Study On Tissue Culture Rapid Propagation System And Regeneration System Of Zikui Tea Plant

Zikui tea plant was a new line screened from the local tea plant germplasm resource Meitan-taicha（Camellia sinensis‘Meitan-taicha’）in Guizhou,and it was a native purple-leaf tea plant.Zikui tea plant was an ideal raw material for making black tea and green tea,and could be promoted as a high-quality germplasm resource in Guizhou.At present,the shortage of tea seedlings of Zikui had limited its promotion and application.In order to increase the number of seedlings and shorten the breeding cycle of Zikui,in this study,Zikui was used as the material to establish the tissue culture rapid propagation system and regeneration system.The results of the study were as follows:1)Study on tissue culture rapid propagation system of Zikui tea plantAn aseptic system for stem segments with axillary buds was established:an experiment was designed with the combination of 75%alcohol and 20%sodium hypochlorite.The results showed that the best effect was achieved under the conditions of 75%alcohol for 2 minutes and 20%sodium hypochlorite for 13 minutes.After 20 days,the pollution rate was 25.45%,the mortality rate was 6.67%,and the survival rate was 67.88%.Seed embryo germination medium is determined.Different concentrations of 6-BA,IBA and GA₃ were added to MS medium to induce axillary bud germination.The germination rate of axillary buds reached 94.29%in the medium of MS+2.00 mg/L 6-BA+0.90 mg/L IBA+1.20mg/L GA₃,after 45 days,the subsequent sprouts grew rapidly,the axillary buds were robust,and the leaves stretched.Axillary bud proliferation medium was determined:different concentrations of 6-BA,NAA and IBA were added to MS medium to promote axillary bud proliferation.The results showed that the proliferation coefficient reached 4.36 after 50 days in the medium of MS+3.50mg/L 6-BA+0.20 mg/L IBA,and the sprouts were enlarged and sprouted at the incision,resulting in a large number of clustered buds.The optimum medium for seedling growth was determined:different concentrations of6-BA and IBA were added to the MS medium to induce seedling growth.The results showed that the plant height difference was 2.12 cm after 45 days in the medium of MS+2.00 mg/L 6-BA+0.60 mg/L IBA,the sprouts were robust and without withering.The rooting medium of the plants was determined:the unrooted shoots were soaked in60.00 mg/L IBA sterile solution for 8 minutes,then it was inoculated into the basal medium of different concentrations of IBA or NAA combined for rooting.The results showed that the rooting rate after 60 days was 68.57%,the average rooting number was 8.67,the average root length was 2.23 cm in the medium of 1/2 MS+1.60 mg/L IBA,and the overall growth of the plant was good.The hardening time and transplanting medium were determined:the hardening time and the transplanting medium was designed as an experiment.The results showed that when the seedlings were hardened for 6 days under natural light conditions,transplanted to V _{yellow loam}:V _vermiculite=2:1 matrix,the survival rate after 60 days was 65.22%.2)Study on Zikui tea plant regeneration systemAn aseptic system for seed embryos was established:an experiment was designed to disinfect the seeds with a combination of 75%alcohol and 20%sodium hypochlorite.After treatment,the seed embryos were picked.The results showed that the embryos were sterilized with 75%alcohol for 3 minutes and 20%sodium hypochlorite for 10 minutes,the embryo contamination rate was 17.58%,the mortality rate was 9.69%,and the survival rate reached72.73%.The seed embryo germination medium was determined:the MS medium was supplemented with different concentrations of GA₃ for seed embryo germination culture.The results showed that the germination rate of the embryos after 60 days was 93.64%in the medium of MS+2.40 mg/L GA₃,the hypocotyl was long,the root hair was short,and the hypocotyl was easier to cut.Determination of hypocotyl callus and differentiation medium:adding different concentrations of 6-BA and IBA,6-BA and NAA,and 6-BA and 2,4-D to the WPM medium.The hypocotyls were cultured for callus and bud differentiation induction.Hypocotyls were better when the medium was WPM+2.00 mg/L 6-BA+0.10 mg/L NAA.After 60 days,the callus induction rate was 95.19%,and the adventitious bud differentiation rate was 20.74%.The adventitious buds were grown vigorously:the adventitious buds were excised from the callus,and the lower ends of the adventitious buds were cut obliquely.The adventitious buds were grown for strong seedlings under the conditions of MS+2.00 mg/L 6-BA+0.6 mg/L IBA.The sterile seedlings were rooted,hardened and transplanted:The lower end of the rootless seedlings with robust growth and a height of about 5 cm was cut obliquely.Rootless shoots were rooted under the condition of 1/2 MS+1.60 mg/L IBA.The tea plants were rinsed and transplanted into V _{yellow loam}:V _vermiculite=2:1 transplanting medium.