Establishment Of Tissue Culture And Regeneration System Of Zhonghuang No.1 Tea Tree

Zhonghua No.1 tea tree is a perennial evergreen shrub of Camellia.It has high amino content,moderate polyphenols and caffeine content,and strong resistance to stress.It is a special rare yellow tea product selected in China.In traditional production,the propagation rate of cuttings is low,which cannot meet the demand of tea seedlings.In this study,the medium yellow tea tree was used as the material to explore the basic conditions of tissue culture,and the tissue culture and rapid propagation technology system of the stem segment of Zhonghuang No.1 tea plant was established,which was the production of the tea tree germplasm of Zhonghuang No.1.Provide technical support to study and establish the regeneration system of the tea tree hypocotyl of Zhonghuang No.1 to lay the foundation for the genetic transformation of tea trees.The main findings are as follows:?1?Establishment of tissue culture and rapid propagation system of stem segments of Zhonghuang No.1 tea plantThe stem segments of Zhonghuang No.1 were used as explants to explore the effects of sampling time,disinfection conditions and plant growth regulators on axillary bud germination,proliferation,seedling growth and rooting.The results showed that the lowest rate of young stems taken in April,June and August was the explants taken in April.The aseptic system of stem tissue culture showed that the stem segment was soaked in 75%alcohol for 60 s and 0.1%mercury immersion for 12 min.After 20 days,the explant survival rate was 91.3%,and the contamination rate was 2.7%.The optimum medium for axillary bud germination and proliferation culture showed that the sterile stem segment was inoculated into the axillary bud germination medium MS+2 mg.L^-16-BA+1 mg.L^-1GA₃,and the budding rate was 91.7%after 40 days of culture.After the axillary buds were grown to 2-4 cm,the shoots were inoculated into the proliferation medium to induce cluster buds.The optimum axillary bud proliferation medium formula under the conditions of the study was MS+4mg.L^-16-BA+1 mg.L^-1IBA,cultured for 45 days,the coefficient of appreciation was 3.8.After60 days,the shoots grew to 2-4 cm,and the shoots were separated for strong seedling and rooting culture,see?3?.It is concluded that the establishment of the medium-cultivation rapid propagation system of Zhonghuang No.1 has improved the rate of tea seedling reproduction and shortened the seedling cycle.?2?Establishment of tissue culture regeneration system of Zhonghuang No.1 tea tree hypocotylThe seeds of Zhonghuang No.1 tea tree were used as explants to explore the disinfection conditions and treatment methods of explants,and the effects of plant growth regulators on seed germination,callus induction and differentiation,seedling growth and rooting.The results showed that cotyledons and embryos were soaked with 75%alcohol for 1.5 min and0.1%mercuric chloride for 20 min to remove cotyledon seed embryos with low contamination rate and high survival rate.After 20 days,explant survival rate was 90%.The pollution rate is 6.7%.The germination conditions of the embryos showed that the sterile embryos were inoculated into the germination medium MS+1 mg.L^-1GA₃ for 15 days,the germination rate was 98.8%,the embryos were germinated and the hypocotyls were longer,60 days later.The hypocotyls of sterile tea seedlings grown in germination medium were used as explants.The callus induction and differentiation conditions of hypocotyl callus showed that the optimal medium for induction and differentiation of hypocotyl callus was MS+2 mg.L.^-16-BA+0.3 mg.L^-1IBA,the callus is firm and the color is green,the adventitious bud is good,the recovery rate is 95.5%,the differentiation rate is 17.5%,and the bud length is 2-3 cm.The buds are separately seeded and rooted,see?3?.?3?Zhonghuang No.1 tissue culture seedlings strong seedling and rooting cultureThe adventitious buds obtained from the callus differentiation of the shoot buds and hypocotyls induced by the stem segments of Zhonghuang No.1 tea tree were cultured in a strong seedling.The plant growth regulators of different concentrations of 6-BA and IBA were studied to grow the tissue culture seedlings.And the impact of rooting.The results showed that the optimum medium for the seedlings was MS+1 mg.L^-16-BA+0.5 mg.L^-1IBA,and the average height of the seedlings cultured for 45 days was 5.3 cm.The rooting conditions of tissue cultured seedlings showed that the plants were immersed in 50 mg.L^-1IBA for 5 min,then transferred to 1/2MS+1.5 mg.L^-1IBA medium,and the rooting rate reached82.5%,and the average rooting rate was 5-6.Strip,main root length,more lateral roots.It is concluded that the hormone concentration conditions can promote the growth and root differentiation of Zhonghuang No.1 tissue culture seedlings.?4?Zhonghuang No.1 tissue culture seedlings refining and transplantingThe effects of different refining time and transplanting substrate on the survival rate of tissue culture seedlings were studied by using medium yellow seedlings as the material.The results showed that the seedlings were opened at room temperature for 5 days under natural light conditions,and transplanted to a matrix of 2:1 in nutrient soil and vermiculite.The survival rate of transplanting was 66.7%,indicating that the survival rate of tea seedling transplanting was improved under this condition.