pens with accession number and crop year Take few samples at a time from the drying room to minimize reabsorption of water Pour and vacuum seal the seeds in semi automatic can sealer with 20 PSI Check any packing deficiency Replace if there is any deficiency For Active Collection 1 6 7 Prepare and label the aluminum foil packets bulk and pre pack using computer generated sticky labels with the following information accession number variety name crop year Take out few samples at a time from the drying room to minimize reabsorption of water Prepare the required pre pack samples and weigh the rest for bulk storage Record the amount Place the weighed bulk samples immediately in the foil bag Add small packet of activated silica gel with perforation pricked just before sealing to serve as check for possible air seepage during storage Seal the bag using high temperature constant heat sealer 4 with 1 cm sealing width Check for packing deficiency Reseal or replace if there is any deficiency For Duplicate Storage 1 Pack duplicate samples in labelled small aluminum packets similar with pre pack samples Packing procedure for
141. ore than 18 to collaborators outside IRRI e TO send rice germplasm outside the Philippines an import permit is needed from the requesting country All shipments are checked by IRRI s Seed Health Unit A Philippine phytosanitary certificate accompanies shipments Hot water and fumigation treatments are undertaken as prescribed by recipient authorities Germplasm characterization and evaluation e The genebank does not contain a museum collection of germplasm The conserved materials are characterized not only to distinguish different varieties but also to facilitate preliminary selection of germplasm by end users e Morphological and agronomic characters are scored in small field plots during June November using a standard descriptor list Almost all characteristics are recorded using coded qualitative scores Passport data country site and location of collection permit selection of germplasm on a geographical basis We use a range of molecular markers isozymes RAPD AFLP and microsatellites for classification of germplasm used in diversity studies e IRRI scientists have screened thousands of accessions for pest and disease resistance and tolerance to different abiotic stresses Biosystematic studies of the wild species and molecular studies of genetic diversity provide data on species taxonomy and genetic relationships Data management The International Rice Genebank Collection Information System IRGCIS operates under ORACLE
142. ot be used as a guide to provide control measures It is therefore very important to monitor the field regularly all throughout the cropping season from whorl maggots at seedling stage hoppers and stemborers and rice bugs at the reproductive stage The most common diseases that occur are the viruses and leaf diseases The virus diseases such as tungro are critical so spread of the disease should be controlled Eradicate the vector and rogue out and bury or burn the infected plants Field inspection is done by Seed Health Unit to assess severity and presence of these diseases One protocol to prevent unexpected outburst of diseases is to place all incoming materials in an isolated area or quarantine area The quarantine officer and field inspector should also regularly inspect this d Rats This pest is destructive as it attacks all stages of the rice plant Unlike in a single variety field where the damage is easily noticed in this type of cropping several accessions may show no damage but other entries may have already been lost due to rat preference It is therefore recommended to start the control program as early as possible e Maintain field sanitation Clear the surroundings the levees and destroy possible breeding places e If the population is very high and your budget permits construct peripheral rat fences as in the active barrier system ABS e Place rat bait in strategic places all over the field and the surroundings Susta
143. pe Label each one Buffer System Buffer System II and Buffer System III Then apply a thin coating of 50 glycerol to each gel mold 2 Prepare the gels in the following manners see Table 8 1 Table 8 1 Buffer System Buffer System I Buffer System Ii Weigh 77 g of starch Weigh 58 5 g of starch Weigh 56 g of starch Transfer to a 1000 ml Transfer to a 1000 ml Transfer to a 1000 ml Erlenmeyer flask Erlenmeyer flask Erlenmeyer flask labeled with labeled with labeled with Buffer System Buffer System ll Buffer System Ill Add 27 5 ml of Add 45 ml of Add 20 ml of System gel buffer system Il gel buffer system Ill gel buffer Add 522 5 ml Add 405 mi Add 380 ml distilled water distilled water distilled water 3 Cover the flasks with rubber stoppers Stir and preheat using a hot platestirrer until the gels thicken Transfer these into a microwave oven and heat until they boil Deaerate the solution in each flask with a tap aspirator Pour each solution into the properly labeled gel molds prepared earlier Solid particles and air bubbles can be quickly removed with forceps Allow them to solidify and cool Cover each gel with plastic wrapping film Gels can be left overnight inside the refrigerator Crude Extract Preparation for Electrophoresis 1 2 3 Prepare a spot plate by labeling each depression with an accession number of the sample Get 10 young leaves 5 10 day old seedlings of each rice accession in the petri dish Nee
144. products Assembling the glass plate sandwich Adapted from Sequi Gen GT Nucleic Acid Electrophoresis Cell Instructional Manual 1 Thoroughly clean both Sequi Gen GT integral plate chamber IPC glass plate and outer glass plate with liquid soap Rinse plates with dH20 Always wear gloves while handling the glass plates Fingerprints will cause bubbles to form during gel casting 2 Place the outer glass plate flat on a lab table Wipe the entire surface of the outer glass plate with fresh binding solution see Table 8 9 Spread evenly with Kimwipe tissue Allow to dry for 5 min 3 Place the IPC flat on the lab table with the glass plate facing upward Apply 1 ml of Sigmacote Spread evenly over the entire surface of the glass plate with Kimwipe tissue Allow to dry 54 Position one 0 4 mm side spacer along each edge of the IPC glass plate The bottom edges of the spacer and the IPC glass plate should be flush and the long edge of the spacer should be next to the plastic lip of the IPC panel Place the front of the outer plate on to the IPC and spacers with the coated surface facing down With both hands stand the IPC outer glass plate sandwich on the lab table with the outer glass plate facing away from you Slide the clamps over the sides of the IPC assembly The ever of the clamp should be on the IPC panel side of the assembly and facing away from the unit and perpendicular to the IPC panel for the clamps to slide
