Aseptic Seed Germination And Rapid Clonal Propagation Of Bletilla Striata

Bletilla striata is a terrestrial orchid native to Asia.It is has been cultivated as an ornamental plant because of big,beautiful flowers.Also,B.striata has been one of important herbs and has been used traditionally for treatments of various ailments,such as tuberculosis,hemoptysis,gastric and duodenal ulcers,trauma,sores,and cracked skin on the feet and hands.Modern pharmacology research has revealed that B.striata has biological activities of anti-inflammation and antitumor.In addition,the underground tubers contain high level of polysaccharides that have multiple biological functions,such as antioxidation,anti-inflammation,and promotion of wound healing.Because B.striata polysaccharides are nature biopolymers and have no cytotoxicity,they have been widely used not only in foods and cosmetics,but also as biomedical materials.Because its use has been widely expanded,B.striata has been over collected in the past decade,which has led to sharp reduction of its wild resources.In the future,B.striata must be artificially cultivated to meet increasing commercial demands.Large-scale cultivation of B.striata depends on enough supply of young plants.A mature B.striata plant can produce several flowers every year,and after pollination,each flower can develop into a capsule containing tens of thousands of seeds.However,B.striata mature seeds contain few nutrient reserves and embryos are undifferentiated,and few of them can germinate in nature.Although B.striata can be vegetatively propagated by tuber-dividing,the rate is very low.Efficient propagation of B.striata must rely on plant tissue culture.In this study,we systematically investigated the effects of seed maturity,basal media,plant growth regulators,illumination conditions,storage temperatures and times on germinability of B.striata and the effects of plant growth regulators on induction,multiplication and shoot initiation of protocorm-like bodies(plbs),and established an efficient seedling production protocol via aseptic germination and a rapid clonal propagation protocol of the species.The main results are following:1.Germinability of B.Striata increased as seeds matured.The fully mature seeds germinated within one week of incubation on basal CF medium and germination rates were over 95%,even 100%within two weeks.B.Striata seeds matured after 130 days of pollination,when capsules were pale yellow or yellow.2.Low salt media such as CF,1/2MS and WPM media were superior to high salt media like MS and B5media in supporting seed germination and shoot growth.3.0.05-3?M BA significantly promoted germination of immature seeds,but not mature ones.1?M NAA had no profound effects on germination of both immature and mature seeds.Presence of 2-3 ?M BA in medium stimulated multiplication of protocorms and formation shoot clusters,but inhibited further growth of the shoots.4.Dark culture did not affect germination of both immature and mature seeds.5.Dry mature seeds remain excellent germinability during 7 months of storage at 25? or 12 months of storage at-8 ? or-80?.6.About 30%of lateral buds and shoot tips isolated from young shoots produced protocorm-like bodies within one month of culture on CF medium supplemented with 5 mg/1 BA and 2 or 5 mg/1 NAA.7.Protocorm-like bodies multiplied by 6-fold when they were cut into pieces and subcultured on CF medium containing 0.2 mg/1 BA and 1 mg/1 NAA for one month.The newly-formed protocorm-like bodies initiated shoots as incubation lasted for three months.8.Either seed-derived shoots or protocorm-like body-derived shoots rooted very well on medium enriched with 1 g/1 activated charcoal and 0.5 g/1 casein hydrolysate;within 3 months of culture,each of the shoots developed into a strong young plant with 3-5 roots,4 or 5 leaves and a mini tuber at the base.