Studies On Regeneration System And Genetic Transformation Of Sinningia Speciosa

Sinningia speciosa, perennial herbaceous bulbous flowers belonging to Gesneriaceae, isoriginated from Brazil and widely cultivated as an indoor ornamental flower plant during thespring and summer around the world. It likes warm conditions, is not tolerant of lowtemperature, and can’t overwinter below five centigrade degree. Therefore, cultivatingcold-resistant variety is expected to achieve non-greenhouse overwintering, expand theplanting area, reduce the production cost. Sinningia speciosa flowers about160-210daysafter sowing, and the vegetative growth period is long. Cultivating early-flowering varietycan come into the market earlier and improve the value of the commodity.In this paper, cold-resistant regulatory gene AtCBF1and early flowering gene AtLEAFYwere introduced into Sinningia speciosa respectively by Agrobacterium tumefaciensLBA4404. The results obtained are also following:（1） We established the regeneration system of gloxinia. The young leaves of Sinningiaspeciosa growing exuberantly in pots, were sterilized with alcohol and HgCl2, and putted intothe bud induction medium for adventitious buds differentiation. Then, adventitious budscutted stem apexes were used for subculture proliferation. Cutted shoots could be used to root,and regenerated plantlets survived after transplantation. Obtaining the optimal medium ofdifferent culture stages: bud induction and subculture medium, MS+0.5mg·L^-16-BA+0.1mg·L^-1NAA; rooting medium,1/2MS+0.2mg·L^-1NAA.（2） The plant expression vectors of AtCBF1and AtLEAFY were constructed. We ampliedthe CBF1gene using Arabidopsis thaliana leaves cDNA as the template. The plantexpression vector pBI121-AtCBF1was constructed by using restriction enzyme sites BamH Iand Sac I through the ligation of AtCBF1and pBI121. We amplied the LEAFY gene usingArabidopsis thaliana buds cDNA as the template. The plant expression vectorpBI121-AtLEAFY was constructed by using restriction enzyme sites Xma I and Sac I through the ligation of AtLEAFY and pBI121. The constructed expression vectors were transformedinto Agrobacterium tumefaciens LBA4404.（3） We used petioles and leaves of Gloxinia with red and purple flowers as materials toinduce callus and adventitious buds. Both induction medium MS+1.0mg·L^-16-BA+0.1mg·L^-1NAA and MS+2mg·L^-16-BA+0.2mg·L^-1NAA could induce callus and adventitious budsdifferentiation, but the former is faster than the latter in buds differentiation. We confirmedthe leaves selection pressure of20mg·L^-1Kan by kanamycin （Kan） concentration gradientexperiment using Gloxinia aseptic seedlings leaves as material. And we confirmed the rootingselection pressure of10mg·L^-1Kan by kanamycin （Kan） concentration gradient experimentusing Gloxinia aseptic seedlings adventitious buds as material.（4） The leaves and petioles explants were inoculated with Agrobacterium tumefaciensLBA4404containing CBF1/LEAFY gene. Then the explants were induced to generateadventitious shoots and roots. Finally, we obtained regenerated candidate transgenic plants.We extracted genomic DNA from candidate transgenic plants and recovered CBF1transgenicplants, identified by PCR. And cold resistance of Sinningia speciosa CBF1transgenic plantswere preliminarily identified. Identification of LEAFY transgenic Gloxinia plants isunderway.