http://www.bio-protocol.org/e1484
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1. 1 solution 1 for 100 ml 0 1 mol L citric acid 10 ml of 1 mol L 1 v v Triton X 100 1 000 ul Fill up with water conductivity gt 18 MQ cm to a total volume of 100 ml 4 Otto 1 solution 0 5 for 100 ml 0 1 mol L citric acid 10 ml of 1 mol L 0 5 v v Triton X 100 500 ul Fill up with water conductivity gt 18 MQ cm to a total volume of 100 ml 5 Otto 2 solution for 100 ml 0 4 mol L Nas2HPO g2H20 74 1 ml saturated solution 0 4 ug ml DAPI 200 ul of 2 mg mL stock solution 0 2 ul ml B mercaptoethanol 200 ul Fill up with water conductivity gt 18 MQ cm to a total volume of 100 ml Acknowledgements This protocol is based on previously published work Matzk et al 2000 Schmidt et al 2014 Our research in this area was supported by the University of Zurich the Marie Curie project IDEAGENA a Syngenta Ph D Project of the Zurich Basel Plant Science Center and a grant from the Staatssekretariat fur Bildung und Forschung in the framework of the COST Action FA0903 We thank Manuel Waller for help with flow cytometry and Anja Herrmann and Margarida Sofia Nobre for careful reading of this protocol References 1 Koltunow A M and Grossniklaus U 2003 Apomixis a developmental perspective Annu Rev Plant Biol 54 547 574 2 Matzk F Meister A and Schubert 2000 An efficient screen for reproductive pathways using mature seeds of monocots and dicots Plant J 21 1 97 108 3 Ru2. 11 Jun 05 2015 Turn plates 180 and repeat step A6 The distal part of the arm of the mixer mill has a larger amplitude than the proximal part Turning the plates compensates for the different amplitudes of the mixer mill Control whether the seeds are crushed by visual inspection Seeds are crushed if the solution turned milky Figure 2 t t p i i y p 3 gt m b 4 Figure 2 Crushed and non crushed seeds The green arrows indicate successfully crushed seeds and a milky solution The white arrow points to a non crushed seed The red arrow shows a tube without a seed If the solution is clear and the seed is visible in the cluster tube it was not crushed and cannot be measured 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 If not all seeds are crushed repeat steps A7 9 until all seeds are crushed Shake down the solution to remove drops from the lid either by gently knocking the plate flat against the table or by a quick spin 5 sec in the centrifuge Add 80 ul of Otto 1 1 to each tube Be careful to not touch the tube or the foam with the tip to not cross contaminate the samples Close the tubes and mix by inverting 40x manually and avoid generating foam by fast movements Now the volume is 160 ul and the concentrations are 0 1 M citric acid and 0 5 Triton X 100 Place a filter plate onto a clean 96 well V bottom plate Figure 3A Label the plates Transfe
3. 0 4x 374 55 z2 6g 359 1 623 18 23 Bix 554 70 226 2a 301 3 01 ox 751 18 1 29 214 240 2 405 Figure 4 Sexual offspring n n of diploid Boechera stricta This histogram shows the fluorescence intensity on the x axis and the number of counts for each intensity on the y axis The peaks are manually annotated and labeled according to ploidy inferred from the internal or external standard Green represents the nuclei of the embryo The 4x and 8x peaks are from endoreduplicated embryonic nuclei autopolyploidization Brown represents the endosperm nuclei The ratio of the 3x 2x peaks is 275 99 187 4 1 47 indicating sexual development The ratio of the 6x 4x peaks is 1 48 Using the autopolyploid peaks for ratio calculations provides an internal control bio protocol All Data Points http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 0 200 400 600 800 1000 Region FL1 FL1 FLA Pet Mean HPEY CV Count Total 3x 235 60 4 06 5 10 4 007 46 45 6x 449 47 1 68 4 29 1 139 13 20 9x 673 36 3 09 3 05 510 5 91 12x 903 36 1 56 3 43 274 3 18 Figure 5 Apomictic offspring 2n 0 of triploid Boechera gunnisoniana Green represents the nuclei of the embryo The 3x 6x and 12x peaks represent autopolyploidization As apomictic Boechera spp are typically pseudogamous Rushworth et al 2011 we expect a fertilized endosperm Table 1 In this case the endosperm is 9x 9x 3x ratio is 2 86 indicating fertilization by a