145. quality with the ultimate goal of obtaining the maximum genetic composition with maximum viability potential This is handled by cropping season and involves the following processes Table 6 1 Countries requiring import permit labels and their authorized channel COUNTRY REQUIREMENTS AUTHORIZED CHANNEL Angola Import Permit As provided by the consignee Australia Without Import Permit Shipment should be addressed to the Australian 71 Bangladesh Benin Brazil Brunei British Solomon Islands Burundi Cambodia Cameroon Central African Rep Table 6 1 con t COUNTRY Colombia Congo Costa Rica Fiji Islands Guyana Import Permit valid for 3 mos from date of issue Import Permit Import Permit label green and yellow tag Import Permit valid for 6 mos from date of issue Import Permit valid for 1 yr from date of issue Import Permit Import Permit Import Permit Import Permit REQUIREMENTS Import Permit Import Permit Import Permit Import Permit Import Permit Plant Quarantine Office as provided by the consignee The Director General c o IRRI Bangladesh Rice Research Institute BRRI House 39 Road 23 Block J Banani Dhaka 1212 Bangladesh As provided by the consignee Empresa Brasileira de Pesquisa Agropecuaria EMBRAPA Centro Nacional de Recursos Geneticos CENARGEN Setor de Areas Isoladas Norte Parque Rural Caixa Postal 10 2372 Brazil Director of Agricu
146. rage Bulk sample for active collection if amount is greater than 60 g otherwise prepare planting materials For the wild rices 1 x 50 species for base collection 2 5 x 20 seeds for prepack 1 x 50 seeds for duplicate storage 23 14 15 bulk sample for Active Collection b For temporary storage insufficient seeds 5 to 120 g and samples with viability ranging from 50 to 85 for japonica and 50 to 89 for indica 1 60 g planting materials 1 or 2 x 100 grain sample for viability testing 1 to 5 x 10 g samples for paper pre packs c For planting low viable seeds lt 50 viability prepare 60 g planting material d For embryo rescue insufficient seeds lt 20 grains For wild rice hand threshing verification and cleaning are done at one time Ten grain sample is taken for viability testing and the cleaned samples are transferred to paper envelopes for final drying Place the cleaned samples again in the drying room while waiting for the viability and seed health test results for the final drying Encode all related information Viability testing This is the most important aspect of seed quality Viability is a measure of how many seeds are alive and can develop into normal plants It is usually expressed as germination Viability is determined before the seeds are packed and placed in the storage and at regular intervals during storage This will serve as a guide to the regeneration of an accession In
147. rginine b naphthylamide AAA 85 Le EA AL MA EE EO DE AE EE OR AE eee 68 85 OER EE EA TE RA ER EI EE 68 85 MORE MA Ee kee Ee 68 85 NADP RE EE Ee eege OO OE tear are 68 85 Mad ae RE EE IE LE EE OAR EE 104 MET E 79 N El EE IE ON N ENE AA EE EES 8 International Rice Genebank ee se ee ER AA ee Re GR AA AA ee ee AR Re ee ee ee ee ee ke ee 8 International Rice Germplasm Center ee ee AA ee Re Ge AA Ge ee Re ee Ge ee ee ee ee 8 E Elle Er EE ee ee oe Ag ea ve a ee ae EE ee ee ge 79 NaMoO42H20 Ee SE Gla ee A GaSe age vee eg ed ee 104 Nanogram i008 8 ean EE il hinted ido 68 lee Oe ie OR AG ele ig Ge ee NR N 73 Ven ER EN A 68 85 Ete ES OAR EE OE AE OE AE ER N EE a erase 79 Plant Genetic Deen HEEN ee AEN 79 National Program cece ccc iii gee SE ee Ee es ee Ee De his Ee Pe ee NEEN 47 National Seed Storage Laboratonm canon cnn ncn rancia 10 30 Nature importance Ee se dee Ee We EES Ve Re ee NEE id 35 depending occ EE ee edi dean ee ae Ae ey ae 35 El KE EE 79 NC Brady Laboratory RR EE feet ER EN 73 N dimethyl formamide EE 85 ye e ME AR EE EE N ER EE EE N N 31 GT ER ME EE EE EE HE OE EG 31 NGO EE 8 Genetic conservation tan 8 Neoealedonm et gtt zeng tick OE OE dale aio 7 8 83 N W Caledonia MAAK EE N RE EE ER ONE RE 7 83 New Delhi 1100 ea EE DE N 79 New Ia ll IE EE EE AE 5 Newly received germplasm AAA 46 PLOCOSSIOO ER RE EE OR e OE OE TE 46 EE RE KO AO ER EE EE EE on EE ORR ON N 64 68 RUE EE OE EE as ORE
148. riety name A local or vernacular name of the sample Former designation Designated name given to the seed by the original source which is later renamed as cultivar by the country doing the selection Seed source Institute agency which donated the seed to the genebank It includes donor identification number Donor code Number assigned by the collector donor Country of origin Country from which the sample originally came Temporary ID An identification assigned to incoming samples in lieu of accession number Scientific name Genus and species name Herbarium specimens taken Indicates presence of herbarium specimen Date germinated dd mm yy Actual date of seed germination Re identified name Verified name given a particular species after full characterization Seed File Information This information will guide the evaluator to authenticate the sample being characterized Population composition 1 homogeneous 2 heterogeneous Lemma and palea color Color of the lemma and palea of mature grains is assessed as 010 white 020 straw 042 gold and gold furrows 052 brown tawny 053 brown spots 054 brown furrows 080 purple 082 reddish to light purple 090 purple spots 091 purple furrows 100 black 999 mixture Lemma and palea pubescence Ocular inspection of mature grains using hand lens and classified as 1 glabrous 2 hairs on lemma keel 3 hairs on upper portion 4 short hair
149. roup and storage conditions Table 3 1 Prepare the seeds Locate and remove the seeds in the cold storage Allow to warm to room temperature before opening Do the germination test following the guidelines see viability testing procedure Compare the result of this test with that of the previous test and decide whether regeneration is necessary Regenerate the accession if the germination has declined to 85 of the initial germination Seed availability monitoring The weight of the seed should be monitored each time seeds are removed from the storage for whatever purpose When an accession in store is less than 60 g 28 in the Active Collection regenerate the accession Facilities Significant fluctuations in the environment during the handling process and storage pose some problems These changes must be noted as soon as possible and immediate remedial measures implemented Some control measures are 1 Daily monitoring of temperature and relative humidity in the work area drying room and cold storage 2 Digital monitor system attached to the drying room and storage areas a chart recorder is also connected to note the fluctuation in temperature and relative humidity during night time 3 The independent refrigeration system back up running alternately on a monthly basis to ensure that it remains in good working order 4 A time switch defrost cycle to maintain the equipment in good shape 5 Door interlock system in th
