三褶虾脊兰无菌播种快繁技术研究

A rapid micro propagation technology for Calanthe triplicate and generation of aseptic seedlings

【目的】建立三褶虾脊兰(Calanthe triplicata)无菌播种快繁技术体系。【方法】以三褶虾脊兰种子为外植体,采用种子→原球茎→完整植株途径,探讨不同组合(0.5mg/L6-BA、0.1mg/LNAA和1.0g/LAC)、附加物[椰汁(CM)、土豆泥、香蕉泥]及基本培养基(花宝1号、1/2花宝1号、MS和1/2MS)对三褶虾脊兰种子萌发、原球茎诱导、增殖、分化、生根的效果,筛选适宜的培养基。【结果】成熟种子在1/2花宝1号+0.5mg/L6-BA+0.1mg/LNAA+200mL/LCM+1.0g/LAC+20.0g/L蔗糖培养基中培养150d左右可形成原球茎,萌发率超过80%以上;诱导原球茎在1/2MS+100mL/LCM+2.0mg/L6-BA+0.1mg/LNAA+1.0g/LAC+20.0g/L蔗糖培养基上的增殖效果最好,增殖率为4.5;原球茎接种于花宝1号+0.1mg/L6-BA+0.1mg/LNAA+100.0g/L土豆泥+1.0g/LAC+20.0g/L蔗糖培养基上分化培养40d后,再转入花宝1号+0.1mg/LNAA+100.0g/L土豆泥+2.0g/LAC+20.0g/L蔗糖培养基上培养40d,可诱导形成完整植株。在花宝1号培养基中添加NAA和土豆泥或香蕉泥,原球茎生根率均达100.0%,但土豆泥的成本最低。【结论】建立了比较完善的三褶虾脊兰无菌播种和快繁技术体系,为其种质资源保护、种苗繁育以及产业开发提供技术支持。

[Objective]The experiment was aimed to establish the system of aseptic sowing and rapid propagation technology for Calanthe triplicata. [Method]The Calantbe triplicata seeds were used as explants to study the effects of different hormone combinations （0.5 mg/L 6-BA, 0.1 mg/L NAA and 1.0 g/L AC）, additive combinations （coconut milk, mashed potato and mashed banana） and different culture media （Hyponex No.1, 1/2 Hyponex No.1, MS and 1/2MS medium） on seed germination, induction, multiplication, and differentiation and protocorm rooting by the way of seed→protocorm→the whole plant. [Result]After 150 d of culture, the seeds were found to germinate and developed into protocorm with above 80% germination rate in 1/2 Hyponex No.1 medium containing 0.5 mg/L 6-BA ＋0.1 mg/L NAA ＋ 200 ml/L CM ＋ 1.0 g/L AC ＋ 20.0 g/L sugar. The induced protocorm showed best multiplication （multiplication coefficient 4.5） in 1/2MS medium containing 100 ml/L CM ＋ 2.0 mg/L 6-BA ＋ 0.1 mg/L NAA ＋ 1.0 g/L AC ＋ 20.0 g/L sugar. After sub-culturing, the protocorm in Hyponex No.1 medium （containing 0.1 mg/L 6-BA ＋ 0.1 mg/L NAA ＋ 100.0 g/L mashed potato ＋ 1.0 g/L AC ＋ 20 g/L sugar） for 40 days and then transferring subculture into Hyponex No.1 medium （containing 0.1 mg/L NAA ＋ 100.0 g/L mashed potato ＋ 2.0 g/L AC ＋ 20.0 g/L sugar） for 40 days, the young plantlets were induced and were able to develop into the whole plant. [Conclusion]The technology for rapid propagation of Calanthe triplicata was established and aseptic seedlings were transplanted successfully which showed good growth with high vigor. The technology developed will help in protecting germplasm resources, seedling propagation and its industrial applications.