Seed germination and tissue culture propagation technique of Anoectochilus calcareus Aver.

ObjectiveIn order to provide references for industrializing production of tissue-cultured seedlings of Anoectochilus calcareus Aver., tissue-cultured seedlings of A. calcareus were obtained by axenic sowing in vitro.MethodWith A. calcareus seeds as explants, MS or 1/2MS as basic culture medium, effects of different hormone combinations on aseptic sowing, protocorm proliferation, multiple shoot differentiation,promoting growth and rooting were studied. ResultResults showed that, 0.1%mercuric-chloride for 20 min showed better disinfection effect on A. calcareus capsule,with 100% of disinfection rate, and the seed germination was not affected. Adding banana puree to medium benefited seed germination. The optimal medium for seed germination was 1/2MS+6-0.5 mg/L 6-BA+0.2 mg/L NAA+60 g/L banana puree. In proliferation culture of protocorm, low concentration of 6-BA and NAA was beneficial to protocorm proliferation. The optimal medium for protocorm proliferation was 1/2MS+0.2 mg/L 6-BA+0.2 mg/L NAA+60 g/L banana puree. The optimal medium for multiple shoots differentiation was MS+2.0 mg/L 6-BA+0.5 mg/L NAA+60 g/L banana puree+0.1% activated carbon, protocorm could differentiate into more multiple shoots, showing growth vigor. The optimal medium for promoting growth and rooting of seedlings was MS+0.5 mg/L 6-BA+2.0 mg/L NAA+60 g/L banana puree+0.1%activated carbon. ConclusionA lot of high-quality tissue-cultured seedlings of A. calcareus can be obtained by aseptic sowing, proliferation and differentiation of protocorm. Therefore, aseptic sowing is ideal way to realize industrial production of A. calcareus seedlings.