| An efficient transformation system for cabbage hypocotyl explant was developed using Agrobacterium tumefactions strains EHA105 and LBA4404 respectively. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings, growth condition and status of Agrobacterium suspension, preculture of explants,6-BA, acetosyringone and silver nitrate had a significant influence on transformation frequency and plant regeneration. The presence of the Bt genes in those selected plants in kanamycin medium were confirmed by PCR and Southern blotting, the frequency of regeneration from hypocotyl was analyzed. The RT-PCR was done to detect the Bt gene expressed in different tissues. Leaf section bioassays of insect resistance showed that Bt transgenic cabbage significantly enhanced the level of resistance against the larvae of insect diamondback moth. The main results were as follows:1. The system for regeneration frequency of cabbage was as follows:the medium was MS+BA 2.0 mg/L+NAA 0.05 mg/L+Ag NO3 5 mg/L, the age of seedling was 6d to 7d, the better explant was hypocotyl. The time spent both on preculture and coculture period were 2 days respectively. And the regeneration frequency was more than 80%.2. Based on the high frequency transformation, mediated by Agrotactterium tumefaciecin, 369 putative transgenic CryIAc hybird cabbage plants,412 CryIC hybird cabbage,750 CryIAc inbred incompatible line cabbages were obtained. The rooted ratio of both hybird cabbage and inbred incompatible line plants were 60% and 10% respectively.3. In selective rooted medium containing 5mg/L kanamycin,5 rooted CrylAc hybrid plants,5 rooted transgenic CryIC hybrid cabbages and 3 rooted transgenic CryIAc inbred incompatible line cabbages were obtained.4. The PCR analysis demonstrated that 4 CrylAc and 4 CryIC hybrid plants and 3 CryIAc inbred incompatible line cabbages were confirmed.90% rooted selective plants were positive.5. The Southern blotting analysis showed that the target CryIAc gene was integrated into the cabbage genome of some putative transgenic plants.6. The RT-PCR analysis of some CryIAc transgenic plants showed that the CryIAc gene expressed differently in root, stem, young leaves and mature leaves tissues.7. The bioassays of Bt transgenic cabbages resistant to insect indicated that all the transgenic leaves showed little damage (<10%), the growth of larvae was severely inhibited on detached transgenic Bt cabbage leaves; and the average weight of insect on Bt leaves is significantly less than the ones' on wild type cabbages; the corrected mortality of larvae is more than 80%, significantly higher than the control. All the transgenic plants showed the partial to total control of larvae. The plants among the transgenic CrylAc hybrid cabbage showed the similar control, and the plants among the transgenic CrylC hybrid cabbages showed the similar resistance to the diamondback moth, the plants between the CrylAc and the CryIC transgenic hybird cabbage showed no difference in resistance to the larvae, yet the transgenic CrylAc plants between the hybrid cabbage and inbred incompatible line cabbage showed a little difference in leaves damage area. The dead diamondback moths both on wild type cabbage and Bt cabbage were observed under the light microscopy. The results demonstrated that the size of dead larvae placed on wild leaves was bigger than the one on transgenic leaves; and the midgut of insects died on wild type cabbages was not damaged, but the one on the transgenic leaves was affected by the Bt protein. |
