Study On Rapid Propagation In Vitro Of Thuja Sutchuenensis Franch.

Thuja sutchuenensis Franch.,an evergreen tree of the genus Thuja of the Cupressaceae,is an endemic,rare and endangered gymnosperm and relict plant in China.It was discovered first time in April 1892 but has been extinct for over 100 years,The World Conservation Union（IUCN-SSC）added it as a Critically Endangered plant after it had been rediscovered in 1999.T.sutchuenensis is mainly distributed in limestone mountains at an altitude of 700 m～2200 m in Chengkou County and Kai County,Chongqing,China.It’s wild population is mainly concentrated in Chengkou Dabashan National Nature Reserve.Field investigation of T.sutchuenensis population revealed that its age structure is severely discontinuous,the number of seedlings and young trees is insufficient and the survival rate is low.It’s also found that the seed bearing capacity of T.sutchuenensis is extremely low,the number of seeds is seriously lacking and the abortion is serious.Therefore,it does not have the basic conditions for mass propagation through seeds.Cutting propagation also has many problems,such as limited cutting resources,age effects and position effects.On the whole,T.sutchuenensis population is in a state of extreme decline,the number of it is very limited and the survival condition is also severe.So the rescue breeding of T.sutchuenensis is urgently needed.Therefore,for rare and endangered tree species which have difficulty in sexual reproduction and extremely lack of propagation materials,plant tissue culture can overcome these problems and become an important way to expand their populations.This study used T.sutchuenensis as experiment materials,the rapid propagation system was established by initiation culture,adventitious buds induction,subculture of buds and rooting culture with the technique of plant tissue culture by using the stem tips,scale leaves and scaly stem segments of young and tender branches of T.sutchuenensis growing in the very year as explants.It has laid a foundation for the population propagation and protection of T.sutchuenensis.The main results are as follows:1.The initial cultivation of T.sutchuenensisThe experiment materials were young branches of T.sutchuenen.in conservation and breeding base in Chongqing Dabashan Nature Reserve.This study researched the effects of the seasons of extraction（April,October）,explants（stem tip,scale leaf,scaly stem segment）,surficial disinfectants（0.1%HgCl₂,0.15%HgCl₂,2%NaClO,5%NaClO）and times of disinfection（8,10,12,16 min）and different plant antibacterial PPM(0.5,1.0,1.5,2.0 mg·L^-1),Yipeiling(50 mg·L^-1、100 mg·L^-1、150 mg·L^-1)on the acquisition of sterile materials of T.sutchuenenisis.The test results show that April is the best season of extraction and the stem tips are the best explants.Disinfecting explants with 75%ethanol for 1 min and washing with sterile water for 3 times,and then disinfecting with 0.15%HgCl₂ for 16 min and washing with sterile water for 5 times,and then inoculating them into 1/2 SH basic medium containing 0.15%PPM was the best surface disinfection method for explants.When the stem tips were inoculated into start-up medium（1/2SH+2 mg·L^-11 Trans-ZT）,the new shoots at the leaf tip grew well,reaching a length of 2.47 cm;continue to grow and reproduce under this initial culture condition,the reproductive coefficient was 5.22times,explants grew well and had a certain number of sterile seedlings.2.Induction of cluster buds of T.sutchuenensis.The stem tips of the sterile seedlings of Thuja sutchuenensis Franch.were used as explants.The study researched the effects of different plant growth regulators 1/2 SH+2mg·L^-1 Trans-ZT+NAA(0、0.5 mg·L^-1),1/2SH+2 mg·L^-11 6-BA+NAA(0、0.5 mg·L^-1),1/2SH+2 mg·L^-1 KT+NAA(0、0.5 mg·L^-1),1/2SH+2 mg·L^-1 TDZ+NAA(0、0.5 mg·L^-1),different basic media（1/2SH,MS,WPM,DCR）and different concentrations Trans-ZT(1.0、1.5、2.0、2.5、3.0、3.5、4.0mg·L^-1)on cluster buds induction of T.sutchuenensis.The results showed that the best basic medium was 1/2SH.Using cytokinin Trans-ZT alone has a better induction effect on bush buds than mixed using with auxin NAA.The best adventitious buds induction medium was 1/2SH+2 mg·L^-1Trans-ZT,whose rate of buds was 100%and the proliferation