Creation Of New Germplasm Of Strawberry Overexpressing Citrus Phytoene Synthase Gene (citPSY)

Strawberry (Fragaria ananassa Duch), a perennial herbaceous plant in the Rosaceae family, is a major berry crop around the world. With short juvenile phase, strawberry is easy to bearing fruits with special flavor and enriched anthocyanin. Citrus has long juvenile phase, which lead to the difficults in validating gene functions involved in metabolism of pigment in citrus. Strawberry genetic engineering is an alternative efficient strategy to solve the problem. Phytoene synthase gene (citPSY) is a rate-limting gene in the carotenoids biosynthesis. To overexpress the citrus citPSY in strawberry via agrobacterium-mediated transformation system, we aimed to explore the interactions of secondary metabolites in the future, especially the regulation of carotenoids and anthocyanins in strawberry, and to provide more clues in plant regulation mechanism in carotenoids production.The major results are summarized as follows:1. The strawberry regeneration system has been optimizedThe high efficient media for’Allstar’leaves regeneration is MS+1.5mg/L TDZ+0.2mg/L IBA, and 2-4 weeks of dark culture is benefit for regeneration. The callus regeneration frequency is as high as 97.1% and shoot regeneration frequency is 55.2%. The average number of regenerate bud per leaf is 2.09.2. An efficient strawberry transgenic system has been established and a few transgenic plants were obtainedThe optimized parameters for agrobacterium-mediated transformation had been establised as following:The selecting pressure was under 10mg/L Kan and the optical bacteriophage was 250 mg/L Cef. The leaf disks was precultured for 2 d firstly.The bacterium concertration for inoculation was OD600=0.3-0.5, which was re-suspended by MS before using.The suitable inoculation time was about 7 min. Coculturating for 3 d was optimum.After coculturation, leaf disks were transferred to the inducing medium containing 250 mg/L Cef for 2 w. After that, these disks were transferred to the inducing medium containing 10 mg/L Kan and 250 mg/L Cef.3. PCR assayingForteen regenerated plants were obtained from the transformation above, which were seleceted with 10 mg/L Kan and acquired Kanamycin resistant. Genomic DNA of above regenerated plants were extracted and assyed by PCR reation with the untransformed plant as negative control and plasmid DNA as positive control. Three out of these 14 plants showed identical bands to the plasmid control, and the untransformed plant has no such band, which indicated that the citPSY gene has been integrated into the strawberry genome.