大叶种胡椒实生苗茎尖培养和合子胚培养研究

利用各种表面消毒方法对采自海南岛三个地区的胡椒大田植株的外植体进行消毒试验,由于内源性污染,除胡椒成熟种子外,其它各种大田外植体的表面消毒均未能成功。以胡椒成熟种子无菌萌发的实生苗茎尖作外植体,在1/2MS(MS或B5)+1.5mg/LBA+0～0.2mg/LIAA(或NAA)上可实现丛生芽增殖。茎尖水平或竖直接种方法显著影响茎尖的增殖;水平接种茎尖的生长和增殖效果优于竖直茎尖接种方式。茎尖增殖率随BA浓度的增加而提高,但BA浓度大于2.0mg/L时会使苗芽的质量降低,愈伤组织产生严重,苗芽细小,抽出不明显,颜色发黄甚至变白。附加或不附加100mg/LAdSO4对丛生芽增殖没有明显影响。生根培养基以1/2MS+1.0mg/LIBA+0.5～1.0mg/LIAA为最优,生根率可达100%;在细沙∶土∶椰糠(1∶1∶1)的基质中常规炼苗,成活率可达98%以上。液体纸桥法对胡椒种胚进行培养,在不附加任何生长调节物质的培养基(MS、B5或SH)上只产生单苗,而在附加不同种类和不同浓度的生长调节物质的培养基上则诱导形成愈伤组织,但未能实现分化;以胡椒无菌萌发的实生苗胚轴和叶片切段作外植体进行培养,较易诱导产生愈伤组织,但难以实现分化。

In vitro culture of shoot tips from seedlings and zygotic embryos of Piper nigrum

Experiments on surface-sterilization methods were carried out with various explants collected from field-grown black pepper (Piper nigrum Linn.)c.v.Daye(Lampong Type),extensively cultivated in China. Due to endogenous contaminants,contamination of all types of explants except for seeds was not yet effectively controlled. In vitro clonal propagation of black pepper with shoot tips excised from aseptic seedlings through multiple-shoot multiplication methods is successfully achieved. The best establishment and proliferation of shoot tips was obtained on 1/2MS(MS or Bs)basal medium supplemented with 1.5 mg/L BA and 0～0.2 mg/L IAA(or NAA). Excised microshoots were rooted in vitro on 1/2MS in the presence of 1.0 mg/L IBA and 0.5～1.0 mg/L IAA with the optimum rooting results. Plantlets had been successfully acclimatized and transferred to the greenhouse conditions. Complete plants were grown from mature and immature zygotic embryos of black pepper incubated on filter paper bridges in test tubes containing liquid SH(MS or B5)basal medium with not any growth regulators,and calli were induced with different combinations of auxins and cytokinins. Subculture of those calli onto the multiplication medium and differentiation medium led to browning and death finally,and no plant regeneration occurred. The morphogenetic potential of other explants such as leaf pieces and hypocotyl segments from aseptic seedlings was also investigated in vitro. Callus induction was relatively easy on MS(1/2MS,B5 or SH)basal medium fortified with a wide range of auxin-cytokinin combinations,but most attempts to regenerate plants from the calli were unsuccessful due to serious browning occurred during the subculture onto the multiplication medium and differentiation medium.