The Establishment Of Regeneration System Of Black Raspberry And The Transformation Of Agrobacterium-Mediated Salt-tolerant Genes

With leaves, petioles, internode segments,and tip shoot with destroyed growth points of black raspberry (R.occidentalis L.) as explants, this experiment was to induce the regeneration buds and establish the regeneration system of black raspberry. MS as the basic medium, this research explored the effect of different concentration ratios of auxin, cytokinin, gibberellin on the regeneration frequencies of explants, the most appropriate hormone ratio for black raspberry to regenerate, and the most suitable explants. On the basis of the regeneration system, this research then studied the genetic transformation of black raspberry. With the genetic engineering technology that the efficient vectors of Agrobacterium (Agrobacterium tumefaciens) mediate the explants of black raspberry, such as tip shoot with destroyed growth points, internode segments and petioles, we transmitted the transcription factor HS which regulates the expression of antiretroviral genes such as salt-resistance genes, drought-resistance genes, antioxidant genes into the different explants to get resistance plants. The results are as follows:1. Black raspberry and Malus robusta (M. robusta) as the materials, the tissue-cultured seedlings were exerted stress treatment in different media and salt concentrations. After 60 days, the salt resistances of black raspberry and Malus robusta in 1/2 MS medium were about 0.4% and 0.2% respectively, while in MS medium and MS+1BA+0.01NAA+2GA medium the salt resistances of black raspberry were about 0.6% and 0.8% respectively.2. Induce regeneration buds with the leaves, petioles, internode segments, tip shoot with destroyed growth points of black raspberry as explants. MS as the basic medium, and with different ratios of 2,4-D,NAA,IBA,IAA,6-BA,KT,GA, it was found that the petioles, internode segments, and tip shoot with destroyed growth points all regenerated in varying degrees when the concentrations of 6-BA and NAA were 5-6mg/L and 0.05mg/L respectively, which proved that black raspberry wasn't strict with the concentration of hormone within a certain range and MS+6-BA2mg/L+NAA0.1mg/L+GA2mg/L was the most appropriate differentiation and regeneration medium for black raspberry. On analysis of differentiation and regeneration percentages of the explants such as leaves, petioles, internode segments, tip shoot with destroyed growth points in the same condition, it was found that the most appropriate explants was tip shoot with destroyed growth points that it had the lowest death rate, the quickest and largest differentiation rate. There were also buds' regeneration in the cuttings of stems and petioles. It was also appropriate to use stems and petioles as explants when there were few materials.3. It was so easy for black raspberry's test-tube plantlets to root that it could be induced in 1/2 MS medium without any hormone. But the roots were weak. NAA had a significant effect on rooting. The most appropriate rooting medium is MS+NAA0.1mg/L.4. 5. The screening medium for black raspberry was MS+6-BA2mg/L+NAA0.1mg/L+GA2rng/L. Screened from different herbicide concentrations it was found that the herbicide concentrations for differentiation and rooting were 4mg/L and 6mg/L respectively.5. In comparison with different pre-cultured durations, solution concentrations, infection durations, co-cultured durations, co-cultured types and co-cultured media, it was screened that the most appropriate transformation condition was that explants, such as the leaves, petioles, internode segments, tip shoot with destroyed growth points, were pre-cultured in regeneration medium MS+6-BA2mg/L+NAA0.1mg/L+GA2mg/L for 2 days, then infected by Agrobacterium for 12 min. After cultured in darkness in MS +6-BA2.0mg/L+NAA0.1mg/L +GA2mg/L+AS100μm/L for 3 days, they were inoculated in MS+6-BA2mg/L +NAA0.1mg/L+4mg/L herbicide+500mg/L carbenicillin. The resistance buds got from pressure screening were transgenetic mutants. Until the mutants were as tall as 1cm, they were moved into the rooting medium MS+NAA0.1 mg/L+6mg/L herbicide +500mg/L carbenicillin. After 35 days or so they could be transplanted into sterile substrate.6. In comparison with different transplanting conditions, it was found that the most favorable measure for resistance seedlings of black raspberry was like this: the transformation seedlings already growing roots were taken out from the incubator, trained at room temperature for 3 days. Then they were transplanted into cups filled with sterile vermiculite after medium adhering to the roots was removed and washed away by sterile water. After cultured in room temperature for 7 days while watered with distilled water and kept from sunshine, the transformation seedlings were transferred into an incubator equipped with wetting devices, watered with distilled water and shined with dispersion sunshine for 14 days. Then the seedlings were removed from wetting devices gradually and shined directly with sunshine. When they have grown new leaves they were watered with nutrition solution. After 14 days or so they were transplanted into the soil.7. On the observation of rooting situations for transformation plants in rooting medium with 6mg/L herbicide, non-transformation plants without herbicide, and non-transformation plants with 6mg/L herbicide, it can be identified preliminarily that the obtained resistance plants were trans-HS gene plants. With regard to molecule detection, salt resistance and measure of relevant physiological indexes of transformation plants, they will be the topic of subsequent research.