4. andard of the known ploidy median of the peak the ploidy of the unknown sample can be calculated we bio protocol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 All Data Points 200 endosperm 100 0 200 400 600 800 1000 FL1 Region FL1 FL1 FL1 Pct Mean HPCY cy Count Total embryo 282 35 6 45 9 19 6 201 62 01 endosperm 789 65 0 34 4 94 144 1 44 Figure 7 Unidentifiable endosperm peak A All Data Points B All Data Points eae ee 50 0 Region FL1 FL1 Region FL1 FL1 Mean HPEY Mean HPEY Count Total 3x 316 47 1 46 476 297 62 3 54 5 14 1 923 25 64 Solanum 693 02 3 05 2 64 1 212 16 16 Solanum 635 57 0 16 3 36 1 481 19 75 2x 233 89 4 24 4 55 1 619 21 59 2x 236 69 2 29 6 34 199 2 65 Figure 8 Diploid A and triploid B Boechera plants with internal standard Solanum Red marks the internal standard having a higher DNA content than the sample It is therefore on the right hand side of the histogram Recipes 1 Stock solutions 1 mol L citric acid Saturated 0 54 mol L at 20 C NasHPO 2H20 solution 2 mg ml DAPI in water ultrapure water conductivity gt 18 MQ cm 2 Otto 1 solution 0 01 for 100 ml 0 1 mol L citric acid 10 ml of 1 mol L bio protocol ot O htto www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 0 01 v v Triton X 100 10 ul Fill up with water conductivity gt 18 MQ cm to a total volume of 100 ml 3 Otto
5. bio protocol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 Determination of the Developmental Origin of Seeds Containing Endosperm Using Flow Cytometric Analysis Christian Sailer Anja Schmidt and Ueli Grossniklaus Institute of Plant Biology amp Zurich Basel Plant Science Center University of Zurich Z rich Switzerland For correspondence christian sailer access uzh ch grossnik botinst uzh ch Abstract Seeds derived from a diploid sexual plant typically contain a 2n embryo n n and 3n endosperm a ratio characteristic for most flowering plants However this ratio is altered in apomictic species which reproduce asexually through seeds Koltunow and Grossniklaus 2003 Apomixis is usually a facultative trait and encompasses several developmental steps 1 apomeiosis avoidance of meiosis 2 parthenogenesis embryo development without fertilization and 3 functional endosperm formation autonomous without fertilization or pseudogamous requiring fertilization If all three steps occur this process results in maternal offspring 2n 0 which is genetically identical to the mother plant clonal Moreover sexual and apomictic pathways can occur in the same plant and sometimes they cross over producing polyhaploid offspring n 0 resulting from meiosis and parthenogenesis or BII hybrids 2n n resulting from apomeiosis and fertilization Rutishauser 1947 The different types of offspring can be determine
6. d in a flow cytometric seed screen FCSS in which the relative chromatin content of stained nuclei is determined by measuring their fluorescence intensity This allows a comparison of the ploidy of the endosperm to the ploidy of the embryo and thus an inference of the pathway by which a seed was formed Matzk et al 2000 This method is particularly useful to characterize the developmental origin of seeds in apomictic plants or reproductive mutants of sexual species Here we present the protocol for an FCSS in Brassicaceae that has specifically been adapted to plants of the genus Boechera However in principle this protocol can be applied to any species producing seeds that contain endosperm Materials and Reagents Citric acid monohydrate Sigma Aldrich catalog number 331 14 Triton X 100 Sigma Aldrich catalog number X100 NasHPO 2H20 Merck KGaA catalog number 1 06580 1000 4 6 Diamidin 2 phenylindo DAPI Life Technologies Invitrogen catalog number D1306 B mercaptoethanol Sigma Aldrich catalog number M6250 Sheath fluid Ilso Diluent Beckman Coulter catalog number NPE 629967 Cleaning solution Beckman Coulter catalog number 629969 ao oe P Ss on 1 bio protocol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 8 Shutdown solution Beckman Coulter catalog number 629968 9 Stock solutions see Recipes 10 Otto 1 solution 0 01 see Recipes 11 Otto 1 solution 1 see Recip
