Transformation Of Physalis Linn. With IaaM Gene Using Agrobaterium And Creation Of Genetic Diversity Of Parthenocarpy

This research focuses on transplanting the iaaM gene into Physalis Linn. mediated byAgrobacterium. The level of IAA of Physalis Linn fruit was increased, then, and parthenocarpicPhysalis Linn. plants were achieved, The trandgenic Physalis Linn. plants are not only newparthenocarpic germplasm but also new experiment materialsfor Physalis Linn. parthenocarpicmolecular study, in addition, it provides a new way of Physalis Linn. parthenocarpic molecularbreeding. By using the established regeneration and transformation system, one of the transgeniccallus had been tested positive through PCR and Southern blotting. The main research work andresults were as follows:1. A primary tissue culture system for callus induction and plant regeneration had beenestablished by using different growth regulators on medium MS. Using different concentrationproportion of various hormones which were based on MS culture medium, high efficiently shootregenerating cultivar was selected. The optimal regeneration medium was MS+TDZ 1mg/L+NAA0.5mg/L+Suger 20g/L+Agar 8g/L and roots well on MS+NAA 0.5mg/L+Suger 30g+Agra8g/L with nearly 90% rate. At the same time, the factors which would influence shoot regenerationsuch as the different explant types, genotype and hormones were studied. The results showed that:the leaves' regeneration ability was best, TDZ in vitro influenced the leaves' regeneration abilityand the concentration of TDZ for keeping high abstract regeneration ability was different accordingto different cultivars.2. The genetic transformation receptor systems for five different Physalis Linn. genotype wereestablished. The susceptibility of Physalis Linn. to kanamycin were also studied. Research on genetransformation system has found the suitable Km concentrations for bud and root inducingselection and the time for Agrobacterium tumefaciences infection and incubation. The criticalconcentration of kanamycin for shoot regeneration of Physalis angulata L. was 70mg/L and rootregeneration was 20mg/L. As a kind of antibiotic, 350mg/L Cefotaxime had no distinct influence onregeneration frequency and quantity of shoot regeneration of Physalis angulata L., but it was able torestrain the growth of Agrobacterium tumefaciens.3. On the establishments of a regeneration system of fastgrowing variety of Physalis angulataL. gene transformation system was optimized by GUS coloation analysis. That the leaves explantswere pre-cultured for 24 hours was best for transformation by Agrobacteriurn tumefaciens. Thesuitable concentration of Agrobacterium tumefaciens carrying iaaM and the time for transformingwas OD600=0.6 and 8 minutes. Co-cultured for 2d on the medium with AS 100μmol/L afterinfection and delayed cultivation for 2 days were suitable for transformation. Subsequently it wasbetter to select the regenerating materials consecutively under the critical concentration of 70mg/Lkanamycin. One centimeter transgenic regeneration shoot was transformed into MS+0.5mg/L NAA with 20mg/L kanamycin. The transformed plants were cultured on bud inducing medium andformed Km resisted bud which were subjected to molecular sequence examination with PCR andSouthern blotting technology. Identified by PCR and Southern-blotting, iaaM gene had beenintegrated with the genome of Physalis angulata L., the rate is 2, 8.2% and foreign gene wasinserted multiple in most transgenic lines.