The High-quality Propagation Of Bletilla Striata Seedlings And Screening And Identification Of Growth-promoting Endophytes

Bletilla striata is one of the precious Chinese traditional medicinal crops in China.It is perennial herb of Orchidaceae and its tubers can be used as medicine.In recent years,the clinical application of B.striata has been expanding,the demand has increased rapidly and the price has also risen sharply.Using tissue culture technology can breed a large number of high-quality seedlings quickly,shorten the seedling cycle obviously and is an effective method to solve the needs of seedlings.The research on the diversity of endophytes in medicinal plants has become a hot topic.Isolation of endophytes from medicinal plants is not only conducive to the full development and utilization of endophyte resources,but also a new way to obtain strains with important research value.In this paper,plant tissue culture technology was used to select the best medium to promote the germination of seed and the induction of pseudobulb.The best diameter of pseudobulb for transplanting tissue culture seedlings of B.striata was explored.The isolation and identification of endophyte from B.striata was carried out.A endophyte B.velezensis F was screened out in this experiment which had the best growth promoting effect.The main results are as follows:1.Mature uncracked capsules of B.striata from Ningbo of Zhejiang were used in the experiment to screen and optimize the best medium to promote the germination of seed.The best medium was MN+100 mg/L KH₂PO₄+8 g/L Agar+100 g/L Bananas+30g/L Sucrose.Seedlings obtained after aseptic germination of the above seeds were used in the experiment to screen the best medium to promote proliferation and expansion of pseudobulb.The best medium of expansion was 1/2 MS+1.0 mg/L 6-BA+0.2 mg/L NAA+100 g/L Potatoes+7 g/L Agar+30 g/L Sucrose+2 g/L Hyponex No.5.The best medium of proliferation is 1/2 MS+1.0 mg/L 6-BA+0.2 mg/L NAA+100 g/L Potatoes+7 g/L Agar+30 g/L Sucrose+3 g/L Hyponex No.5.The transplanting experiment of B.striata with different diameter of pseudobulb showed that when the diameter of sterile seedlings pseudobulb of B.striata reached 6 mm,the transplanting could be carried out.At this time,production cost can be saved and the survival rate of seedlings was as high as 98.44%.2.Seven strains of endophytic bacteria were isolated from the healthy roots,stems,leaves and pseudobulb of tissue culture seedlings of B.striata.Through the seed germination experiments of rape and rice,one endophytic bacteria F with the best growth promoting effect was screened out.The strain F was identified as Bacillus velezensis through morphological observation,physiological and biochemical identification,16S rDNA and homology analysis of gyrB gene sequence.The strain has been preserved in China Center for Type Culture Collection（Preservation number:CCTCC NO:M 2019838）.3.Watering roots of B.striata with fermentation broth of B.velezensis F can promote growth of B.striata and the best concentration was OD₆₀₀=0.5.The average induction rate of pseudobulb was 127%,and the average diameter of pseudobulb was7.592 mm.4.The analysis by LC-MS showed B.velezensis F could produce plant hormone KT,SA and 6-BA,and their concentrations were 20.87 ng/L,56.91μg/L and 30.40 ng/L.