| Scutellaria tsinyunensis C.Y.Wu et S.Chow,as a rare and endangered plant in Chongqing,its population and individual number are decreasing,natural regeneration is hindered,and the living conditions in the field are extremely worrying.Under natural conditions,there are sexual reproduction obstacles in S.tsinyunensis,so asexual reproduction has become the main mode of reproduction in the field population,but it is also restricted by many factors such as allelopathy and autotoxicity,which leads to its asexual reproduction being inhibited.Therefore,only through artificial propagation to increase the number of individuals,expand the range of population and study the restriction mechanism of allelopathic autotoxicity on S.tsinyunensis,can we alleviate the endangered situation of S.tsinyunensis and put forward effective protection countermeasures.However,the way of cuttage propagation in artificial propagation has great damage to the original population of S.tsinyunensis,and it can not provide a large number of the same experimental materials for the study of allelopathic autotoxicity,so the above problems can be completely solved only by selecting the artificial propagation method of tissue culture.Therefore,the main difficulty of this paper lies in the establishment of tissue culture system of S.tsinyunensis and the study of allelopathic autotoxicity,the comprehensive analysis of the potential endangered mechanism and the corresponding protection countermeasures.In this paper,the wild S.tsinyunensis was used as material,and the rapid propagation system of S.tsinyunensis in vitro was established by exploring the conditions of cluster bud induction,subculture,induced rooting and seedling cultivation.The tissue culture seedlings of S.tsinyunensis were used as materials,and the regeneration system of S.tsinyunensis was established by screening the effects of different media on the dedifferentiation of S.tsinyunensis stem,the effects of different explants on the formation of callus and the effects of plant growth regulators on the dedifferentiation of S.tsinyunensis stem.The tissue culture seedlings used as materials,and the allelopathic autotoxicity of S.tsinyunensis were studied by exploring the effects of water extracts from stems and leaves and root exudates on the growth and physiological activities of S.tsinyunensis.The main conclusions are as follows:(1)MS+0.5 mg·L-1 6-BA+0.5 mg·L-1 NAA was the best medium for cluster bud induction and subculture of S.tsinyunensis.The rate of cluster bud induction was 100%.After 30 days of culture,the propagation coefficient was as high as 9.55.The average propagation coefficient of four successive subcultures was 9.11.The growth of cluster bud was good.The best medium for inducing adventitious bud rooting was 1/2MS+0.1mg·L-1 IBA+0.1 mg·L-1 NAA,the rooting rate is 100.00%,the number of roots is 24.92,the length of roots is 3.11 cm,the root system is developed,the growth of test tube seedlings is robust;after test tube seedlings are cultured,59 plants survive 30 days,the survival rate is 98.33%.(2)Compared with WPM and B5 medium,MS medium is the most suitable medium for inducing stem dedifferentiation of S.tsinyunensis,with the induction rate as high as76.67%;Compared with leaves and petioles,the dedifferentiation effect of stem segments was the best,and the induction rate was 86.67%;MS+1.0 mg·L-1 6-BA+0.5 mg·L-1 NAA is the best medium for inducing stem dedifferentiation of S.tsinyunensis.The callus induction rate is 89.58%.The callus can be differentiated from bud induction medium(MS+0.5 mg·L-1 6-BA+0.5 mg·L-1 NAA).(3)The results showed that the growth of the tissue culture plantlets was inhibited obviously and the root growth was hindered by the water extracts of stems and leaves of different concentrations of S.tsinyunensis.The plant height,root length,fresh weight and fresh weight of the tissue culture plantlets were all inhibited by allelopathy,which were lower than that of the control group.The MDA content of the tissue culture plantlets increased,and the allelopathy was promoted,while the decrease of soluble protein content was decreased,and the allelopathy was inhibited.The activities of POD and SOD in tissue culture seedlings increased first and then decreased.In general,the water extract of stem and leaf of S.tsinyunensis had allelopathic autotoxicity,and the inhibition of stem water extract was stronger than that of leaf water extract.(4)The root exudates of different concentrations of tissue culture seedlings of S.tsinyunensis were used to treat the tissue culture seedlings of S.tsinyunensis.The growth of the tissue culture seedlings was slow and the root growth was inhibited.The plant height,root length,plant fresh weight and root fresh weight of the tissue culture seedlings were all reduced,and the inhibition of the root length was the strongest,which was manifested as the allelopathy inhibition.The MDA content of the tissue culture seedlings of S.tsinyunensis was gradually increased,and the allelopathy effect was manifested as the promotion.The soluble protein content content of the tissue culture seedlings of S.tsinyunensis was gradually decreased,and the allelopathy effect was manifested as the inhibition.The activity of POD and SOD in tissue culture seedlings increased first and then decreased,but both of them were higher than that in the control group.In conclusion,the root exudates of S.tsinyunensis tissue culture seedlings have allelopathic autotoxicity effects on S.tsinyunensis tissue culture seedlings.Combined with the previous studies,a comprehensive analysis of the results of this experiment shows that the endangered mechanism of S.tsinyunensis is the result of the interaction of the intraspecific competition of allelopathic autotoxicity,the breeding characteristics of low natural fruit setting rate,the seed characteristics of low germination rate of nutlets,the status quo of isolated"island"distribution,the interspecific competition of associated plants and the interference of human activities.In this regard,the countermeasures of local protection and ex situ protection of S.tsinyunensis were put forward.The existing wild habitat should be protected and monitored for a long time.The breeding base of S.tsinyunensis should be established to realize rejuvenation and field return,and effectively expand the range and quantity of its wild population.In addition,we should further study the allelopathic autotoxicity of S.tsinyunensis,clarify the process of allelopathic autotoxicity caused by allelochemicals,analyze the mechanism of allelochemicals affecting the growth and population expansion of S.tsinyunensis,and then put forward protective countermeasures. |
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