| The establishment of efficient regeneration system is the prerequisite of wheat transformation, and the selection of explants is quite important. Wheat immature embryo is considered to be the best explant material source for calli development and shoot regeneration because of its high cultural characteristics and better regeneration potential. But, in the Agrobacterium-mediated transformation of wheat, the transformed tissues become severely necrotic after co-cultivation, which prohibits the process of genetic breeding and functional genome study. Therefore, it is very important for the improvement of wheat transformation to overcome Agrobacterium-induced tissue necrosis and optimise the transformation system of immature embryos. Immature embryos of different wheat genotypes were infected by Agrobacterium harboring HMW-GS genes 154X, 154Y, 1Dy12.1*t and 1By8 in this study. Some genotypes with higher culture potential and transformability were screened out, and the target genes above were introduced into wheat by employing suitable culture media and culture condition in the case of inhibiting the cell necrosis and death.Seven wheat genotypes with better culture potential were screened out from 41 genotypes after infected by Agrobacterium, including CB037, 06SKW28, Han6172, Yumai66, Xinchun9, Lunxuan987, CA8694, all of whose frequencies of embryonic calli were higher than 10.0% and frequencies of resistant plantlets regeneration were higher than 2.0%. Different hormones were added in medium to test different genotypes in the process of embryonic calli induction. CB037 and Bobwhite reached the highest embryonic calli induction frequency and resistant plantlets regeneration frequency in the 2 mg﹒L-1 dicamba containing medium, but for Shi4185, Yangmai12 and Yangmai 15, the medium with 2 mg﹒L-1 2, 4-D showed better effect. The result indicated that hormones were specific to wheat genotypes. The results of modification of callus initiation medium indicated that the calli growth state can't be improved by adding plant growth regulators like 2-HNA, PAA, GGR and antioxidants like AgNO3, Vc and Cysteine, organic additives such as VA and Asp. On the contrary, the medium including the simplest MS components without organic components and vitamins, replacing 2 mg﹒L-1 2, 4-D with 2 mg﹒L-1 Dicamba was the best choice because it can inhibit cell necrosis effectively and be suitable for more genotypes. Fragmentation of immature embryos improved the embryonic calli induction frequency and resistant plantlets regeneration frequency after infection by Agrobacterium for both genotypes with high and low culture potential. The resistant plantlets regeneration frequency reached 46.67% and 21.05% for Bobwhite and CB037 after fragmentation, and increased from 0.00% to 9.76% for Chinese Spring which is not easy to initiate calli and regenerate adventitious bud. The immature embryos of Kenong199 were very sensitive to the Agrobacterium infection, and GUS gene transient expression frequency was 76.7%. Growth temperature had great impact on immature embryo regeneration. The study showed that the environmental temperature at 15-20℃(night) and 20-30℃(day) during grain-filling stage after flowering was the most suitable condition to collect the immature embryo for Agrobacterium-mediated transformation, and higher or lower temperature had adverse effect on the immature embryo regeneration after cocultivation with Agrobacterium. By using the modified media and transformation protocol, wheat immature embryos were transformed by the Agrobacterium carrying HMW-GS genes coding 154X, 154Y, 1Dy12.1*t, 1By8, and 496 plantlets with high resistance to G418 were obtained. Of them, 467 plants were employed to perform PCR detection and 109 plants were proved to be positive from 11 tested wheat varieties, PCR-positive rate ranging from 0.46% to 4.24%. The SDS-PAGE analysis showed no target protein expression in the seeds of all transgenic plants, but a specific protein band was detected in one transgenic plant, which was needed to be identified further.
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