7. e Here we describe the procedure using sampling robotics which ensures that the time interval from staining to measuring is constant The volume ratio of Otto 2 to Otto 1 solutions should be about 4 1 In our experience 25 35 of the data has to be discarded due to unidentifiable endosperm peaks Figure 7 This can go up to 45 in material from natural populations The results show higher reproducibility if the median is used for ratio calculations 4 independent measurements CV lt 0 01 Use internal standards whenever possible Internal standards are mixed to every single sample Figure 8 For Arabidopsis thaliana we have successfully used Solanum lycopersicum var San Marzano and for Hieracium pilosella we have used Bellis perennis Choosing internal standards The DNA content of an internal standard should differ enough from the DNA content of the sample species to avoid any overlapping ploidy peaks between the standard and the sample species Values can be found in literature Ideally an internal standard has a lower DNA content than the sample always the very left peak in the histogram or a higher DNA content than any expected ploidy peak of the sample species always the very right peak of the histogram If internal standards are unavailable use external standards plants of Known ploidy The external standard should be the first and the last sample measured in a set of samples on one plate From the position of the external st
8. es 12 Otto 1 solution 0 5 see Recipes 13 Otto 2 solution see Recipes Equipment 1 1 2 ml Cluster tube and rack sterile Thermo Fisher Scientific catalog number 07 200 320 2 Storage plate cap strips Thermo Fisher Scientific catalog number AB 0981 3 3 mm stainless steel beads Schieritz amp Hauenstein catalog number 22 455 001 1 4 Nunc Fritted deep well plates Thermo Fisher Scientific catalog number 278011 5 96 well V bottom plates SARSTEDT AG catalog number 82 1583 001 6 200 ul tips SARSTEDT AG catalog number 70 760 002 7 1 mltips Rainin Mettler Toledo catalog number RC 1000 10 8 Single channel pipettes Gilson catalog number P10 P1000 9 Multichannel pipette 10 100 ul Thermo Fisher Scientific catalog number 4661130 10 Multichannel pipette 30 300 ul Thermo Fisher Scientific catalog number 4661 140 11 Mixer mill Retsch model MM300 12 Centrifuge Eppendorf model 5810R 12 UV lamp part of 13 flow cytometer 13 Flow cytometer Beckman Coulter Cell Lab Quanta SC serial number ANO20010 14 Flow cytometer robotics Beckman Coulter MPL Cell Lab Quanta SC serial number AN90022 Procedure A Isolation of nuclei by bead beating 3 4 h 1 2 3 4 Place a single 3 mm stainless steal bead into each cluster tube Figure 1A Place cluster tubes into 96 wellrack Figure 1B Label the tubes and mark positions of samples as schematic drawing in the lab book Fix the silique o
9. n a sticky tape and slit it open with a dissection or hypodermic needle Figure 1C D Add a single mature but still green seed Figure 1C D into each cluster tube In Boechera spp endosperm degrades upon seed maturation If a fully mature seed of the species under investigation contains endosperm the mature seed can be used This can be checked by microscopic analyses of cleared seeds at various stages The method of clearing depends on the species and should be chosen and adapted based on published protocols bio protocol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 oldest cia N s Figure 1 A A single 3 mm stainless steel bead in a cluster tube B Cluster tubes containing single beads in the appropriate 96 well rack C Optimal seed stage for Boechera spp seeds The top silique has seeds that are too young to be used too few cells and the bottom silique has seeds at an optimal stage D The top silique has seeds that are too old they start to turn brown coinciding with degradation of the endosperm The lower silique has seeds at an optimal stage E Close up of C F Close up of D Lines indicate that part of the picture was removed in the close up 5 Add 80 ul of Otto 1 0 01 to each tube 6 Close the tubes using the storage plate cap strips 7 Crush the seeds by shaking the samples for 3 min at 30 Hz using the mixer mill bio protocol 8 http www bio protocol org e1484 Vol 6 Iss