150. rs 2 3 4 Take out the seeds from the oven and place in dessicator for 1 hr 5 Take the final weight 6 Calculate the percent moisture lost using the formula MC Initial wt of sample Final wt of sample x100 Initial wt of sample Packing Seed packing is done to keep each accession separate and to prevent absorption of water from the surrounding atmosphere after drying The packing medium is dictated by the action taken on the prepared samples Packing materials that are impermeable to water are suitable for long term use for the Active and Base Collections Only O sativa and O glaberrima materials with viability gt 90 are packed for long term conservation except for some materials which exhibited consistently lower viability potential such as japonica glutinous and large seeded materials which have 85 viability cut off The genebank manager makes the final decision to accept or not to accept the materials for long term conservation 25 For the Base Collection moisture resistant rust proof aluminum cans 2 with 60 g capacity are used Since seeds in the Active Collection are frequently retrieved and sub sampled specially made re sealable laminated aluminum foil bags 3 240 x 155 mm have been used since 1992 In this case minimal time is consumed when opening and resealing the foil bag without any additional cost Small packets of the same material are also used to prepare pre pack samples that are readily available f
151. s 5 long hairs velvety 999 mixture Sterile lemma length Measurement is made on each of the two sterile lemmas Six classes are recognized on the basis of 5 grain samples 1 short not longer than 1 5 mm 8 medium 1 6 2 5 mm 5 long 2 5 mm but shorter than the lemma 7 extra long gt the lemma 9 asymmetrical and 999 mixture Vegetative stage Seedling height Ten seedlings are measured for height at the 5 leaf stage 20 DAS Height is taken from the base of the shoot to the tip of the tallest leaf blade using a coded measuring instrument as follows 1 short lt 30 cm 2 intermediate 30 59 cm 3 tall gt 59 cm and 999 mixture Blade pubescence Aside from ocular inspection at late vegetative stage rub fingers from the tip down on the leaf surface Presence of hairs on the blade surfaces are classified as 1 glabrous smooth including ciliated margins 2 intermediate 3 pubescent or 999 mixture Blade color Eight broad classes of blade color are recognized at late vegetative stage 060 green 061 light green 063 dark green 080 purple full 085 purple margins 086 purple tips 089 purple blotch purple mixed with green and 999 mixture Leaf blade texture Based on ocular inspection at late vegetative stage texture is classified as 1 herbaceous having little or no woody tissues soft 2 coriaceous leather like in appearance tough hard 999 mixture Ba
152. s can also be used as a basis in deciding the number of entries to be planted Plant Establishment Field O sativa O glaberrima Refer to Section 2 on Seed Multiplication In cases where the amount of seeds of O glaberrima is limited crop establishment can be done as in O sativa except that after transplanting the field should be kept drained Nursery wild species Refer to Section 4 on Germplasm Nursery and Screenhouses For wet season field establishment please take note of changes in fertilizer rate use a lower nitrogen N and a wider spacing 30 cm to offset the expected increase in vegetative growth Morpho Agronomic Characterization Morphological and agronomic characters of plants are best scored at different growth stages of the crop thus characterization is done at three different stages vegetative reproductive and at post harvest stages Post harvest characteristics are scored in the laboratory from the panicle samples that are taken at harvest time There are characters that are unique for particular species as shown in the sample data sheets used for characterization of O sativa O glaberrima and wild species see Appendices 5 1 5 2 and 5 3 respectively Descriptor and descriptor states Accession identification International Rice Genebank Collection IRGC Accession no A unique identification 34 number assigned to a sample once it has satisfied the minimum storage requirements of the genebank Va
153. sal leaf sheath color Color of the outer surface of the leafsheath at early to late vegetative stage is classified as 060 green 080 purple 081 light purple 084 purple lines and 999 mixture Leaf angle 1 9 999 The angle of openness of the blade tip is measured against the culm on the leaf below the flagleaf at late vegetative stage 1 erect 5 horizontal 9 drooping and 999 mixture Ligule shape Five classes are taken at late vegetative stage for O sativa O glaberrima 35 and at early reproductive stage for the wild species 0 absent 1 acute to acuminate 2 2 cleft 3 truncate and 999 mixture Ligule color Five classes of ligule colors are recognized at late vegetative stage 000 absent liguleless 011 whitish 080 purple 084 purple lines and 999 mixture Collar color Collar color at late vegetative stage is 000 absent collarless 060 green 061 light green or 080 purple and 999 mixture Auricle color Auricles at late vegetative stage are 000 absent auricleless 061 light green 080 purple and 999 mixture Reproductive stage Number of days from effective seeding date to 80 heading For wetland culture use the date on which sowing on a wet seedbed or soaking of seed was made For direct seeded rice use the effective seeding date to indicate the date when rain or other moisture become available to the seed for germination For the wild species the
154. se of materials that have low viability during routine monitoring after a period of storage or accessions with insufficient stocks for either distribution or for long term conservation e A special seed multiplication for accessions that are frequently requested or with special characters that breeders and researchers usually use It also includes seed requests that need more than the standard amount the genebank normally distributes i e 10 g 1 Seed preparation and assignment of plot numbers Registered accessions e Request seeds of the selected accessions Two computer generated lists will be produced per species lists of accessions with prepared planting materials lists of accessions with no planting materials but with other sources e Prepare the planting materials using the following procedure 1 Accessions with prepared planting materials withdraw the prepared samples 2 Accessions with no prepared planting materials a write the accession number and the corresponding seed source on the seed envelope b withdraw the seeds 1 c arrange the materials by ascending number to facilitate verification or comparison with the seed file d secure the seed file and compare the grain characters to authenticate purity and identity of materials e select the seeds based on seedfile verification f for O sativa and O glaberrima e determine the number of rows based on the available seeds using the following guidelines insufficient with very f
