| B-1, 3-glucanase gene was transferred into cultivars LF3 and LF10 (good quality, high yield but susceptible to disease), and HMW-GS 5+10 genes were delivered into NE7742, LF10 and LF8 (good quality, high yield), and NKH9 (high yield but poor quality) via particle bombardment and pollen tube pathway.The culture effect of embryo in 12 genotypes showed that there was genotype difference in frequency of callus differentiation and culture capacity except the frequency of callus initiation. NE7742, LF3 and L6239 would be the proper materials for gene transform because of the high culture capacity. The different culture effect was found in three types of explants (immature embryo, young spike, and mature embryo) used in the study. However, the frequency of callus initiation in immature embryo and mature embryo was almost the same, but higher than young spike. There was difference in frequency of differentiation among three explants, and the immature embryo ranked the first, young spike next, and mature embryo the last, but immature embryo > mature embryo >young spike when comparing on the basis of cultural capacity. Therefore, immature embryo used as explant in gene transfer would be better than young spike and mature embryo.The result of B-1, 3-glucanase gene transferred into LF3 and LF10 via particle bombardment showed that three transgenic plants with B-1, 3-glucanaes gene integrated have been obtained and confirmed by PCR and PCR-Southem hybridization. The transfer frequencies of LF10 and LF3 were 0.88% and 0.90%, respectively. Three transgenic plants from NE7742 and LF10 that gene HMW-GS 5+10 delivered into were positive and proved by PCR. And the transferred frequencies were 2.63% in NE7742 and 1.83% in LF10. Three of 1 3 T; plants were positive and confirmed by PCR and PCR-Southem hybridization.In gene transformation via pollen tube pathway, B-1, 3-glucanase and HMW-GS 5+10 genes were delivered into LF10, LF3, NE7742, LF8 and XKH9. Five transgenic plants integrated B-1, 3-glucanase gene were obtained and proved by PCR and PCR-Southern hybridization. Four transgenic plants, 3 from LF3 and one from LF10, presented the same hybridization band as positive CK confirmed by PCR and PCR-hybridization. The transferred frequencies were 1.55% in LF3 and 0.53% in LF10. The DNA sample of transgenic line 99K1354 from LF10 was provided with the hybridization band the same as the positive CK. It indicated that gene B-1, 3-glucanase had been integrated into the wheat genome.Nine transgenic plants from NE7742, LF10, LF8 and NKH9 transferred via pollen tube pathway were obtained and confirmed by PCR and PCR-Southern. One of them was line 21K867 (D4) from NKH9 which expressed the integrated HMW-GS 5+10 genes. The line was selected by SDS-PAGE with half kernel in its early generations. The transferred frequencies of NE7742, LF10 and LF8 were 2.27%, 2.44% and 1.64%, respectively.The pollen did not germinate 3 minutes after self-pollination, and started to germinate and formed pollen tube at 5 min. Pollen tubes could be check on the upper part of ovary at 20min and entered into ovary at 30 min, and several approached the embryo sac 40 min after self-pollination. After then, sedimentation of callus was found inside pollen tubes 60 min after pollination. The treatment of stigmas excised after self-pollination showed that wheat started seed setting 20 min after self-pollination. And the seed setting rate tend to stable 40 min after self-pollination. Therefore, it seems that the proper transferring time of foreign gene via pollen tube pathway would be around 30-60 min after self-pollination.Both Kanamycin and Geneticin (G418) can be used as selective regents of mark gene NPTII. However, the selection effect of Geneticin was better when compared with Kanamycin because of the higher drastic toxicity in Geneticin. Even though Geneticin was removed from media, a part of callus treated with Geneticin turned into brown in color and die in continue culture in vitro afterward. The suitable selective concentration of Geneticin an...
|
PDF Full Text Request