Establishment Of Strawberry Regeneration System And Transgene Of Early Flower Gene PcFT

Strawberry is one of the most important herbaceous commercial fruit crops in the world and the best commercial acinus planted in city circle, which has short growing period, enjoyable flavor and special smell. Because of low temprature and short day length desired during the period of flower differentiation, strawberry is commonly planted in plastic greenhouse. However, the limitation of harvest time of strawberry, which is between November in the first year to May in the next, have effected commercial production. A major challenge ramains in the accommodation of bud differentiation time, make it fruit as required to improve the commercial benefit.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 (genotype, explant, hormone concentrations, dark culture) 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 leaf transgenic system of strawberry, with which the transcription factor(Agrobacterium concentration, infectin time, preculture time, coculture time, selection method) early flowering PcFT gene was successfully transformed into the strawberry genome.The major results can be summarized as below:1 .Research of strawberry anther regenerationThe high efficient media for anther of strawberry 'HY' is MS+1.0 mg/L TDZ + 0.4 mg/L IB A, which has the highest regeneration rate when the is single nucleus keep to the side of dust. The regeneration rate is 12.3% and haploidy differentiation rate is 1.11%.2. Establishment of a high efficient regeneration system from leaf explants of strawberryThe high efficient media for 'HY' is MS+1.5 mg/L TDZ + 0.4 mg/L IBA and four weeks of dark culture is benefit for regeneration of strawberry leaves, under which the regeneration frequency is high up to 67.8%.The most appropriate treatment for 'Toyonoka' is MS+1.0 mg/L TDZ + 0.6 mg/L IBA and three weeks of dark culture is benefit for regeneration of strawberry leaves, under which the regeneration frequency is high up to 78.7%.Further research of transgenic system is based on 'HY' regeneratin system for the higher difficulty of regeneratin and commercial importance than 'Toyonoka'.3. Construction of plant express vector pBI121-PcFT4. An efficient strawberry transgenic system has been established.In the Agrobacterium -mediated gene transformation, the selecting pressure is 10mg/L Kam. And the optical bacteriophage is 400mg/L Cef.At first, the leaf disks should be precultured for 3 days in the inducing medium. While Agrobacterium concentration for inoculation is OD₆₀₀=0.3-0.5, the inoculation time of about 10 minutes is very suitable, then cocultured for 3 days. The cocultured leaf disks were transferred to the inducing medium containing 400mg/L Cef. After two weeks, the disks were transferred to the inducing medium supplementing with 10mg/L Km and 400mg/L Cef.5. PCR assaying and chilling-resistance testTen 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 PcFT gene as specific primers. One plant showed specific positive band same as the plasmid control, while untransformed plants have not any band. The result indicated that the PcFT lgene has been integrated into the strawberry genome.