155. see Table 8 9 for preparation of MgCl and dNTP mix Component Stock Volume Final Volume 40 Concentratio ul Concentratio rxn ul n n SUPH O 14 1 564 53 PCR buffer 10x 2 5 1x 100 MgCl 15 mM 1 7 1 0 mM 68 dNTP mix 5 mM 0 5 0 1 mM 20 Primer reverse 10 uM 0 5 0 2 uM 20 Primer forward 10 uM 0 5 0 2 uM 20 Commercial Puiu 0 2 tu 8 Taq Polymerase Note Homemade Taq Polymerase of 1 unit final concentration can be used Mix the cocktail by flicking the tube and spin down to collect the mixture 4 Add 20 ul of cocktail to the genomic DNA and mix gently by flicking the tube 5 Overlay the mixture with 1 drop 10 ul mineral oil Spin down to collect the mixture for tubes only 10x PCR buffer Stock Concentration Final Concentration Amount Tris HCl 1 M 100 mM 2 5 ml KCL 2 M 500 mM 6 25 ml Gelatine 2 0 1 1 25 ml Amplification 1 Place tubes plate in a thermal cycler and allow amplification to proceed with the following temperature profile Temperature C Time min No of cycles 94 5 1 94 1 35 55 1 72 2 72 5 1 Hold temperature 4 C 2 After amplification remove tubes plate from thermal cycler and add 12 5 ul 3x STR loading buffer see Table 8 9 Store at 4 C for further use Note PCR conditions for microsatellite markers will vary depending on the individual PCR machine and the actual primer used Annealing and denaturation temperatures and MgCl should be adjusted in order to obtain amplified
156. sing Although rice is considered as a self pollinating species a study conducted by Reafio and Pham 1998 Appendix 2 1 has shown that outcrossing can occur and as the exsertion of the stigma increases the chances of cross pollination also increases It should also be noted that to facilitate management of plots materials are sorted by maturity thus chances of simultaneous flowering are high for adjacent entries Also mark the end of the plot about 5 m wide Make sure all the bamboo stakes labels face the levees This will facilitate field observation later as the inspector evaluator can walk on the levees Seedling distribution This operation includes transfer of seedlings from the seedbed to the field Extra care should be done to prevent interchanging plot numbers and arrangement problems before these seedlings are transplanted e Distribute the seedlings in S type orientation wherein lower plot numbers start from the left going to right and across strips and blocks This technique will help in renumbering of plots in cases of lost tags e Untie the seedlings and attach the tags to the bamboo stakes to serve as the plot labels e Fix the seedlings at the base of the bamboo stake making sure that they are intact and the roots in contact with the soil to prevent further stress due to delays in transplanting e Lay out the entries with insufficient seedlings separately in smaller plots arranged by ascending plot number e Take note
157. soil e Split application of nitrogen is preferred as this element is mobile and can easily be lost a First top dressing is done at about 21 days after transplanting DAT or at maximum tillering for all nurseries This is done after first weeding at the rate of 30 0 0 kg N P K b Second top dressing is done by nurseries This is approximately at panicle initiation PI and since we are dealing with diverse germplasm wherein PI is not simultaneous early maturing and japonicas should be fertilized earlier at the rate of 30 0 0 kg NPK Other nurseries can also be fertilized but only at 20 0 0 kg NPK and the next 10 kg N applied about 10 days after on a selective basis spot application Amount of fertilizer kg ha 1 Recommended rate RR kg x 100 nutrient in fertilizer 16 Irrigation and water management As mentioned earlier the field should have a good irrigation system and excellent drainage to permit good water control During land preparation the water level should be maintained to keep the soil soft prior to transplanting Maintain 3 5 cm level of water during the early stage of crop to control early growth of weeds unless the snail population is high This will enhance effectivity of applied pre emergence herbicides Irrigation is done intermittently throughout the cropping season so as not to submerge all the materials If most of the entries are already mature and irrigation is still required to supplem
158. sphate 10 units dehydrogenase Just before mixing with agar solution MTT 10 mg mI PMS 1 mg mi Mix with 25 ml 2 agar 0 5 g 25 ml dH20 formerly brought to 80 C boiling and kept at 60 C Immediately pour into the stain box The starch gel is later placed on the agar layer Protect from light Incubate for 25 min at room temperature Shikimic acid 25 mg mi Tris HCI buffer 0 5 M pH 8 5 NADP 5 mg ml Distilled water Just before use add MTT 10 mg mI PMS 1 mg mi 50 mg 20 ml 2 ml 1 mi 1 mi 1 ml 1 ml 1 mi 10 ml 1 mi 36 ml 1 ml 1 mil 83 Protect from light Incubate for 30 min at 50 C Reference Glaszmann J C B G de los Reyes and G S Khush 1988 Electrophoretic variation of isozymes in plumules of rice Oryza sativa L a key to the identification of 76 alleles at 24 loci IRRI Res Pap Ser 134 Table 8 3 Substrate staining salt and co factor stock solutions Stock solution Qty mg ml Qty 25 mI dH O dH20 mg g 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetra zolium bromide thiazolyl blue 10 250 0 250 MTT Glucose 6 phosphate 10 250 dehydrogenase G 6PDH units units DL isocitric acid 100 2500 2 500 a naphthyl acetate 50 1250 1 250 in acetone in acetone in acetone B naphthyl acetate 25 625 0 625 B nicotinamide adenine dinucleotide 10 250 0 250 NAD B nicotinamide adenine dinucleotide 25 625 0 625 NAD for Mdh
159. supH20 volume to MgCl 500 mM supH O volume to NaOH upH O volume to AgNO H2CO 37 upH20 NaOH 5M Formamide Bromphenol blue Xylene cynole FF upH2O volume to Tris EDTA Na Boric acid upH20 volume to Amount 74 55 g 500 mI 254g 25 ml 0 75 ml 25 ml 100g 500 ml 29 3 ml 2000 mI 0 2 ml 95 ml 50 mg 50 mg 100 ml 108 g 939 55 Y 1000 ml 95 Table 8 9 con t Solution TBE buffer 1x TBE buffer 0 5x TE buffer pH7 8 10x TE buffer 1x Tris HCI 1M Chemical Composition TBE buffer 10x upH20 volume to TBE buffer 1x upH20O Tris 500 mM EDTA 50 mM supH O volume to TE bufer 10x upH2O volume to Trizma base HCl upH20 volume to Amount 200 ml 1800 ml 1000 ml 1000 ml 1 ml 1 ml 100 ml 10 ml 90 ml 60 55 g 21 ml 500 ml Table 9 1 Stock solutions for culture media Solution Chemical Components A Ammonium nitrate NH NO3 Potassium nitrate KNO3 Potassium phosphate KH2PQO dH O volume to B Calcium chloride 2 hydrate CaCls 2H O dH O volume to C Magnesium sulfate 7 hydrate MgSO 7H20 dH O volume to Amount 8 250 y 9 500 y 0 850 g 500 ml 2 200 g 500 ml 1 850 g 500 ml 96 Potassium iodide Kl Sodium molybdate 2 hydrate NaMoQ 2H 0 dH O volume to 0 415g 0 125 g 500 ml E Cupric sulfate 5 hydrate CuSO4 5H O 0 125 g Cobalt chloride 6 hydrate CoCl 6H20 0 125 g OHIO vol
