风兰丛芽高效诱导及植株再生研究

为了建立高效、稳定的风兰(Neofinetia falcata)丛芽诱导体系,以非共生性萌发所获得的风兰愈伤组织和原球茎(PLB)混合物为材料,改良的MS为基本培养基,附加不同植物激素及天然附加产物,进行单因素试验,选取适宜风兰生长的各因素种类和质量浓度范围,进而通过正交及完全组合试验,研究不同植物激素种类及其质量浓度和天然附加产物对其愈伤组织诱导和增殖、丛芽发生及植株再生的影响。结果表明,较适宜的非共生性萌发培养基为改良的MS+活性炭(AC)0.5 g·L^-1,在该培养基中种子培养75 d即可获得大量原球茎;原球茎在改良的MS+6-苄氨基嘌呤(6-BA) 0.5 mg·L^-1 +萘乙酸(NAA) 0.5 mg·L^-1 + 苹果泥30 g·L^-1 + AC 0.5 g·L^-1中培养70 d,诱导出的愈伤组织大量增殖,随后由原球茎产生大量丛芽,愈伤增殖系数6.84,丛芽发生率98.96%;将诱导出的丛芽转接至相同培养基中,以“芽繁芽”方式增殖,其增殖系数可达4.36。无菌苗生根则在改良的MS + NAA 1.0 mg·L^-1 + AC 0.5 g·L^-1 + 苹果泥70 g·L^-1 中进行,生根率100%。生根苗移栽至消毒后的碎松树皮中,保温保湿培养90 d后,成活率100%。本研究建立了风兰体外高效快速繁殖体系,为进一步开展其人工繁育和栽培提供了技术支持和理论基础,同时也为其他兰科植物的离体快繁提供了技术参考。

Study of Regeneration and Efficient Induction of Buds in Neofinetia falcata

To establish an efficient and stable induction system of adventitious buds from Neofinetia falcata and select the most suitable regeneration protocol. Mono-factorial experiment was conducted to select the dominant growth regulator and their optimal concentrations using protocorm-like bodies and callus obtained from non-symbiotic germination and modified basal MS medium with various plant hormone and natural additives. Furthermore, complete combination test and orthogonal experiment were used to investigate the impacts on callus induction and proliferation, growth of adventitious buds and plant regeneration by various plant hormones with different concentration and natural additives. The results indicated that the most suitable modified medium for non-symbiotic germination was MS medium with 0.5 g·L^-1active carbon, with which lots of protocorm-like bodies were obtained from the seeds of Neofinetia falcate after culturing for 75 d. A mass of calluses was inducted from protocorm-like bodies in the modified MS medium with 0.5 mg·L^-16-BA, 0.5 mg·L^-1 NAA, 30 g·L^-1 mashed apple and 0.5 g·L^-1active carbon after 70 d. Hereafer, lots of adventitious buds differentiated from protocorm-like bodies, the proliferation coefficient of callus was 6.84 and the differentiation rate was 98.06%. The adventitious buds were proliferated as a pattern of “buds to buds” when sub-cultured on the same medium as differentiation and the proliferation coefficient reached up to 4.36. The sterile seedlings were 100% rooted when cultured on the modified MS medium with 1.0 mg·L^-1 NAA, 0.5 g·L^-1 active carbon and 70 g·L^-1 mashed apple. The survival rate of these rooted seedlings was 100% after transplanted into sterile pine bark fragment for 90 d with suitable temperature and humidity. Our study establishes an efficient regeneration system of N. falcata in vitro, which provides a technology reference and theoretical basis for its artificial breeding and cultivation. Our results also provide a technology reference of rapid propagation in vitro for other Orchidaceae plants.