Establishment Of Plant Regeneration And Transferring Bt And Ca-eIF4E Genes Into Pepper (Capsicum Annuum L.)

Pepper (Capsicum annuum L.), one of the most important vegetables, is planted in areas multiplied rapidly for its abundant nutrition and special flavor. However, with the development of pepper industry, it was severely attacked by many pests year by year. Consequently, it damaged growers economically. Plant genetic engineering opens a new way for breeding pest-resistant cultivars, for example, by transferring the insect-resistant or virus-resistant genes into pepper. In recent year, many researchers had made great efforts on pepper genetic transformation, but little success was achieved.The main problem in pepper genetic transformation lies in the difficulty of shoot regeneration from explants. We made a detailed study on the effect of genotypes, explant types, seedling ages, and medium components on plant regeneration, and established the system of pepper regeneration in vitro. Then we study some influences factors on pepper transformation, and established the system of pepper genetic transformation.The toxin protein encoded by crylA(c), derived from Bacillus thuringiensis, could kill lepidopters specifically but was harmless to higher animal and human being. Ca-eIF4E was a eukaryotic initiation factor 4E (eIF4E) gene of Capsicum annuum L. We could improve pepper resistance to viruses such as potyvirus by regulating Ca-eIF4E expression level in pepper. We transferred crylA(c) and Ca-eIF4E into pepper respectively, and PCR detection on transgenic plants showed the genes were successfully integrated into pepper genome.Now the major results summarized from this research were as follows:1. As for adventitious bud induction of pepper, cotyledons were optimal explants compared to hypocotyls; the regeneration capabilities of cotyledon explants varied with age stages. Cotyledons which were expanding immediately gave the highest rate of differentiation, and that was recommended as explants; the capabilities of different genotypes for regeneration were various. Among 7 cultivars tested, Chufeng (CF) was the best; hormone played an important role in adventitious bud induction. 6-BA+IAA was the most effective, 6-BA+PAA was better, and Zt has less effect. Chufeng, Sujiao NO.5, Xiangjiao NO.21 and Qiemen got 100% of adventitious bud induction on 6-BA 5.0 mg/L + IAA 0.2 mg/L; Ag+ showed polarity action on adventitious bud induction. The more Ag+ was added, the stronger polarity action occured. The polarity action in AgNO₃ was stronger than in STS's. AgNO3 would inhibit bud induction of pepper, while silver thiosulfate(STS) would promote or inhibit bud induction depending on STS concentration or pepper genotype.2. There was something effect on shoot elongation when adventitious buds were induced in different time. When adventitious buds were induced for 21 days, the percentage of shoot elongation was higher than that of 14 days; GA3 promoted shoot elongation. With its concentration increased, the percentage of shoot elongation increased. But 3.0 mg/L GA3 would inhibit shoot elongation. The suitable concentration was 1.0-2.0 mg/L; effect on shoot elongation was influenced by whether using the same kinds of hormone or not during shoot elongation and adventitious bud induction. Taking one with another, the effect on shoot elongation, Zt+GA3 was the best, 6-BA+IAA+GA3 was better, and 6-BA+PAA+GA3 was the worst; B5 organic constituents were better than MS organic constituents on shoot elongation: nutrient juice extracted from young pepper seedlings did not showed positive effort on shoot elongation as reported.3. Shoot rooted on medium containing IAA, IB A or NAA. IB A was the best, and 1/2MS supplemented with 0.5 mg/L IBA was the best for rooting.4. Cloned Ca-eEF4E gene by RT-PCR.5. Fivty mg/L kanamycin would completely inhibit bud induction from cotyledons of SW NO.5 and Qiemen, while 75 mg/L kanamycin was needed to completely inhibit bud induction from cotyledon of Chufeng, Bianhong NO.l and Tedatianjiao; the effect of Cefotaxime (Cef) or Carbenicillin (Cb) on bud induction was similar to Ag+, and the effect of Cef was much stronger than Cb's.6. Pre-culture of explants on bud induction medium for 1 day (before infection with Atumefaciens) and co-culture on bud induction medium for 3 days were favorable for the transformation; for the transformation system by decapitation regeneration system, kanamycin inhibited plants rooting obviously, so it was not advisable to use kanamycin for selection. Pre-culture of explants on MS medium for 0 day and co-culture on MS medium for 1 day were favorable for transformation; the time of infection v/ithAgrobacterium tumefaciens had effect on transformation rate, and I min was better than 5 min.7. Genomic DNA was extracted from some putative transgenic plants, and the results of PCR analysis showed that crylA(c) or Ca-eIF4E was integrated into the pepper genome.