| Strawberry is one of the most important commercial fruit crops in the world. Because of the genetic limitations associated with high heterozygosity and polyploidy, it is laborious, costly, and time-consuming to improve strawberry quality and yield by the conventional methods of breeding, which usually results in a low frequency. A major challenge remains in commercial strawberry cultivars for the lack of good varieties with resistance to coldness, insects and disease. Recent advances and applications in genetic engineering technology provide a new path for strawberry breeding and selection by way of incorporating foreign genes into plant genome for desired agronomic traits. However, an efficient tissue culture system is thought to be crucial to the success of plant genetic engineering, since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors, one being the regeneration ability of the infected tissue, and the other, the infection efficiency of Agrobacterium.In this research, we have studied the effects of a series of cultural factors (genotypes, basic media, hormone concentrations, sucrose and agar concentrations, leaf ages and explants) in the inducing adventitious shoot of strawberry, respectively. Then on the basic of high efficient regeneration system obtained above, we established an efficient and quick agrobacterium-mediated transgenic system of strawberry, with which the transcription factor CBF1 gene was successfully transformed into the strawberry genome. The major results can be summarized as below:1. The strawberry regeneration system has been established successfully. Regeneration frequency varied dramatically with genotype of strawberry, ranging from 94% to 0%. Of four strawberry cultivars, the regeneration frequency of Dasrlect was the most highest, whereas the strawberry Liyinv was observed to be very difficult to regenerate. The combination of 3mg/L 6-BA and 0.1mg/L 2,4-D were found to be the most efficient of Dasrlect. And 2, 4-D was better than IAA, IBA and NAA, all of 2, 4-D, IBA and NAA with high concentration were not beneficial to induce shoot of Dasrlect. In addition, it had no obvious effects on the shoot formation of Dasrlect with TDZ at the levels tested in this study. It was suitable to induce shoot with 0.7% agar and 3% sucrose. And the regeneration frequency was significantly enhanced by the MS+B5 basic medium. The regeneration capacity of leaf was higher far than petiole and root. Leaf age can affect also the regeneration frequency and 28～35 days was the best. The leaf disks were cultured with the adaxial surface in contact with the regeneration media. The maximum regeneration frequency of petiole only got 40%, whereas the roots did not induced any advenvious bud.2. An efficient strawberry transgenic system has been established. In the agrobacterium-mediated gene transformation, the selecting pressure is 20mg/L Kan. And the optical bacteriophage is 500mg/L Cef. At first, the leaf disks should be precultured for 2～4 days in the inducing medium. While bacterium concentration for inoculation is OD6000.3-0.5, the inoculation time within 3～5 minutes is very suitable, then cocultured for 2 days. The cocultured leaf disks were transferred to the inducing medium containing 500mg/L Cef. After two weeks, the disks were transferred to the inducing medium supplementing with 20mg/L Kan and 500mg/L Cef . 3. PCR assaying and chilling-resistance testTwenty eight regenerated Kanamycin resistant plants were obtained from the transformation above, and they were assayed by PCR reaction with the untransformed plants as negative control, plasmid DNA as positive control and the sequence of CBF1gene as specific primers. Ten plants showed specific positive band same as the plasmid control, while untransformed plants have not any band. The result indicated that the CBF1gene has been integrated into the strawberry genome.Electrolyte leakage test of leaf demonstrated that the freezing tolerance of the transgenic plants, whic... |
