Studies On Tissue Culture Of Tea Seed And Somaclonal Variation In Regenerated Plants

Camellia sinensis (L.)O. Kuntze is a significant economic plant. Although a mass of research is about tissue culture of tea plant, the data related to somaclonal variation of tissue culture process haven’t been reported. In this paper, we studied the tissue culture of tea seeds from different varieties and the genetic variation of in vitro regenerated plants by ISSR analysis. The main results are as follows:1. The effects of low temperature storage of tea seeds on differentiation of adventitious bud and adventitious embryoThe results of this study showed that low temperature (4℃) storage of tea seeds was beneficial for the differentiation of adventitious buds of cotyledon and cotyledonary petiole. The optimal storage time of tea seeds from most tea varieties for adventitious buds differentiation was about14days. When vaccination was immediately performed after tea seeds were plucked, the differentiation rate of adventitious embryos was the highest. Generally, tea seeds have a short after-ripening period, which may be a process of intrinsic physiological substances and embryogenic state undergoing changes; and during this period, some bipolar cells transform into unipolar cells. According to this study, low temperature (4℃) storage of tea seeds could accelerate the after-ripening process.2. The difference of differentiation on adventitious buds and adventitious embryos from tea seeds of different varietiesThe research results indicated that induction rate of adventitious buds and adventitious embryos were different because of tea seeds from different varieties, and each kind of tea seeds had their own optimal culture medium for differentiation of adventitious buds and adventitious embryos. After14days storage at low temperature, Anhua Yuan Ye had higher induction rates of adventitious buds and adventitious embryos, the highest induction rate of adventitious buds is100%in C6, and the highest induction rate of adventitious embryos is66.7%in C4; Donghu Zao had higher induction rate of adventitious embryos is100%in C4; Zhu Ye Qi and Bi Xang Zao had the highest induction rate of adventitious buds is100%in C8; but Zhu Ye Qi could barely induce adventitious embryos.3. The effects of different hormone levels on explant differentiation and embryoid re-differentiationAfter comparing different culture medium with different kind of hormone or different concentration of the same hormone, the data showed that adding TDZ, BA+IBA, BA+NAA in culture medium could promote the differentiation rates of adventitious buds and adventitious embryos; culture medium containing2,4-D was suitable for inducing the differentiation of adventitious embryos from tea seeds without storing at low temperature or with short time storing at low temperature; but culture medium containing2,4-D rarely induced the differentiation of adventitious bud from tea seeds.In different culture medium, the re-differentiation situation of embryoid was different. When the culture medium was added with TDZ, it could induce normal shoots and help buds to grow better. In the culture medium adding both BA and IBA, the induction rate of adventitious buds and re-differentiation rate of adventitious embryos were higher. When the culture media were added with TDZ or both BA and high concentration of IBA, embryoid firstly dedifferentiated into callus; then, the differentiated into buds; however, there-differentiated embryoid wasn’t induced. Also, the results showed that the high concentration of IBA could induce the differentiation of abnormal buds, but the lower concentration of IBA could induce the differentiation of normal buds. If both BA and low concentration of IBA were added to the culture medium, abnormal embryoid could be induced directly.4. The ISSR analysis of genetic variation for in vitro regenerated plantsISSR approach was used to analyze the genetic variation of in vitro regenerated plants. The results showed that the genotypes of regenerated plants derived from different hormone levels were different. And the genetic variation extent of regenerated plants due to different hormone levels was different as well. If the culture medium was added with2,4-D or IBA, the extent of genetic variation in regenerated plants was higher, but this induced-variation didn’t happen to every explant. The extent of genetic variation in regenerated plants was different when the explants were from different parts of the same seed, which may be related to the growth and development stages of the explants. Different tea varieties were differently sensitive to hormone at different stages. TDZ played a certain role in the process of inducing genetic variation, the research reports on its mechanism of action in the domestic and foreign are few, looking forward to further study.