Polyploidy Induction And Cytological Analyses Of Russian Wild Ryegrass

Russian wild rye(Psathyrostachys juncea (Fisch.) Nevski)is a rare diploid species in Triticeae which is fit to be developed with chromosome doubling. polyploidy were induced via in vitro culture and chromosome were analyzed with C-banding and 45S rDNA FISH.Efficient regeneration system was established from three explants of two varieties, young spikes, immature and mature embryos. Young spikes with the length of 1~3cm and immature embryos after pollinated 14~17d were ideal for callus induction. Immature embryos was pretreatment at 4℃for 1~5d, mature embryos soaked at 10℃for 48h. 2,4-D had significant effect on callus induction. ABA in media was necessary when callus was induced from spikes with length longer than 4cm and mature embryos. CH in media promoted the growth of callus but had no impact on differentiation. A number of Non-embryogenic callus were transformed into embryogenic callus after subculture for more than 2 times. Differentiation rate of embryogenic callus reached 80% . NAA and 6-BA had significant effect on regeneration. The key points of efficient regeneration system of Russian wild rye is explants screening, callus induction and embryogenic callus transformation.Chromosome doubling were induced with colchicine from germinating seeds, buds and callus. Ploidy of colchicine treated plants were identified by chromosome counts in somatic cells. Mixploid plants were induced from seeds treated with 1500mg/L colchicine and 1.5%DMSO for 4 h. the percentage of tetraploid cells was 24.4%. The chromosome doubling efficient was higher when buds were treated with 500mg/L colchicine and 1.5%DMSO for 48h. the percentage of tetraploid cells was 42.65%. There were some haploid, triploid, aneuploid cells in colchicine treated plants, with percentage of 8.88%. Effect of treating callus with 100mg/L colchicine and 1.5%DMSO for 72h was the best for polyploidy induction. The tetraploid cell percentage was 53.58%, including 8 plants with 80% tetraploid cells. Callus can not regenerate after treated with Pendimethalin. Morphology comparation showed that the length and wide of tetraploid plant leaves were increased. The length of guard cells increased by 13.52% than diploid. The distance between stomotals increased significantly.The photosynthesis and chlorophyll fluorescence of induced autotetraploid and diploid P.juncea were compared. Light and CO2 response curves were estimated by linear regression and Farquhar model. Saturating and compensation light intensity and Maximum photosynthetic rate of tetraploid plant were higher than diploid, but stomatal conductance was lower than diploid. Saturating and compensation CO2 concentration was higher than diploid too. Photosynthetic limited factors (Vcmax, Jmax and TPUmax) of tetraploid is more efficient. Results showed that the photosynthetic system of tetraploid was more efficient in high light intensity and high CO2 concentrations than diploid. Fluorescence parameters were surveyed under water stress. Fv/Fm of tetraploid was higher, but qP and qN was lower than diploid. The photosynthetic apparatus of diploid was protected by exhausting the extra light energy through heat dissipation. Chlorophyll fluorescence parameters was sensitive to water stress. But the drought resistance of autotetraploid need further experiment.Chromosome number were counted in somatic cells of two varieties. Mean percentage of diploid cells (2n=14) was 86.25%. Chromosome was analyzed by Sequential C-banding and 45S rDNA FISH. Chromosome can be distinguished from each other according to their respective bandtype. Caryotype of P.junceais 2n=2x=14=8m+6sm, belongs to 2A. Chromosomes can be divided into 3 groups according the bandtype. C-banding pattern polymorphisms were observed among different individuals, and within homologous chromosome pairs. There were 6 main 45S rDNA sites located in the end of short arms of 3 pairs of diploid chromosome. They were chromosomeⅠ,ⅢandⅤwhich can be speculated as NOR chromosome. There were also weak hybridization signal on some long arm of chromosome. 12 main 45S rDNA sites were located on trtraploid chromosomes.