10. n unreduced sperm cell All Data Points Region FLT FL1 Mean HPCY 3x 186 54 5 67 6x 382 63 515 9x 570 85 4 34 12x 700 56 1 67 Figure 6 B hybrid offspring 2n 2n of triploid Boechera gunnisoniana This histogram is almost identical to that shown in Figure 6 However the red peak 3x is much smaller than the 6x peak indicating that this is contamination from maternal sporophytic tissues due to scraping the seeds from the silique As B gunnisoniana produces both unreduced female and male gametes the 2n 2n embryo peak 6x will be double the size of that from maternal nuclei 3x These data were published by Schmidt and colleagues 2014 bio protocol Notes http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 Colder samples show higher fluorescence intensity The settings are changed in distinct ways using different software The software is usually provided together with the flow cytometer Therefore we do not provide details about the software used However adapting the protocol to a new species or new tissue always requires some optimization first Please refer to the user manual and or obtain training from the person in charge of the instrument Staining intensity changes over time after the Otto 2 solution has been added It has to be ensured that the time interval from adding the Otto 2 solution to measuring the sample is constant or that the measuring takes place once the staining intensity is stabl
11. o equilibrate see Notes Load or change the detection settings so that the diploid peak is at channel 200 relative fluorescent units on a linear scale see Notes Add 160 ul of Otto 2 solution either manually or by using the flow cytometer s robotics Measure the sample and record the data Stop collecting data either after a total count of 6 000 this takes about 7 8 min or after a certain time period e g 5 min Treat all additional samples accordingly Manually annotate the peaks see Figures 4 6 Discard the samples in which the endosperm does not give a clear peak It is essential to see the peak of the endosperm see Notes Save your data in a table that provides at minimum the sample name mean median and coefficient of variance Analyzing the data 33 34 Compute the ratio of endosperm to embryo If the ploidy of the mother plant is available compute the ratio of embryo to mother plant we i ot ol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 Ratio endosperm embryo Offspring type embryo reduced central cell fertilized 1 5 sexual n n l with reduced pollen 3n unreduced central cell apomictic 2n 0 fertilized with reduced pollen 5n unreduced central cell o fertilized with unreduced apomictic 2n 0 pollen 6n Representative data All Data Points Region FLA FL1 FLA Pct bean HET cy Count Total 2x 187 40 4 32 525 3 711 37 11 Jx 275 99 3 62 3 51 620 a2
12. r the solution with the extracted nuclei into the fritted deep well plates either by using a multichannel pipette or by pouring Be careful to keep the order of the samples Centrifuge 5 min at 150 x g at room temperature 20 C to pellet the nuclei Carefully remove the plates from the centrifuge The pelleted nuclei are very loose Remove the fritted deep well plates If they are thoroughly washed with tap water and rinsed with deionized water they can be reused Remove the supernatant with a pipette tip placed at the edge of the V Figure 3B Do not pipette fast otherwise nuclei will be sucked up The pellet should be of white to light brown appearance Add 30 ul of Otto 1 0 5 to resuspend the nuclei Adding the buffer by fast pipetting is sufficient to resuspend the nuclei Samples can now be stored for up to 72 h at 4 C until used bio protocol http www bio protocol org e1484 Vol 6 Iss 11 Jun 05 2015 Figure 3 A A fritted deep well plate on a 96 well V bottom plate B Cartoon representing the placement of the tip to remove the supernatant Measuring DNA content ploidy ca 7 8 min per sample depending on the concentration of the nuclei 20 26 27 28 29 30 31 32 Start the flow cytometer and the UV lamp Please refer to the user manual of the flow cytometer or get an introduction from the person in charge While the UV lamp heats up 30 min place the samples at room temperature t
13. tishauser A 1947 Untersuchungen Uber die Genetik der Aposporie bei pseudogamen Potentillen Experientia 3 5 204 205 4 Rushworth C A Song B H Lee C R and Mitchell Olds T 2011 Boechera a model system for ecological genomics Mol Ecol 20 23 4843 4857 5 Schmidt A Schmid M W Klostermeier U C Qi W Guthorl D Sailer C Waller M Rosenstiel P and Grossniklaus U 2014 Apomictic and sexual germline development differ with respect to cell cycle transcriptional hormonal and epigenetic regulation PLoS Genet 10 7 e 1004476 10
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