Studies On The Transformation Of Tomato With The CsZCD Gene

Systematic research for the tomato tissue culture system and agrobacterium-mediated genetic transformation system were optimezed,and zeaxanthin cleavage dioxygenase (CsZCD) gene was successfully transfered into tomato explants and transgenic plants were obtained.The results were as followings:1. The seed germination was studied by different disinfection methods,seed medium and planting methods.The results showed that they were surface sterilized by treatment C which included first with 70% alcohol disinfection of tomato seeds for 1 min. and then 2% of sodium hypochlorite disinfection 10min. final rinsed with sterile water for five times.Then the tomato seeds were inserted the mediumâ…¡which include 1 / 2 MSB5,1% sucrose ,0.6% agar with pH 5.8. The seed germination rate of'Fudan','Yasheng'and'Zheza 905'is 83.33% 82.00% and 80.67% respectively.2. Plant hormone ratio, seedling age, medium Vc and AgNO3 content of medium were the main factor. The best regeneration medium of tomato cotyledon explants include MSB5,3%sucrose ,0.6% agar ,BA2.0 mg/L, NAA0.1 mg/L, AgNO32.0 mg/L and Vc100mg/L with pH5.8. The cotyledon callus rates of'Zheza 905','Yasheng','Fudan'were 92.67%, 95.67%, 93.33% respectively. While the medium for stem explants includes MSB5,3.0%sucrose , 0.6% agar, 2.0 mg/L BA, 0.2 mg/L NAA, 3.0 mg/L AgNO₃ and 100mg/L Vc with pH5.8. The stem callus rate of'Zheza 905','Yasheng'and'Fudan'were 79.33%,81.33%,87.00%, respectively.3. Tomato cotyledon explants is more sensitive than stem explants to kanamycin, for tomato cotyledon favorable selection concentration of kanamycin is 50mg·L^-1,and for tomato stem favorable selection concentration of kanamycin is 60mg·L^-1.The growth of agrobacterium was inhibited by cefotaxime which the concentration above 400 mg·L^-1.4. The impact factors of the transformatoin rate of tomato cotyledon, rank as age> Pre-culture> bacterium concentration> co-culture > infection time. The best combination of the five factors were seedlings 8-9 days, pre-cultured for 2 days, the concentration of bacteria (OD₆₀₀ = 0.6), were cultured for 3 days, infection time 20mins. The impact fanctors of the transformatoin rate of tomato stem segments, rank as pre-culture> bacterium concentration> Age> coculture> infection time. The best combination of the five factor is bacteria (OD₆₀₀ = 0.6), seedling age 4-5 days, 4 days co-culture, pre-culture for 3d, infection time 15mins.5. CsZCD gene in the regenerated plants of'Zheza 905'and'Yasheng'have been successfully detected by PCR.