The Establishment Of Rapid Propagation And Regeneration System Of Three Variety Rhododendron In Vitro

Rhododendron is famous woody ornamental plants in the world.It has high ornamental value because of its large flowers,bright colors,various varieties,long flowering period and other characteristics.Its breeding methods are mainly seed propagation and cutting,but have disadvantages of long cycle,prone to mutation and hard of rooting.The cultivation of Rhododendron adventitious shoots through plant tissue culture can maintain the excellent species of Rhododendron,and have characteristics of high reproduction coefficient and fast reproduction speed.It is possible to obtain a large number of high-quality seedlings with excellent species characteristics in a short period of time,which have important application value for industrial production of Rhododendron seedlings.It can provide a reasonable,feasible theoretical basis and supporting technical means for the rapid propagation,seedling breeding,and genetic transformation of Rhododendron.This study focuses on R.simsii,R.latoucheae,and R.delavayi,conducts tissue culture of their in vitro stem segments and leaves.Conduct research on rapid propagation and regeneration in vitro from the aspects of aseptic line establishment,in vitro proliferation,in vitro regeneration,and rooting,in order to obtain transplanted seedlings after in vitro culture,establish aseptic lines,rapid propagation systems,and regeneration systems for three species of Rhododendron.The results of the study are as follows:(1)The best disinfection methods of R.simsii leaves were: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 10 min,sterile water rinsed 3 times,1 min each time.The best formula medium for callus induction was WPM+0.5 mg/L 2,4-D + 0.1 mg/L NAA+0.1 mg/L IBA,with induction rate of 92.65%;the medium formula for callus proliferation: WPM+1 mg/L ZT + 0.5 mg/L NAA,the proliferation rate was 91.43%,and the proliferation coefficient was 2.08;the optimal medium for adventitious bud induction was WPM+0.5 mg/L TDZ + 0.1 mg/L NAA,with induction rate of 91.32%;he adventitious bud proliferation medium was: WPM + 1 mg/L ZT + 0.5 mg/L IAA,with a proliferation rate of 95.32 and a proliferation coefficient of 9.42,and the adventitious bud rooting medium formula: WPM+1 mg/L IBA + 0.5 mg/L NAA,with a rooting rate of 86.43%.The best disinfection methods for the disinfection of R.simsii stem segment were: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 15 min,and sterile water rinsing 3 times for 1 min each time.The optimal combination of media to induce axillary bud germination in stem segments was WPM + 2 mg/L TDZ + 0.5 mg/L NAA,with germination rate of 85.63%;the combination of media for axillary bud proliferation: WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA;the proliferation rate was only 35.07,and the proliferation coefficient was 5.34;the rooting medium for adventitious buds was: WPM + 1 mg/L IBA + 0.5 mg/L NAA,with a rooting rate of 47.76%.(2)The best disinfection methods for R.latoucheae leaves were as follows: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 5 min,sterile water rinsing 3 times,1 min each time.Optimal induction medium formulation for calluses: WPM + 0.5 mg/L 2,4-D + 0.1 mg/L NAA+ 0.1 mg/L IBA,induction rate of 86.07%;calus proliferation medium formulation: WPM + 2 mg/L ZT + 0.1 mg/L NAA,the proliferation rate reached 90.03%,and the proliferation coefficient reached8.97,but in the subsequent adventitious bud induction,the adventitious bud was not differentiated,and callus were browned and died during culture process.The best disinfection methods for the disinfection of s R.latoucheae stem segments were: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 5 min,sterile water rinsing 3 times for 1 min each time.The optimal combination of media for inducing axillary bud germination in stem segments was: WPM + 1 mg/L TDZ + 0.5mg/L NAA,with a germination rate of 83.89%;The medium-base combination of axillary bud proliferation: WPM + 1 mg/L TDZ + 0.1 mg/L NAA,with a proliferation rate of 85.72%and a proliferation coefficient of 6.23,and the rooting medium for adventitious buds was WPM + 2 mg/L IBA + 0.5 mg/L NAA,with rooting rate of 78.23%.(3)The best disinfection methods for R.delavayi leaves were as follows: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 5 min,sterile water rinsing 3 times,1 min each time.The optimal medium for callus induction was: WPM + 1 mg/L 2,4-D + 1 mg/L NAA + 0.5mg/L IBA,with an induction rate of 91.58%;The optimal proliferation medium for calluses: WPM+ 2 mg/L ZT + 0.1 mg/L NAA,the proliferation rate was 90.47%,the proliferation coefficient was7.92,and in the subsequent adventitious bud induction stage,the callus failed to differentiate the adventitious bud and died browned during the culture process.Adventitious budding was directly induced with sterile leaves germinated by axillary buds,and the adventitious bud induction medium was formulated as WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA,with an induction rate of 86.06%;The medium formula for adventitious bud proliferation: WPM + 0.5 mg/L ZT + 0.1 mg/L IAA,with a proliferation rate of 86.32% and a proliferation coefficient of 9.42,and a clanditious bud rooting medium formula: WPM + 1 mg/L IBA + 0.5 mg/L NAA,with rooting rate of 86.43%.The best disinfection method for the disinfection of stem segment of R.delavayi was: 75% alcohol disinfection for 30 s,0.1% Hg Cl2 disinfection for 10 min,sterile water rinsing 3 times,1 min each time.The optimal combination of media to induce axillary bud germination in stem segments was:WPM + 1 mg/L TDZ + 0.1 mg/L NAA,with an axillary bud germination rate of 50.12%;axillary buds of Rhododendron equine had no proliferative effect on the medium,which required further study;the rooting medium of adventitious buds was: WPM + 1 mg/LIBA + 0.1 mg/L NAA,with rooting rate of 28.06%.