The Study On Sacb Gene Transformation, Transgenic Plants Regeneration And Establishment Of Conditions For Pcr Detection In Medicago Sativa

In this study, three varieties of alfalfa (Hetian, Gannon NO.3 and Longdong)were used as acceptor. Agrobacterium-mediated method was used in the research of transforming of SacB gene in alfalfa. Conditions of transformation and regeneration and browning elements during regeneration were explored. Resistant plants were selected. PCR tests of transformed tissues were made and the PCR test conditions were optimized. Appropriate conditions of PCR amplification were established. The main research contents and results were as follows:1. Established transformation system of three varieties of alfalfa.Hypocotyls of Hetian alfalfa, Gannon NO. 3 and Longdong were made as explants. Agrobacterium-mediated was used as transformation method to transform SacB gene. Effects of Kan on the growth of alfalfa tissues , Kan screening and selection concentration , the concentration of bacteria, infection time and pre-culture time were studied systematicly. The results showed that: Kanamycin was not significant on seed germination. Callus differentiation stage was the most appropriate screening stage. The selection pressure of Hetian alfalfa, Gannon NO. 3 and Longdong was 80mg / L, 60mg / L, 60mg / L. In rooting stage, the selection pressure of Kanamycin of three varieties of was 100mg / L, 80mg / L, 80mg / L. The appropriate OD of bacteria of Gannon NO.3, Hetian alfalfa and Longdong was 0.6,0.5,0.4. Pre-cultrue was not required before transformation for alfalal. Appropriate infection time was 10min.2. Browning of the transforming tissues occurred in summer and winter during regeneration was analyzed and corresponding solutions were put forward.The degree of browning of culture had seasonal variations. The best action time of 2,4-D was 15-20d. The optimal concentrations of sucrose in callus induction, regeneration and rooting stages respectively were 30g / L, 20g / L and 10g / L. Genotype influenced the degree of browning culture, and the order of the degree of browning was variety NO. 3> variety NO. 1> variety NO.2. The appropriate incubation temperature in winter was 25℃day and night and in summer was 23℃during the day and 21℃during the night. The suitable light was about 3000Lux. NAA concentration in the summer than in winter should be appropriately reduced.IBA was suitable for rooting in winter with high rooting rate and less browning.3. PCR reaction conditions were optimized and PCR tests on the transformed tissues were made.The transformants were tested by PCR.The results showed that: the best primer was group a primer. The optimal annealing temperature was 52℃. The optimum dosage of dNTP, Mg²⁺, TaqDNA in 25ul reaction system were 2.0ul, 2.5ul and 0.4ul. Only one of the 50 selected resistant calli was positive callus .The conversion rate was 2% .