Anther And Hybrid Embryo Culture In Panax Ginseng C. A. Mey And The Characteristic Evaluation Based On Ginsenoside Content

Ginseng (Panax ginseng C. A. Mey) is a perennial herb that belongs to Araliaceae family. Althoughginseng breeding was started early in China, but has a slow development speed. The long breeding cycle,unstable variants are the main factors that influence the development of breeding. Based on the paralysis,using properiate methods to obtain abundant and stable genetic types are the first job for improving theginseng breeding. In this work, anther culture, hybridization combined with embryo culture, flowcytometry, proteomic analysis and ultra high performance liquid chromatography method have beenused to establish the ginseng anther callus induction and plant regeneration system, the hybridizationyoung embryo culture system and so on. The main results are as follows:1. The study on ginseng anther culture showed that microspore developed at the middle-uninuclearstage has the highest frequency of callus indction. MS+2,4-D1.5mg/L+KT0.5mg/L+Surgure6%+Agar0.6%is the optimal callus induction medium and MS+TDZ3mg/L+IBA0.2mg/L+GA2mg/L+Surgure3%+Agar0.6%is the optimal callus differentiation medium. There are differences onthe frequency of callus induction among different varieties and growth years. Three years damayaginseng has the lower frequency of callus induction, however, the four and five years sample has higher.Huangguo ginseng has the highest frequency of callus induction, and then followed by damaya, ermayaand so on.2. The study on embryo culture of ginseng, American ginseng and ginseng×American ginsengindicated that the development stage of young embryo, varieties and medium are all direct influence thegermination rate of the young embryo. There is a higher germination rate of embryo of varieties ginsengand ginseng×American ginseng at36-45d after pollination, however, American ginseng young embryohas a higher germination rate at45-54d after pollination. Among different varieties, whether at anydeveloped stage, four years ermaya has a higher germination rate (2.3%-38.9%) than four yeas damaya(1.29%-15.6%), four years ermaya×four years American ginseng has higher germination rate amongdifferent hybridized combination (33.7%). MS+BA1mg/L+NAA0.5mg/L+Surgure3%+Agar0.6%medium is fit for the germination of young embryo obtained from four years ermaya×four yearsAmerican ginseng.3. The ploidy identification of anther callus and plants demonstrated that varieties, medium andsubculture times are all factors that direct impact the ploidy of ginseng anther callus. There is noobvious regularity when placed different varieties on the same medium. The first1-2subculture is thepeak of variation of ginseng anther callus, and the mutation rate has a large increase in this period.When subculture exceed5times, the ploidy variation towards stability, and the ploidy is unaffected byculture conditions when anther plantlet appeared.4. Research on cryopreservation method of ginseng anther callus showed that the optimal processof cryopreservation is first pretreated for14d at4℃, then added in cryoprotectant (5%DMSO＋10%Gly+8%Surgure), afterward, the samples were slowly cooled at the rate of1°Cmin-1from0°C to-80°C, maintained at-80°C overnight, and rapidly immersed in liquid N2. The calli were thawed in a 40°C water bath, washed with MS basic medium, and regrown under the same conditions as the originalcalli. Proteomic analysis showed that the abundance of43protein spots significantly changed (p <0.05)under4°C acclimation, and they were identified using mass spectrometry. The36successfullyidentified proteins can be classified into eight groups by their function according to the annotation usingbioinformatics. Most proteins were involved in stress response and defense, amino acid and proteinbiogenesis and degradation, molecular chaperone, electron transport coupled with adenosinetriphosphate (ATP) synthesis, carbohydrate metabolism, and nucleotide metabolism as well as cellularprocesses and so on.5. The ginsenoside content of Rg1, Re, Rb1, Rc, Rb2and Rd in anther callus, anther plants andyoung embryo plants showed that there are significant differences in different culture conditions andmaterial parts. Damaya ginseng cultured on No.1medium has a significantly higher (0.258%) contentthan cultured on No.20medium (0.112%). The total ginsenoside in ginseng leaf (obtained from embryoculture) cultured on L3medium is8.355%, and significantly higher than others (2.100%-6.660%); thetotal ginsenoside content in callus (0.258%) is significantly lower than in leaf (5.291%).In short, in this work, many ginseng and American ginseng breeding resources were obtained,identified, stored and evaluated with a series of related techniques. This is not only enriched our ginsenggermplasm resources but also can provide experimental and theoretical effort for the ginseng breeding.