| Taxus × media Rehder is a Taxus natural hybrid of Taxaceae with Taxus baccata as the male parent and Taxus cuspidata as the female parent.Taxus × media Rehder grows fast,which is 3-7times the growth rate of domestic yew(Taxus chinensis,Taxus yunnanensis,etc.).Taxus × media Rehder is an evergreen coniferous tree with beautiful tree shape,strong germination,and high pruning resistance.The fruits are riped in autumn,and the seeds are wrapped in bright red arils,matching the green needles,showing a red and green scenery,which has high landscaping and beautification value;the branches and leaves are still upright in winter,which adds vitality to the winter landscape,especially to the northern winter urban landscape.In terms of medicinal value,Taxus × media Rehder has a higher content of taxol than other plants in Taxus,but it is currently damaged by some criminals,resulting in wild resources scarce extremely.Therefore,the establishment of an efficient and rapid propagation system and the search for taxol-producing fungi has important practical significance,which are no longer at the expense of taxus resources.In this study,the stalk segment of Taxus × media Rehder was used as material for cutting propagation and tissue culture propagation,and a rapid propagation system of Taxus × media Rehder was established.At the same time,endophyte were isolated and identified,and paclitaxel-producing bacteria were initially selected.The main findings are as follows:(1)IBA,NAA and ABT-1 were used as rooting agents for cutting propagation of Taxus ×media Rehder,and the optimum rooting agent and concentration were selected.The results showed that IBA was superior to NAA and ABT-1 in rooting effect of Taxus × media Rehder,and 400 mg / L is the optimum concentration,with a rooting rate of 79.17%.(2)Tissue culture was performed with the buds and stems of Taxus × media Rehder in current year as explants.The results showed that When MS was used as the basic medium,The plant growth regulator combination suitable for the start culture of Taxus × media Rehder is NAA1.0 mg / L + 6-BA 0.1 mg / L and the WPM medium was the most suitable basic medium for initiation culture.The optimal medium for subculture is MS(double organic matter)+ 1.0 mg /L NAA + 0.1 mg / L 6-BA,average bud length reaches 2.07 cm,and tissue culture seedlings grow robust.(3)In this study,78 endophytic fungi and 40 endophytic bacteria were isolated from stems,leaves,bark and seeds of Taxus × media Rehder.The number of endophytes isolated from different parts varies greatly.Among them,endophytic fungi are most abundant in bark and stem segments,and endophytic bacteria are most abundant in bark and seeds.From the level of the family to which the isolate belongs,the dominant family of endophytic fungi are Pleosporaceae,Subspora,Sphaeropsidaceae,Cryptaceae and Nectriaceae,accounting for 71.79% of the isolated endophyte strains;Microbacteriaceae,Planococcaceae,Micrococcaceae,Pseudomonasceae and Rhizobiaceae are the predominant families of endogenous bacteria,accounting for 67.50% of the isolated endogenous bacterial strains.(4)The isolated endophyte DNA was used as a template,the PCR reaction was conducted to amplify HMGR gene,DBAT gene and BAPT gene,which are indispensable enzymes in the paclitaxel synthesis pathway,the related genes were amplified by Alternaria alternate(Number:1J3),Peyronellaea sp.(Number:TM-21),Dothiorella gregaria(Number:2J8),Alternaria longipes(Number:2J2)and Cladosporium pseudocladosporioides(Number:TM-31)respectively,and it is speculated that these five endophytes have the ability to produce paclitaxel. |
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