Studies On Systems Of High Efficiency Regeneration Of Wheat Plants From Immature Embryos And Exogenous DNA Transfer By Electroporation

With the development of biotechnology, gene engineering has played an important role in improvement quality of wheat, which was paid attention by breeding experts in the world. Compared with the other crops, wheat was hard to establish higher efficient regeneration system and the genetic transformation of wheat was the stage of establishment and improvement, which were main factors that hinder the development of gene engineering of wheat. Wheat is the one of chief crops in China, the lack of water often limited the increase of yield, and improvement drought resistance of wheat has been considered. Therefore 3 aspects had been studied:① higher efficient regeneration system was established by immature spikes and embryos of 10 genotypes of wheat; ② the main parameters in electrotransfer was optimized; ③exogenous DNA from drought resistance wheat was transformed into embyogenic callus of 6-90 by eletroporation and the morphology of transgenic plants were observed, and PAGE of isoperoxidase and soluble protein patterns were analyzed. The main results are the following:1 The result of orthogonal experiment indicated that the optimal induction media was 2,4-D 2.0mg/L+KT 0.4mg/L+NAA 0.01mg/L +CH300 mg/L. Genotypes had not strong influence on frequency of callus induction of wheat, but it influenced frequency of embyogenic callus induction (P<0.01)；There is a significant difference at 0.01 level in the capability of differentiation of embyogenic callus which inducted from immature spikes and embryos of different genotypes; Among 10 varieties (lines) of wheat, '6-90' was the best acceptor genotype.2 Frequency of embryonic callus induction among different ages of immature embryos had significant difference (P<0.05), the best period for embryonic callus induction was 13 -14d. 3 Concentration of sugar in media notablely affected frequency of embryonic callus induction (P<0.05), if the concentration was too high or too low, embryonic callus induction would be affected, even be inhibited. The optimal concentration is 50g/L.4 Low temperature(3-4℃)and hungry treatment (0-7d ) had not strong influence on frequency of embryonic callus induction and frequency of callus induction, they would decrease after 7th day.5 During subculture, with the increase of subculture times, there was a trend that embryonic callus would become non-embryonic callus. Extension of embryonic maintenance had significant difference (P<0.01) between genotypes and subculture times respectively.6 2,4-D could decrease frequency of embryonic callus differentiation. 6-BA was higher than KT in frequency of embryonic callus differentiation, and 6-BA2.0mg/L+ NAA0.02mg/L could increase frequency of embryonic callus differentiation obviously. Frequency of rooting was the highest by adding IAA 0.8mg/L in medium. 7 The best method for transplant is that put the vermiculite on humus among 6 methods, the survival rate reached 95.65%, and the plants grow well.8 FITC compound were transformed into embryonic cell-group by electrporation, fluorescence in some embryonic cells was observed in Opton Fluorescence Microscope. Through this test, we obtained a better condition of electroporation. Set voltage was 300v, capacitor was 975 uf and a single electric pulse.9 Exogenous DNA from drought resistance were transformed into embryonic cell-group of 6-90 with optimal parameters of electrporation. and then, the morphology of transgenic plants was observed, the results showed that some stamens became plumed stigmas, and there is not ovary or thin ovary in some flowers, spikes became longer, and some plants owned several spikes. PAGE analysis indicated that there was a obviously different between transgenic plants and CK in isoperoxidase and soluble protein pattern. The results proved that transgenic plants have had extensive variations.