Studies On Tissue Culture And Rooting Mechanism In Cuttings Of Camellia Sinensis(L.)O.Kuntze

Two technical approaches for in vitro clonal propagation have been developed in tea plants.The experiment takes the Camellia sinensis cv.“Anjihuangcha”,Camellia sinensis cv.“Shanchayihao” as materiales.Initiation of culture,shoot multiplication system,plantlet regeneration system and rooting were investigated.Shoot multiplication system,plantlet regeneration system and rooting were investigated.The experiment takes the Camellia sinensis cv.“Longjing 43” as materials.The dynamic changes of related physiological and biochemical indexes were investigated,analysing the relationship between these indexes and the cutting rooting.Besides,the molecular biological characteristics of gene CsARRO-1 were analysed.The results are listed as follows:1.Establishment of tissue culture and rapid propagation system of tea plantStudy on explant establishment method,primary screening,subculture and rooting culture medium.(1)Explant establishment.The explants,sampling sites,sampling season,and the effects of different antioxidants on the proliferation of explants were investigated.The results showed that the semi lignification shoots cultured in greenhouse were suitable for sterilization,and the best germination effect was obtained at 3~5 months.(2)Primary screening medium.The effects of different concentrations of auxin and different antioxidants on the proliferation of axillary bud of tea tree were investigated: In vitro shoot primary culture medium best Camellia sinensis cv.“Shanchayihao” MS+6-BA 2.0 mg/L+ NAA 0.1 mg/L+GA3 3.0 mg/L+PVP 4 mg/L.Camellia sinensis cv.“Anjihuangcha” best primary culture medium is MS+6-BA 2.0 mg/L+IBA 0.1 mg/L+GA3 3.0 mg/L+PVP 4 mg/L.(3)Screening multiplication medium.The effects of different hormone combinations such as hormone type,concentration and ratio on the axillary bud subculture of tea plant were studied.The results showed the best subculture culture medium for Camellia sinensis cv.“Shanchayihao” is MS+IBA 0.1 mg/L+6-BA 3 mg/L+GA3 1.0 mg/L,Camellia sinensis cv.“Anjihuangcha” is: MS+TDZ 0.02 mg/L+IBA 0.1 mg/L.The long-term use of hormone TDZ is not conducive to the growth of Camellia sinensis cv.“Shanchayihao” seedling to MS+NAA 0.1mg/L+6-BA 1mg/L+GA31.0mg/L;MS+NAA 0.1mg/L+TDZ 0.005mg/L to Camellia sinensis cv.“Anjihuangcha”seedling leaves culture medium for 20 days to help spread and elongation of tea seedlings.(4)Screening rooting medium.The effects of hormone type and concentration on Rooting of tea plant tissue culture seedlings were investigated.For rooting,Camellia sinensis cv.“Shanchayihao” shoots were treated with 500mg/L IBA solution for 5 minutes and transferred directly to hormone-free 1/2 MS medium.Camellia sinensis cv.“Anjihuangcha” in 1/2MS+6-BA 0.5mg/L+NAA 0.2 mg/L+0.1 IBA mg/L,culture medium,root induction effect is better.The addition of activated carbon to 0.2~0.3 mg/L helps to promote root growth.2.Study on the mechanism of adventitious root formation in tea cuttings.The effect of exogenou hormones on cutting survival rate was studied with one-year-old and semi-woody branches of the Camellia sinensis cv.“Longjing 43”.The mechanism of adventitious root formation was analyzed in terms of physiology,biochemistry and molecular biology.(1)The rooting rate of 30 min treated with 300 mg/L NAA was higher than that of water treatment.(2)The activities of three enzymes,IAAO,POD and PPO,related to rooting,were increased in callus formation and root primordium induction stage.The enzyme activities of hormone treated cuttings were basically higher than those in clean water treated groups.(3)The 4 endogenous hormones in cuttings showed different correlations with adventitious roots.IAA is most closely related to adventitious roots.(4)The cDNA gene was cloned from the Camellia sinensis cv.“Longjing 43” cuttings of Longjing.The cDNA full-length was 1129 bp,with 915 bp ORF encoding 305 amino acid.There was one internal transmembrane structure.The expression level of CsARRO-1 was studied with qRT-PCR.It was found that the expression of CsARRO-1 increased after hormone treatment in the cuttings,and the expression of the roots in each phase was higher than that in the water control.