阳春砂愈伤组织诱导与植株再生

Callus Induction and Plantlets Regeneration from Amomum villosum

【目的】通过愈伤组织的诱导与植株再生,建立阳春砂的离体再生系统。【方法】以1 g/L升汞和1.84 kg/L的浓硫酸分别处理阳春砂种子;取阳春砂无菌幼苗的叶片及茎切段为外植体,接种于以MS为基本培养基、分别添加了不同生长素、细胞分裂素的培养基中,进行愈伤组织诱导和再生培养。观察:①不同处理方法对种子萌发的影响;②不同培养基诱导阳春砂愈伤组织的优劣;③不同剂量AgNO3对愈伤组织分化的影响;④不同培养基对芽分化的影响。【结果】①以升汞消毒的种子均未观察到萌发,而经浓硫酸处理的种子萌发率达8.4%。②幼苗叶片不能形成愈伤组织,茎切段能有效诱导愈伤组织;添加0.5 mg/L 2,4-二氯苯氧乙酸(2,4-D)、0.5 mg/Lα-萘乙酸(NAA)和1.0 mg/L 6-苄基腺嘌呤(6-BA)的培养基适合愈伤组织的诱导。③在该培养基中添加6 mg/L AgNO3可使诱导的愈伤组织更易于再分化。④添加0.1 mg/L NAA和2.0mg/L 6-BA,再分别添加0.5 mg/L呋喃氨基嘌呤(KT)或0.1 mg/L N'-苯基-N'-1,2,3-噻二唑-5-脲(TDZ)可诱导愈伤组织的芽分化,丛生芽移至MS培养基上可自然生根。【结论】以阳春砂种子萌发后幼苗的茎切段作为外植体,在一定培养条件下可实现愈伤组织的诱导与再生,建立其离体再生系统。

[Objective] To set up the planflet regeneration system of Amomum villosum （AV） by the proliferation and differentiation of callus. [ Methods ] The seeds of AV were treated with 1 g/L mercuric chloride and 1.84 kg/L concentrated sulfuric acid. The different sterile segments of seedling used as explants were cultured in MS medium with different somatotropins and cytokinins to induce callus and plantlets regeneration. The influences of pre-treating methods on the sprouting of seeds, MS medium on callus induction, various dosages of AgNO3 on callus differentiation, and MS on bud differentiation were observed. [ Results] The sprouting rate of seeds sterilized by mercuric chloride was zero while that of the seeds sterilized by concentrated sulfuric acid was 8.4%. The leaves of seedling cannot develop into callus while stem segments can develop into callus. MS medium added with 0. 5 mg/L 2,4-dichlorophenoxyacetic acid （2,4-D）, 0.5 mg/L a-naphthalene acetic acid （NAA） and 1.0 mg/L 6-benzyl adenine （6-BA） was suitable for the callus induction. Moreover, 6 mg/L AgNO3 added into the above culture medium can promote the differentiation of callus. When added with 0.5 mg/L kinetin （KT） or 0.1 mg/L thidiazuron （TDZ）, MS medium including 0.1 mg/L NAA and 2.0 mg/L 6-BA was suitable for the sprouting differentiation of callus and natural roots can form from the axillary buds when cultured in MS medium. [ Conclusion] With stem segments of seedling as the explants, callus formation and plantlet regeneration can be achieved under certain cultured conditions and a in-vitro regeneration system can be established.