160. t N ER EE EE RE 17 1 1 2 Prepare oc it AE ee e Deeg 64 EA EE EE N EE RE EE EN 85 Ede ER RE AR ME EE AE EE 56 1 75 dadarose Me ie S Ee EE EG EE 56 CTT Lull io ei EE ee 56 10 UpH2O EE EK RE iii 60 lune Wu ER N EE RE ER debated aged EE IR EE N 85 100 aa ER AE OE EE EE EE 60 e ele EE ee es 74 LULL Ly ER reer RE ER errr reper EE OE EE freee 60 100 ER OE ER EN OE OE OR EE EE ES 58 85 DO Gh ER EA AE EE 85 UU TU EE c 39 ar EE EE ER OR OE EE EN 39 Uu Te RE N N OE NE N 39 MA AN EE AA eee 56 58 60 85 58 TOX PCR EE EE A EO EA EE AR EENet 60 64 e EE N N N OE a et ON Ee E TE E 39 EER OO a N EO N N EE 31 ORE EE RA OK ON EE OE N ne ees 73 ld AR AR AE RA EA ER EE EE NEG 13 125 EN 73 EE EE EE RR EE EE N wares 15 kaders ER EE EE N N 39 15 POM ANNIING RR EE EE A 64 V5 MM ER MA RR EE EO N GRA ON NG 85 em EE EE RE EE EA 8 17 1520 di EE EE EE EE EE A 56 AE OE EE TE OE EE N 56 RR EE EE EE ta 17 EE N 39 100 MERE EE EE HR OE OOR OR EE ER 85 E Wl E E 39 be EE ER EE EO o tere SONES 58 60 64 85 IX NEE 60 IX EE EE OE EE Onda ino nro oda 60 2 2 dus ac 17 blocks 2e a encore EE 17 5 diphenyltetrar OE EO EE EE 85 20 elle AA EE AE OR A RE ER 28 60 EER EEN eens as 56 58 64 OCA 56 AO 58 64 RE AI ae ee ee 10 200 ll Te EE AE EE EE EL N EE RE HE HA EE 60 ARE EE EE EE ME EE EE IE DE N 85 2 8 settle Eeer Ge GE A tt 15 AR RE AE RE RE AO EE ET TO E 56 5600C EE ee tne ote Ee ats cia elas Uae ee EE EE RE ED
161. t filter paper at 30 C in an incubator 2 Harvest 1 to 2 mm root tips and pre treat with 002 M hydroxyquinoline for 3 h Protect from light Pre treatment is best started at about 9 00 A M 3 Fix with fresh 3 1 ethanol acetic acid for 24 h at 4 C 4 Store in 70 ethanol at 4 C 5 For staining submerse roots in 2 aceto orcein and store at room temperature for at least 5 days before squashing Slide preparation 1 Transfer one root tip to a clean slide and remove root cap Cut off a small piece and add a drop of 45 acetic acid 2 Place a cover slip with edge raised on top of a razor blade to allow for cell movement during squashing 3 Squash gently with the tip of a rod and remove the razor blade gently when cells are sufficiently squashed 4 Apply enough pressure to flatten cells 5 Temporarily seal slides with Hoeyer s solution Stains and reagents Snow carmine Orcein 4 g carmine 15 ml distilled water 1 ml HCL 95 ml 85 ethanol 1 Mix 4gcarmine 15 ml distilled water and 1 ml HCL 2 Stir boil gently for 10 min 3 Cool and add 95 ml 85 ethanol 4 Filter 2 g orcein 100 ml 45 acetic acid 1 Add 2 g orcein to 100 ml 45 acetic acid 2 Stir boil gently for 10 min 3 Cool and filter 67 Hoeyer s solution 30 ml distilled water 3 g gum arabic 25 g chloral hydrate 3 ml glycerin Pos Pre treating agent Dissolve 3 g of gum arabic in 30 ml distilled water for 24 h
162. ter hydrochloric acid formaldehyde hydrogen peroxide isocitrate dehydrogenase E C 1 1 1 42 integral plate chamber potassium chloride kilobase 62 KH2PQ KI LAP mA Mdh ME mg Mg MgCl MgCl 6H20 min ml mm mM MTT MW NaCl NasCO NAD NADH NADP NasHPOZH O NaOH Na2S205HO ng NH potassium phosphate monobasic potassium iodide aminopeptidase leucine substrate molar milliampere s malate dehydrogenase E C 1 1 1 37 malic enzyme E C 1 1 1 40 milligram s magnesium magnesium chloride magnesium chloride 6 hydrate minute s milliliter s millimeter s millimolar s 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide Thiazolyl blue molecular weight sodium chloride sodium carbonate nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide reduced form nicotinamide adenine dinucleotide phosphate sodium phosphate dibasic 7 hydrate sodium hydroxide sodium thiosulfate nanogram s 10 g ammonium 63 PCR Pgd Pgi PMS POX PVP RAPD RNA rpm rxn Sdh sec supH20 Taq TBE TE TEMED tris UV Ug ul uM upH20 polymerase chain reaction posphogluconate dehydrogenase E C 1 1 1 43 phosphoglucose isomerase E C 5 3 1 9 phenazine methosulfate peroxidase E C 1 11 1 7 polyvinylpyrrolidone random amplified polymorphic DNA ribonucleic acid revolution per minute reaction s shikimate dehydrogenase E C 1 1 1 25 second s sterile ul
163. the bottom of each tube and spin to collect the solution This will be use for pre amplification check 9 Check the pre amplified DNA by running the sample through a 1 2 agarose gel Follow the procedure in the restriction digestion check Selective amplification 1 Prepare Mix 1 by adding the following components in a 1 5 ml microtube enough for 10 reactions 5 ul EcoR1 primer 45 ul Mse1 primer 50 ul total volume Mix gently by flicking the bottom of each tube and spin to collect the solution 2 Prepare Mix 2 by adding the following components in a 1 5 ml microtube enough 59 for 10 reactions 20 ul 10x PCR buffer plus Mg Ju Taq 100 ul total volume Mix gently by flicking the bottom of each tube and spin to collect the solution 3 Prepare the AFLP amplification by combining the following components in 0 5 ml microtubes 5 ul diluted pre amplified DNA 5 ul Mix 1 10 ul Mix 2 20 ul total volume Mix gently by flicking the bottom of each tube and spin to collect the solution Overlay with 1 drop of mineral oil 4 Place PCR tubes in a thermal cycler Amplify using the following temperature profile Time No of cycles Temperature C 94 30 sec 1 65 30 sec 72 1 min 94 30 sec 1 64 30 sec 72 1 min 94 30 sec 1 63 30 sec 72 1 min 94 30 sec 1 62 30 sec 72 1 min 94 30 sec 1 61 30 sec 72 1 min 94 30 sec 1 60 30 sec 72 1 min 94 30 sec 1 59 30 sec 60 72 1 min 94 30 sec 1 58 30 sec 72 1 mi
164. their habitats are destroyed by human disturbance Future crop improvement needs the genetic variation from traditional varieties and related wild species to cope with the many biotic and abiotic stresses that challenge rice production around the world The genebank IRRI has maintained a collection of rice genetic resources since 1962 The collection comprises more than 107 000 accessions mostly landrace or breeding materials of O sativa O glaberrima and wild species and representative species from 8 genera in the tribe Oryzeae Table 1 3 The genebank has been open since 1977 and underwent a major renovation in 1993 and 1994 with the addition of a seed drying room at 15 C and 15 RH It has the following facilities an Active Collection for medium term storage 20 40 years stored at 2 C a Base Collection for long term 50 gt 100 years conservation at 20 C two screenhouses with a combined area of gt 4 000 m2 One is used for the cultivation of low viability or low seed stock accessions of cultivated rice The other is used exclusively for the cultivation of the wild rices in pots or special propagation beds a seed testing and germplasm characterization laboratory a data management laboratory with four workstations equipped with powerful Pentium microcomputers and several servers dedicated to GRC databases applications and office files These computers are connected to the IRRI local area network a conservation
