Optimization Of Regeneration System In Viola×Wittrockiana Gams. And A Preliminary Study Of Polyploid Breeding In V. Tricolor L.

V. tricolor L. is native to europe, for the perennial plant Viola Violaceae, with a heat-resistant, early flowering, more flowers. Which is one of the main hybrid parents for widely cultivated pansy in the current landscape application. Garden pansy (Viola×wittrockiana Gams.) is important parterre flower in winter and spring, whose market are in great demand.We spent a lot of manpower and resources on F1 seed production. Using of male sterility is imperative to reduce production costs. This study want to speed up the breeding process through creating male-sterile lines using transgenic research, Efficient and stable regeneration system is the basis for genetic transformation system, we did a lot of work to optimize the regeneration system; To create strong resistance, high ornamental value new varieties, doulbing treatment with colchicine in V. tricolor L.. the results as following:1. Regeneration has been affected because of the vitrification in tissue culture, different concentrations (basic medium, agarose, sucrose, hormone and activated carbon)were designed for vitrification and proliferation in tissue culture of pansy in this experiment, The results showed that the conditions for maximal proliferation coefficient and minimal vitrification were as following:the MS basic medium with 0.5mg/L 6-BA, 0.05mg/L NAA,30 g/L sucrose and 8 g/L agarose. We observed that high temperature and insufficient light are the main reason. So cooling facilities are needed in culture room, material should be single layer and equidistance, with good light transmission and breathable sealing membrane.2. Effect of basic media, hormones, activated carbon, dark incubation time on the regeneration of inbred line 22-8 stem were studied. The results showed that the highest regeration frequency was obtained on the 1/2MS basic medium with 1.0mg/L TDZ, 0.1mg/L NAA, was 66.7%; activated carbon inhibited regeneration of pansy; suitable dark time was 5 d.3. (1) Two methods were used for chromosome number doubling, one was achieved by soaking swollen seeds, the other was dipping the apex of the young seedlings. We obtained that swollen seeds were sensitive to colchicine, most of the seeds were in death by poison, only a little of plants have survived at a very low concentration of 0.05%. With the concentration increasing, time prolonging, survival rate decreased dealing by titration tip. The highest mutation rate was 71.4% with the same concentration of 0.5% for 2 days; the highest mutation rate was 73.7% with the same time of 3 days, the concentration of of 0.5%.(2) Polyploid identification:With the slow variation of plant growth, these morphological index, as plant height, plant width, leaf index, were significantly lower than the control. Stem diameter, leaf width, flower diameter index were significantly higher than the control, and the leaf color dark green, wrinkled, rough texture, the flowers fold. The variant plants were larger stomata, stomatal density was smaller, larger pollen grains, pollen viability decreased; chromosome number of dipliod was 2n=26, tetraploid was 2n=52; by flow cytometry detection, we obtained six detected tetraploid, two chimera.