| A stable tissue culture regeneration system is essential to establish a genetic transformation system for plants.The current tissue culture systems of tea plant(Camellia sinensis L.)are still unstable and have problems with reproducibility.This renders them unsuitable for the genetic transformation of tea.Therefore,the establishment of an efficient and stable tea plant regeneration system is particularly important for the development of genetic engineering in tea.The stem segment of tea plant has a strong ability to undergo organogenesis.Its requirements for culture medium and growth regulators are simpler than those of other explants.These factors make it a good material to study the regeneration of explants.The rapid propagation of stem segments is the basis for establishing aseptic regeneration systems and ensuring many unified explants.The induction effect of internode stem segments as explants is ideal,this study utilized the internode stem segments of the "Zhonghuang No.1" tea plant cultivar as explants and examined a series of influencing factors,such as the culture medium and plant growth regulators;optimized various conditions of tea plant tissue culture;and established a complete set of tea plant stem tissue culture regeneration system,which laid a foundation for the research of propagation and genetic transformation of improved varieties of tea.The main results are as follows:1.An aseptic system was established for stem segments with axillary buds of the "Zhonghuang No.1" tea plant.Stem segments with axillary buds of "Zhonghuang No.1" were used as explants to explore the effects of disinfection combinations,sampling time,stem segment morphology with axillary buds and culture conditions on the establishment of an aseptic system.The results showed that the best culture combination was to take the young stems and shoots of "Zhonghuang No.1" that were growing in April as explants,soak them in 75% alcohol for 1 min,wash them with sterile water 3-4 times,soak them in 0.1% mercuric chloride for 12 min,wash them with sterile water 4-5 times,and take the stems with short internodes.The stems were cultured in the dark for 7 days and then exposed to normal light culture to obtain sterile seedlings.The lowest pollution rate was 11.2%,and the highest survival rate was 79.2%.2.Establishment of a tissue culture regeneration system of tea plant stem.The results led us to utilize internode stem segments that were 1 cm long.They were inoculated on MS + 70 mg/L adenine sulfate + 3 mg/L 6-benzylaminopurine(6-BA)+0.2 mg/L TDZ medium and cultured under light conditions.It was the best combination for the induction of callus using a tea plant stem segment.The highest healing rate was 97.8%,and the highest budding rate was 72.3%.The callus was inoculated on MS + 1 mg/L 6-BA + 1 mg /L TDZ + 0.2 mg/L IBA medium and cultured under light conditions.This was the best combination for callus induction and the differentiation of tea plant stems,and the highest proliferation coefficient was4.3.The regenerated buds inoculated with MS + 0.2 mg/L 6-BA + 2 mg/L NAA can effectively promote the growth and development of regenerated buds,and the plants were strong and grew well.tea plant seedlings inoculated with MS + 0.7 mg/L 6-BA +0.2 mg/L NAA + 1 mg/L IBA can effectively promote the cultivation of strong seedlings,and the highest average plant was 4.1 cm.After soaking the bottom incision with 200 mg/L IBA solution for 10 min,the tea plant seedlings were inoculated with1/2 MS + 2 mg/L IBA + 0.5 mg/L NAA.After inoculation,culture in the dark for 7 d could effectively promote rooting.The highest rooting rate was 63.3%,and the highest average number of roots was 4.7.After 3 days of introducing the seedlings to the outside climate,the tea plant seedlings were transplanted into a soil matrix with a ratio of nutrient soil: vermiculite: perlite = 2:1:1(v/v/v),and the highest survival rate was 68.7%. |
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