165. to pre packed seeds application for Phytosanitary Certificate MTA and updating of seed stocks Processing of seed requests and preparation of seeds Follow the step by step procedures below for specific requests to be processed Retrieve batch to be processed Print all accessions requested Pick source for distribution Pre packed seeds is the first priority to be selected for distribution depending on purpose and viability Generate the verified list with selected sources for distribution Manual check withdraw seeds from storage room based on the selected source If there is no available pre packed seed sample prepack 10 g sample for distribution for O sativa and O glaberrima and 20 seeds for wild species and pre pack the remaining amount if seed is from cans If the source is in bulk prepare another 2 pre packs for storage Edit deselect seed source for distribution if there are sources to be changed Print proof list of final selected sources Proofread selected sources with the label on the seed packet Update database with newly prepared packed seeds and seedstock Print labels for newly prepared pre pack samples Label newly prepared seed sample Print Phytosanitary Certificate Application Form for seeds to be sent abroad Print final seedlist Print the Material Transfer Agreement MTA for FAO designated germplasm for all seed requests at IRRI or outside Appendix 6 1 No MTA is used for restoration of germplasm to
166. tocol OE Es Gee A AE 47 Seed File Information Es RENEE GP GEE Ee GP ee EG ON GREG SE Ai de SE Ke Ge GE Ge Ee Gegee Ge geed BEER veg 39 Seed H E RE AE AE ER AE N OS RE EN 14 Seed health evaluation i s EER canes 28 Seed Health Testing ER tt added 31 Seed Health Unit eo EERS ERENS RE oa Ge ee gs Ek ee es 10 17 30 46 47 79 Seed multiplications EE EE OE OE OE DE 13 39 ONY Za SAtiVa MR EE EO RR OE T 13 Seed Preparation Hs its BERE BERE es a EG KG SSA EE SEEK SE Ge SEK eee eu EN 14 Seed Processing Area OE EE EO OE EE EARS 35 Seedbedactivitie secs cans eerste Ek Se Se GE OGE EN GE EE Dee sue bee ee RE be ee ed bee ee Gegee Ge ee EN 15 TT ele ORE OE RE EE RE OE N 14 UCI TE ie RE EE EE RE ER EE EE 14 E Il ee 52 seecdtile ia E 14 17 23 25 consUIt EE DE DE A Ee Gee DE GE ca deat aie Pe Guag tense ake ef 17 Seedlist Ee 14 46 47 ACCOMPANYING EE AR OE N NE ORE NE EE ME OS 46 COPY AE EE N EE EE OR EE AE EE OE OE OE EE ER RR 46 Genera ata A RE ED Ee ee De GE Ee RE OG EE EN Ee Gee Ed ED EE ee AG 14 Seedslock EE E EG Pe Ee Se Ge Ed dinate AE vee Ee Re VR EE TE EE Ee de Ed ED 28 47 Selection EDE ED ER A AI RR ER EE GE ee Ee 13 25 39 VEL EER N NE Rd EO NE AR N NE RE L EEA 39 Seplember 1994 EO AE EA EE EER OE ai 10 49 eli REKE AE EE OR EE N N 60 Sequi Gen GT Nucleic Acid Electrophoresis Cell 60 GT EE AEA EE EE EE N OE EE N Ie EE nase 7 83 85 AREA ETE NE ME EN EE OO N agents 79 EE RE ER RE ER OE ER HI OE E P NR 15 DA ODE
167. tosensitive materials use older seedlings e g 30 day old seedlings Weeds There are critical stages of crop growth where weeds have significant effects on yield They should be controlled during the first three weeks after transplanting as they can compete for sunlight and fertilizer applied to rice crop Presence of weed seeds reduces the quality of harvest Combination of chemical cultural mechanical and manual weed control is more practical and economical than any single method alone These practices should be followed e Apply pre emergence herbicide immediately after transplanting to give ample time for the seedlings to recover and grow much ahead of the weeds This should be combined with good water control e Use rotary weeder or hand weeding at approximately 21 DAT or when the weeds start to grow e Hand weed all the remains usually at the base of the hill at about two weeks after herbicide application Spot weeding may also be done at a later stage Application of post emergence herbicide during the later stages is not advisable when dealing with germplasm because of the variation in crop stages which may affect susceptible entries c Insect pests and diseases Germplasm materials are precious so that any loss due to pests should be avoided Therefore an intensive pest and disease control program should be implemented Threshold levels for insect pest populations as indicated in the Integrated Pest Management IPM should n
168. trapure water Thermophilus aquaticus tris borate EDTA tris EDTA N N N N tetramethylethylenediamine tris hydroxymethyl aminomethane ultraviolet microgram s 10 g microliter s 10 micromolar ultrapure water volt s Watt s 64 Chapter 9 Conservation Support Conservation support The Conservation Support Laboratory is located in the basement of the NC Brady Laboratory There are facilities for cytological studies including meiotic analysis and chromosome number determination of wild rice accessions and the tissue culture of low seed stock or low viability accessions Post harvest morphological characterization of materials for biosystematic studies is also conducted in this laboratory In vitro germination Preparing the seeds 1 1 Break the dormancy of the seeds by the following procedures a For Oryza sativa stabilize the seeds from the medium term storage at room temperature for 2 days Put the seeds in small packets and place them inside the oven at 50 for 3 5 days Take the seeds out of the oven and stabilize at room temperature for 2 days Remove the seeds hulls b For the other Oryza species stabilize the seeds from the medium term storage at room temperature for 2 days Follow the dormancy breaking protocols in Section 3 of this manual Seeding on agar medium All steps in this section should be done inside a laminar flow 1 2 3 8 9 Transfer the seeds from each pac
169. ul of reaction mixture Components Stock Final Vol Rxn Concentration Concentration dNTPs 100 mM 0 8 mM 0 2 ul NH rxn 10x 1x 2 5 ul buffer MgCl 50 mM 2 5 mM 1 25 ul Taq 5 u ul 1 u rxn 0 2 ul Primer 10 uM 0 4 uM 1 0 ul SUDH O 17 35 ul DNA 2 ng ul 5 ng ul 2 5 ul DNA amplification 1 Place PCR tubes in a thermal cycler Amplify using the following temperature profile Temperature Time No of cycles C 94 2 min 1 94 30 sec 2 37 1 min 72 2 min 94 30 sec 2 35 1 min 72 2 min 93 30 sec 41 35 1 min 72 2 min 72 5 min 1 Hold temperature 25 C Note Conditions optimized for Hybaid OmniGene thermalcycler 2 After amplification remove the PCR tubes from the thermal cycler Add 3 ul of 10x 52 loading buffer see Table 8 8 to each tube Mix by flicking the bottom of the tube and spin to collect the mixture The mixture is now ready for loading in the agarose gel Electrophoresis 1 Geta gel mold and seal both edges with 1 masking tape Place in a level platform and attach combs 2 Prepare 1 4 agarose by weighing 3 5 g agarose Transfer this to a 500 ml flask and add 250 ml 0 5x TBE buffer see Table 8 8 3 Boil for 6 min in a microwave Allow the solution to cool to 60 C 4 Pour agarose unto the gel mold and allow to solidify 5 Fill the electrode tank with 0 5x TBE buffer 6 Remove masking tape from both ends of the gel mold Mount the gel mold on to the electrode tank making sure no bubbles for
