Optimization Tissue Culture And Screening Of Endophytic Fungi From Ludisia Discolor (Orchidaceae) In Hainan

As an important plant resource,Orchidaceae has great ornamental and medicinal value,and is in great demand in the market at present.However,natural orchid seed germination difficulty,points strains breeding coefficient is low,although its multiply transformation technology 1s adopted to accomplish,but the somaclone domestication low survival rate,growth and development 1s slow,its important reason is that the rocess of life without the articipation of the symbiotic fungi,so the vaccine symbiosis of orchid conservation has the important significance.Ludisia discolor is a traditional li medicine in Hainan,which has unique ornamental and medicinal value.Due to the complex breeding system and habitat destruction caused by artificial logging,wild Ludisia discolos 1s extremely scarce.Therefore,our studies,the object was to optilize the rapid propagation system by aseptic sowing of Ludisia discolor seeds.The endophytic fungi of wild Ludisia discolor were isolated and the community composition of the endophytic fungi was discussed.The sterile tissue culture seedlings were inoculated against the isolated strains,and the effects of the inoculation were observed,so as to screen out the strains that could significantly promote the growth of the tissue culture seedlings,and provide valuable theoretical basis for the rapid breeding and production of Ludisia discolor seedlings,which is of great significance for the conservation of Ludisia discolor germplasm resources.1 Optimization of rapid propagation system of Ludisia discolor(1)Using the seeds of Ludisia discolor as explants,it was found that the best germination medium was KC+20g/L potato,with the earliest germination time,and the germination rate was as high as 88.44%.(2)The rhizome obtained by seed germination was induced to differentiate,and the medium of lyponex No.4 was added as the best induction medium.The differentiation bud rate was 98.30%,and the germination was neat and robust.(3)The optimal proliferation medium was 3.0mg/L KT+0.3mg/J NAA,the proliferation multiple was 5.05 times,and the growth of prolifc ration buds was better.(4)In the strong root culture,0.5mg/L IBA,0.5mg/J NAA and banana 10 g/L are conducive to the growth of blood blue orchid plant height;0.51g/L IBA+0.01mg/L NAA+100g/L Banana is conducive to the rooting of Ludisia discolor,but the optimal combination was 0.5mg/L IBA+0.1mg/L NAA+100 g/L Banana,the average plant height of 8.97,the average number of roots is 3.59.(5)The best substrate for seeding transplanting seedlings is sphagnum moss,the survival rate of transplanting 1s up to 100%,and the plants are healthy.2 Diversity of endophytic fungi in Ludisia discolorEndophytic fungi were isolated from wild Ludisia discolor,and 66 endophytic fungi were obtained.After molecular biological identification,they were classified into 2 orders,4 classes and 18 genera,showing the diversity of endophytic fungi in the leaves.Among them,Collelolrichum is the dominant group,accounting for 33.33%of the total number of strains,follow by Aspergillus,FusariIm and Alteznaria are also isolated.The isolation rate of endophytic fungi in different parts of Ludisia discolor was the highest in the stem,which was 36.26%;the leaf was the third,which was 34.85%;the root was the lowest,28.79%,and both the root and the stem(Colletotrichum)had the highest rate,accounting for 36.84%and 50.00%of the total number ot strains,respectively.In different tissues,the leaf diversity index and evenness index were the highest,2.75 and 1.19 respectively;the similarity coefficient was 0.381,the lowest endophytic fungi in roots and leaves,the similarity coefficient was 0.267,but both showed moderate similar.3 Screening of symbiotic fungiSix strains were found to be symbiotic with the Ludisia discolor tissue culture seedlings.After co-cultivation with G9 strain for 90 days,the average fresh weight could increase rate was 55.01%,the plant height was 8.76 cm,and the stem diameter was 0.48 mm.The number of roots was 3.60;among them,the G6 strain promoted the root number up to 4.40,and the G11 strain(3.94cm)promoted the root length.Strains G6 belong to Alternaria sp.,G9 and Y21 belong to Colletothchum sp.,G11 and Y20 belong to Daldinia sp.,and Y6 belong to Trichoderma sp..