| Tangtsinia nanchuanica S.C.Chen is a herb in the Tangtsinia of the Orchidaceae,with a height of 15-35 cm.It generally grows at the lower forest illumination,the edge of the bush,or on the grassy slope at an altitude of 700-2100 m.It was discovered and named by the botanist Chen S.C.in Jinfo moutain,Nanchuan,Chongqing,China in the1960s.It is an unique single genus and single species plants of China.Its distribution range is narrow,the quantity is scarce.It is a national secondary protected plant wihch has been included in the"China Plant Red Data Book".Tangtsinia nanchuanica has the more primitive feature than the most primitive Cephalanthera in the Subfam.Orchidoideae,and it has great significance of the study of phylogeny and the evolution of plant systems.At the same time,Chen S.C.believes that Tangtsinia nanchuanica is one of the ancient relict plants,which has important value of the study of plant genetic variation,floristic geography,genetic diversity,and global ecological diversity.In addition,Chinese Orchidaceae have been highly praised for their unique style and beautiful posture since ancient times.They have a very high ornamental value,cultural value and economic value,but also can be eaten,medicinal.Cultivation of Orchidaceae in China has a long history,which is mainly cultivated through the methods of ramet propagation,pseudo-bulb propagation,seed propagation and tissue culture.However,due to the rare amount of Tangtsinia nanchuanica,the low seed setting rate and the low survival rate of artificial cultivation,tissue culture becomes an ideal method.So far,there have been few reports on the tissue culture and rapid propagation of Tangtsinia nanchuanica.In this study,the rapid propagation system was established by initiation culture,adventitious buds induction,subculture of buds and rooting culture with the method and technique of plant tissue culture by taking ovaries as explants.At the same time,it laid a foundation for the improvement of the quantity of Tangtsinia nanchuanica and the realization of wild regression by transplanting the seedlings,rebuilding,regenerating and building new populations.The main results of the study are as follows:1.The initial cultivation of Tangtsinia nanchuanicaIn this study,the optimum disinfection method of explants was determined and the aseptic explants were successfully obtained by using the whole plant of Tangtsinia nanchuanica.The results showed that the best disinfection method was to first rinse with 75%ethanol for 30 s,then 0.1%mercury chloride solution for 4-6 minutes,and finally rinse with sterile water for 5-6 times.Sterilized plants were cut,separated and dried with filter paper.Then they were inoculated into MS+2.0 mg·L-11 ZT+0.2 mg·L-11 NAA start-up medium respectively.Most of the survival rate was higher than 80%,the pollution ratio was less than 23%,and more aseptic explants were obtained.2.Induction of adventitious buds of Tangtsinia nanchuanicaSterile explants?petals,stems,ovaries,stigmas,leaves,flower buds,roots and rhizomes?and different media(0.2 mg·L-11 NAA+2.0 mg·L-11 6-BA,2.0 mg·L-11 ZT and0.2 mg·L-16-BA+2.0 mg·L-1NAA,2.0 mg·L-12,4-D)were preliminarily screened by using the aseptic explants of the Tangtsinia nanchuanica.Adventitious buds were used as explants to study the effects of different basic media?MS,B5,N6?,plant growth regulators ZT(0.5,1.0,1.5,2.0,3.0,4.0 mg·L-1)and NAA(0.05,0.10,0.15,0.20 mg·L-1)on adventitious buds induction of Tangtsinia nanchuanica.The results showed that the optimum explants were ovaries,the optimum medium of Preliminary screening was ZT 2.0mg·L-1+NAA 0.2 mg·L-1,which rate of buds was33.33%and the proliferation coefficient was 2.33?times?.Under these conditions,a certain number of rootless seedlings can be obtained,and the average propagation coefficient of the first three generations is 2.44?times?.The optimum ratio of plant growth regulators were ZT 4.0mg·L-1+NAA 0.2 mg·L-1,the reproductive coefficient up to 7.05?times?,and the buds grew well.3.Subculture of adventitious buds of Tangtsinia nanchuanicaThe effects of plant growth regulators:ZT(0.5,1.0,1.5,2.0,3.0,4.0 mg·L-1)?NAA(0.05,0.10,0.15,0.20 mg·L-1)and cytokinin species on the induction of subculture of buds of Tangtsinia nanchuanic were studied by using single shoots of Tangtsinia nanchuanic adventitious buds as explants.The results showed that the optimal ratio of plant growth regulators of subculture of buds of Tangtsinia nanchuanic were 4.0 mg·L-11 ZT+2.0 mg·L-11 NAA,and the average reproductive coefficient of one to four generations was 5.94?times?.The optimum cytokinin was 6-BA,with a high reproductive coefficient?11.6times?and grew well.4.Rooting of adventitious buds of Tangtsinia nanchuanicaThe adventitious buds of Tangtsinia nanchuanica were used as explants to study the effect of medium type?1/2MS,MS,1/4MS?,plant growth regulators:NAA(0.5,1.0,2.0,3.0,4.0 mg·L-1),6-BA(0,0.05,0.1,0.5 mg·L-1)and IBA(0,0.5,1.0 mg·L-1),medium type?1/2MS,MS,1/4MS?,sucrose concentration(10,20,30,40,50 g·L-1),activated carbon concentration(0,1,2 g·L-1),coagulant species?agar and phytagel?,rooting method?general rooting method and two-stage rooting method?on rooting of Tangtsinia nanchuanic.The results showed that the optimum rooting medium was 1/2 MS.The optimal plant growth regulators were 0.5 mg·L-11 NAA+0.05 mg·L-11 6-BA,with an average root length of 3.95 cm and an average rooting number of 16.65.The optimum sucrose concentration of rooting was 40 g·L-1.The optimum activated carbon concentration was2 g·L-1.The phytagel was the best rooting coagulator,and the general rooting method had better effect.5.Transplanting of Tangtsinia nanchuanica plantletsTaking Tangtsinia nanchuanic seedlings as explants,the effects of seedling hardening method?bottle-sealing seedling hardening 5,10,15 d,bottle-opening seedling hardening 1,3,5 d?,transplanting matrix?Mountain orchid stone,Turfy Soil,Mountain orchid stone:PINDSTRUP=1:1,Mountain orchid stone:Turfy Soil=1:1,Mountain orchid stone:Hu's nutrient soil=1:1,PINDSTRUP:moss=1:1,Turfy Soil:perlite=1:1,PINDSTRUP:perlite=1:1?and germicide?75%chlorothalonil,carbendazim,70%thiophanate-methyl,80%mancozeb,40%dimethachlon,50%iprodione,45%thiophanate-methyl and Hymexazol?on the transplanting of Tangtsinia nanchuanic were studied respectively.At the same time,one new population,two rejuvenated populations and one regenerated population were constructed in the wild habitat for wild regression of Tangtsinia nanchuanic.The results showed that the best method for seedling hardening of Tangtsinia nanchuanic were 10 days of bottle-sealing and 1 day of bottle-opening which survival rate was 100.00%;The optimal transplanting matrix was Mountain orchid stone:Turfy Soil=1:1,with a survival rate of 100.00%;The fungicide with the best germicidal effect was carbendazim,with a survival rate of 70.83%and a survival rate of 60%after two months of transplantation in Jinfo Mountain.After 60 days of wild regression,the survival rate of the newly established population was 84.91%,the rejuvenated population were 72.73%and 71.43%respectively,and the survival rate of reconstructed population seedlings was 33.33%,all of which grew well. |
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