170. ume to 500 ml F Boric acid H3BO3 0 310g Manganese sulfate 4 hydrate 1 1159 MnSO 4H gt 0 Zinc sulfate 4 hydrate 2nSO 4H20 0 430 g Solution D 50 ml Solution E 5 ml dH O volume to 500 mI Table 9 1 con t Solution Chemical Components Amount G EDTA disodium EDTA Nas 1 865 g Ferrous sulfate 7 hydrate 1 390 g FeSO 7H O dH O volume to 500 mI H Glycine 0 100 y Nicotinic acid 0 025 y Pyridoxine HCl 0 025 g Thiamine HCI 0 005 OHIO volume to 500 ml Myo inositol 5 09 dH O volume to 500 mI Table 9 2 Amount of stock solution per liter of culture media Stock Solution ITOTODP Sucrose Agar Amount per liter solution Full Strength 1 2 Strength 25 ml 12 5 ml 25 ml 12 5 ml 25 ml 12 5 ml 10 ml 5 ml 10 ml 5 ml 10 ml 5 ml 10 ml 5 ml 20g 10g 6g 6g 1 4 Strength 6 25 ml 6 25 ml 6 25 ml 2 5 ml 2 5 ml 2 5 ml 2 5 ml 5g 6g 97 Preparation of culture media Dissolve sucrose in 300 ml distilled water in a 1 liter volumetric flask 1 2 3 4 5 6 7 Add stock solution A B C F G H and and fill to 1 liter volume with distilled water Transfer the mixture to a 2 beaker and add 6 g agar Mix using a hotplate stirrer Boil until solution is clear Allow the solution to cool to 50 C Adjust the pH of the solution to 5 8 Dispense at 20 ml per 5 x 1 or 8 x 1 I x d test tubes or 30 ml per 125 ml Erlenmeyer flask Autoclave for 20 minutes Table 9 3
171. uring electrophoresis Gently open the origin and insert the filter paper strips side by side starting at the left side working to the right Then make sure to rinse the forceps tips in distilled water and dry before proceeding to the next depression in the spot plate Repeat this procedure until all strips have been aligned along the origin which should be able to accommodate 30 strips This loading procedure will also be done for the other gels Electrophoresis 1 2 Staining 1 2 Replace the plastic wrapping film on the gel s surface Remove the masking tape from both ends of the gel mold tray Inside a refrigerator at 4 5 C mount the gel mold tray on to 2 electrode trays containing electrode buffer solution see Table 8 1 Connect this set up to a direct current source preferably a DC power supply Turn the power supply on and runa 50 mA current for 4 hours through it Review the connections and make sure the current is flowing in the right direction before closing the refrigerator door Do this for the other 2 gels Using the stain recipes provided see Tables 8 2 8 3 and 8 4 for stock solutions prepare the stains for each enzyme to be tested Pour each staining solution in the appropriate staining trays After the 4 hour run turn off the electric current and remove the gel mold tray from the refrigerator Remove all of the plastic wrapping film The gel slab should have a light blue line towards the anodal en
172. urned and buried 32 e If sufficient seeds are obtained old plants are discarded burned and buried into the pit f Excess planting materials seeds seedlings rattooned tillers are collected burned and buried after seeding transplanting and or replanting g Discarded soil used in growing is treated with herbicide and buried in the designated area h Before filling up all the discarded materials the disposal area is treated with a non selective herbicide e g glyphosate i Screenhouse staff are advised to change their working clothes to minimize dispersal of seeds when they leave j Hand threshing and seed cleaning is done in a specified room in the Seed Processing Area All dried leaves straws unfilled grains mixtures and off types are collected burned and buried k Access to GRC Screenhouse is regulated depending on the nature importance of the visit Modifications to these protocols are being constantly developed to determine the best conditions for individual species The screenhouse facilties are continuously being upgraded to suit their specific needs However understanding plant morphology and knowledge on the natural growing habitat of the different species are significantly important in the initial seed multiplication of this germplasm These will provide the necessary informations on how to properly manage the species 33 Chapter 5 Germplasm Characterization and Evaluation Introduction to germplasm c
173. va O glaberrima species 12 colors of lemma and palea are observed when the terminal spikelets are ripened 010 white A1 020 straw 042 gold and or gold furrows on straw background 052 brown tawny 053 brown spots on straw 054 brown furrows on straw 082 reddish to light purple 090 purple spots on straw 091 purple furrows on straw 080 purple 100 black and 999 mixture Stigma color Color of stigma is classified as 010 white 030 yellow 051 light green 080 purple 081 light purple and 999 mixture Stigma color is determined at flowering between 9 a m and 2 p m with the aid of a hand lens Anther length Exact measurements in mm from 5 samples taken at flowering Chromosome number Determined through pollen samples taken at booting stage or from the root tip of germinating seedlings Leaf length Five leaf lengths are measured in centimeters from the topmost leaf blade below the flag leaf on the main culm using a coded measuring instrument 1 very short lt 21 cm 2 short 21 40 cm 3 intermediate 41 60 cm 4 long 61 80 cm extra long gt 80 cm This is taken at reproductive stage For the wild species actual measurements in cm are taken from 5 leaf samples Leaf width Width is measured at the widest portion of the blade on the leaf below the flag leaf using a coded measuring instrument at late vegetative stage 1 narrow lt 1 cm 2 intermediate 1 2 cm 3 broa
174. velope individually to its corresponding assigned row Review distribution by checking the sequence of distribution and the labels e Sow the seeds evenly on the assigned rows for each accession It will be useful to retain few seeds in the envelope and keep them This will help in tracing any errors that might occur subsequently This can also be used in confirming the type of seeds that were used during seeding e Cover the seedbeds with garden or topsoil For the dry bed use the soil from around the beds but for a modified wet bed use well sieved garden soil e Apply a small amount of fertilizer about 5 g m of ammonium sulfate e 21 0 0 or approximately 10 kg N per hectare e Apply granular insecticide e g carbofuran to control ants and crickets This can also control nematodes e rrigate the seedbeds with an overhead sprinkler for the dry bed and by flooding in the modified wet bed Do not submerge the beds to avoid mixing up of seed samples Irrigation can be done intermittently e Record germination rate 7 days after sowing 7 DAS The non viable accessions will be re seeded either in the nursery area or in the laboratory embryo rescue to save these accessions in time e Monitor and control any seedling pests Pulling and pre pulling preparation e Prepare plot tags using wooden pot labels tied with G I wire gauge 22 or 24 This wire will also be used to tie the seedlings Print the plot numbers on the pot label using in
