| Azadirachta Indica is a typical tropical evergreen tree of the genus Melia in the Meliaceae family. For the extensive use of Azadirachta Indica but it’s deeply harm Longicorn, The study aimed to establish rapid propagation system which could be used to build an efficient genetic transformation system.Next, we tried to study the function of cry3A in Longicorn-resistance by the transgenic method. It will provide reference for Longicorn-resistance transgenic breeding of Azadirachta Indica.The results of experiment showed as follows:1. Establishment of regeneration system of Azadirachta Indica(1) The Azadirachta Indica stem with a bud were inoculated to the primary culture to obtain the axillary bud. The stem with axillary bud were inoculated to substitutive medium to induce proliferation to aseptic seedling.We found that the optimum medium for the growth of aseptic seedling were MS+0.5mg/L6-BA+0.5mg/L NAA+30g/L sucrose+3.0g/L vegetable gum.The seedlings were very strong with much more stem and brighter green leaves.(2) The principal branch,lateral branches,leaves,aseptic seedings of Azadirachta Indica were used as explants to induce callus combined with different concentrations of the hormone for the purpose of selecting the best combination of plant hormones medium,and the optimum medium for callus growth were MS+1.0mg/L6-BA+0.1mg/L NAA+30g/L sucrose+3.0g/L vegetable gum. The rate of callus of different explants were97.67%,78.33%,84.33%,92.67%. The results showed that the best rate of callus was principal branch.(3) The optimum differentiation culture medium for seedling growth were MS+1.0mg/L6-BA+0.5mg/L NAA+30g/L sucrose+3.0g/L vegetable gum. The average bud differentiation of each callus shoots was up to7.35.(4) The growth adventitious bud were inoculated to different mediums for root induction, and the optimum medium for root induction were MS+1.Omg/L IBA+20g/L sucrose+3.0g/L vegetable gum. Under this condition, the roots were much longer and rougher, the highest rooting rate was92.59%, the average number of roots per plant was5.33. In transplanting rooted, first domesticated the Azadirachta Indica4-5d after transplanting seedlings in the greenhouse, and the survival rate can reach98%.2. Construction of a plant expression vector1301-Cry3A.3. The study of genetic transformation system by AgrobacteriumIn the process of genetic transformation system in Azadirachta Indica, by Agrobacterium strain LBA4404mediated cry3A genes, for the study of genetic transformation factors. The results showed that:taking callus as explants, pre-cultured5-7d, chose the Agrobacterium strain LBA4404with OD value of0.2to infect callus for20min, co-cultured for3days with AS40mg/L in co-culture medium. Under such conditions, the genetic transformation would be more efficient.Them Azadirachta Indica callus after infection transferred to the selection medium which comprising40mg/L hygromycin culture, and obtained resistant callus.4The results of GUS stainingAfter the Agrobacterium-mediated transformation and co-culture, the Azadirachta Indica callus were then seeded into the screening of medium to obtain resistant calli after10-15d. And then the resistant callus and Azadirachta Indica callus uninfested soaked in the GUS staining dye. The results showed that uninfested callus as negative control, did not stain, but the resistant callus were successfully stained, the surface was blue. It has indicated that Gus gene has been transferred into the genotype of Azadirachta Indica. But in the latter part of the subculture, the Azadirachta Indica callus tissue regeneration capacity declined in vitality and vulnerability, even browning and necrosis, and therefore failed to obtain the transgenic plants. |
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