coefficient was 11.28（times）,and the buds grew well.3.Subculture of adventitious buds of T.sutchuenensisThe effects of plant growth regulators:Trans-ZT(1.5,2.0,2.5,3.0 mg·L^-1)and NAA(0,0.2,0.4,0.6 mg·L^-1)were studied by using single adventitious bud as explants.We inoculated adventitious buds which are successively subcultured more than 3 times into anti-browning medium containing different kinds and concentrations of anti-browning agents.What’s more we researched the effects of different salt and hormone concentrations medium(1/4SH+1.5 mg·L^-1 Trans-ZT+0.15 mg·L^-1 NAA、1/4SH+1.0 mg·L^-1 Trans-ZT+0.1 mg·L^-1 NAA、1/4SH、1/2SH+1.5 mg·L^-1Trans-ZT+0.15 mg·L^-1 NAA、1/2SH+1.0 mg·L^-1 Trans-ZT+0.1 mg·L^-1 NAA)on the browning,weakening and growth of adventitious buds.The results showed that the optimal ratio of plant growth regulators of subculture of buds were 2.0 mg·L^-1 Trans-ZT+0.2 mg·L^-1 NAA,and the average reproductive coefficient of 1-4 generations was 12.40（times）.VC,Na₂S₂0₃ and AC can all inhibit the browning of bush buds to a certain extent,but the 2.0 g·L^-1 AC is the best anti-browning agent,the bush buds have almost no browning.Properly reducing the salt concentration and hormone concentration of the medium can effectively alleviate the weakening of the shoots.The most suitable medium to alleviate the weakening of adventitious buds was1/4SH+0.5mg·L^-1 Trans-ZT+0.05 mg·L^-11 NAA.4.Elongation of adventitious buds of T.sutchuenensisTwo kinds of adventitious buds（scaly leaf type,coniferous type）which grew well in subculture were used as explants to researched the effects of different plant growth regulators GA₃(0,0.5,0.75,1.0 mg·L^-1),NAA(1.0,1.5,2.0 mg·L^-1)on elongation of adventitious buds.The results showed that adventitious buds had the best elongation effect in DCR basic medium without plant growth regulators,and the buds grew well.5.Rooting of adventitious buds of T.sutchuenensisTwo kinds of adventitious buds（scaly leaf type,coniferous type）whish grew well in elongation culture of T.sutchuenensis were used as explants to research the effects of three different rooting methods（general rooting method,rapidly dipped buds base rooting method,porous matrix instead of agar rooting method）on root rate,root length and growth status.The first method:inoculating adventitious buds into DCR basic medium containing different kinds and concentrations of plant growth regulators(0,1.0,2.0,3.0 mg·L^-1 IBA and 1.0,2.0,3.0 mg·L^-11 ABT rooting powder).The second method:dipping the adventitious buds base quickly with 100 mg·L^-1 IBA and 100 mg·L^-1ABT rooting powder for 5～15 s,and then transferring to the DCR basic medium.The third method:replace the agar in the medium with a porous and dark matrix（perlite:vermiculite=1:1）.The results showed that the scaly adventitious buds were not easy to induce roots and the rooting rate of coniferous adventitious buds was significantly higher than that of scaly adventitious buds.The rooting rate of coniferous adventitious buds was 76.67%after it was quickly dipped with 100 mg·L^-1 IBA for 10 seconds and then transferred to DCR medium without plant growth regulators,and the longest root length was 12.65 cm.Adventitious buds grew vigorously and the root system was well developed.The porous rooting matrix can effectively improve the browning of scaly adventitious buds in agar medium,but the highest rooting rate was only 27.78%.6.Transplanting of test-tube plantlets of T.sutchuenensisTaking test tube plantlets of T.sutchuenensis as experiment material,the effects of seedling hardening method（bottle-sealing seedling hardening for 7,12,17 d,bottle-opening seedling hardening for 2,3,4 d）,transplanting matrix（PINDSTRUP,perlite:vermiculite:PINDSTRUP=1:1:1,humus,perlite:vermiculite:humus=1:1:1,perlite:vermiculite=1:1）on the transplant survival rate of plantlets.The results showed that the best method for seedling hardening was 17 days of bottle-sealing and 2 days of bottle-opening whose survival rate was 100.00%;The optimal transplanting matrix was perlite:vermiculite:PINDSTRUP=1:1:1,with a survival rate of 80%.The test-tube plantlets（20 plants）after seedling hardening were transplanted to the experimental base in the school,and all of them survived and grew well after 60 days.