175. veral types consult the seedfile The final purification is done during seed processing Harvesting and Post harvest Activities At about 28 35 days after anthesis the materials are ready for harvesting Do not wait for full maturity as it will affect the storage potential of the seed Kameswara Rao and Jackson 1996a b and c Appendices 2 2 2 3 and 2 4 Start harvesting from the nursery which flowers first usually japonica and early maturing entries in the morning to give time for threshing and blowing in the afternoon Take note that early morning harvesting might not be good since dew is still present and will increase seed moisture content For O glaberrima delays in harvesting will result in shattering and collection of very few seeds Extra care in handling is also needed during harvesting Cut the panicles using a sickle and place them inside cloth bags with corresponding labels Run down the whole field to be able to get all the materials ready for harvest Several passing are needed to collect all the entries as the maturity of the materials varies Record the harvest dates and encodes them in the computer after each round of harvesting Thresh the harvest using a Vogel type thresher designed to be self cleaning to minimize mechanical mixtures Blow the harvest using Almaco blowers where air is blown out while the grains are slowly sieved through removing unfilled grains and stubble After seed blowing transfer the
176. version 8 0 and can retrieve information about origin and morpho agronomic and evaluation traits of germplasm and facilitates selection based on pre determined criteria Information in the genebank database is freely available to users all over the world An integrated information system links all operations associated with germplasm conservation and management We are planning to make IRGCIS accessible through the Internet It can currently accessed through the System wide Information Network for Genetic Resources SINGER at http www cgiar org singer Chapter 2 Seed multiplication of Oryza sativa and O Glaberrima germplasm Seed multiplication of Oryza sativa and O Glaberrima germplasm One of the major operations of the genebank is production of high quality seeds for long term conservation Cost efficiency of regeneration is maximized when the seed quantity is just sufficient to provide enough for Base Collection storage and Active Collection use before the viability drops below a given threshold Thus the frequency of regeneration can be reduced by improved storage facilities as well as the needs for usage It is almost impossible to predict how much seed is needed by other users and therefore it is the task of the genebank manager to determine the optimum amount of seed that should be produced Most genebanks follow the requirements set for specific crops For rice an inbreeding crop the target number of seeds for storage should rang
177. ws needed Some suggestions as to the plant spacing are a Japonica and dwarf varieties 20 cm x 20 cm to 25 cm x 25 cm with 8 10 rows per accession b Tall and medium early or medium late low to medium tillering 25 x 25 cm to 25 cm x 30 cm can be used For low tillering accessions increase the number of rows to obtain the needed amount of harvest c Photosensitive materials and high tillering 30 cm x 30 cm spacing is required Wider spacing is almost a necessity in this type because they tend to produce vigorous vegetative growth if not subjected to short days to induce flowering d For initial seed increase a 30 cm x 30 cm spacing is advisable to be safe as there is limited information about the materials At the IRRI ES where the fields are divided into equal blocks of 2 500 m 25 m x 100 m including bunds or levees the blocks are subdivided by constructing middle levees The active area per sub plot is about 99 m x 11 5 m These subplots can allow two strips Measure about 0 5 m from the side levees at both ends of the field Place abaca twine to straighten the marker bamboo stakes to be placed Place bamboo stakes to mark each plot Using a planting guide of desired row spacing measure the width of the plots by marking the needed number of rows Add one extra row to serve as the divider between plots It will be important to have vacant row in between or a safer distance of about 0 75m to minimize possible outcros
178. y inversion Centrifuge at 13 000 rpm for 5 min 9 Pour off isopropanol and stand tube upside down on a paper towel for a few minutes 10 Add 300 ul of 70 EtOH and centrifuge at 13 000 rpm for 5 min 49 11 12 13 14 15 16 Method 2 1 w 9 OR 10 11 12 13 14 15 16 18 19 Pour off EtOH and stand tubes upside down for a few minutes With a piece of Kimwipes ensure that any film of EtOH is removed from the neck of each tube Dry the DNA pellet in a vacuum dessicator for at least 20 min Take up DNA pellet in a 100 ul supH O Incubate at 37 C for 2 h To remove RNA from the sample add 1 ul RNAse A pre boiled at 100 C for 5 min see Table 8 5 Incubate at 37 C for 30 to 60 min Centrifuge at 10 000 rpm for 10 min Take out 90 ul of the supernatant and transfer to a new tube Add 20 ml 1 5x CTAB preheated at 65 C for 30 min see Table 8 5 to 5 8 g homogenized leaf sample in a 50 ml centrifuge tube Warm in a water bath at 56 C for 20 min with mild shaking Remove tubes from water bath and allow to cool down for 4 5 min Add 20 ml chloroform isoamyl alcohol 24 1 Incubate for 20 min at room temperature with mild shaking Centrifuge at 2 800 rom for 20 min Use a refrigerated centrifuge Collect the supernatant and transfer to a new tube Add 2 ml 10 CTAB see Table 8 6 and mix Add 20 ml chloroform isoamyl alcohol 24 1 Mix by shaking at room temperature for 20 m
179. y should be stored for long term preservation Seed health evaluation determines the extent to which seeds are infected with diseases Since germplasm is distributed worldwide and every country has its own set of quarantine requirements seed health evaluation provides information on whether the materials will be acceptable worldwide Early determination of this information will enable the genebank staff to immediately replace infected samples Seed health testing requires trained staff In this case standard routine seed health testing Mew amp Misra 1994 1 is done by the IRRI Seed Health Unit refer to seed health management guidelines see Appendix 3 5 Materials beyond the allowable limit set by the Plant Quarantine officers are stored temporarily and enlisted for the next multiplication Necessary steps are done to avoid disease recurrence Final drying During the cleaning process seeds take in moisture To attain the desired moisture level the seeds are placed again in the drying room at 15 RH and 15 C for 1 week while waiting for the viability and seed health results With these pre set conditions it is not necessary to determine moisture level of individual accessions Instead a fixed time to attain the desired 6 MC is pre determined Moisture content is determined following the ISTA rules 1 Grind the sample Weigh 5 g sample in metal container with cover Place the weighed sample in oven set at constant 130 C for 2 hou

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