刘福平 陈淳 程小兵 郑明琼

摘  要：為了解培养基中附加6-BA长期继代培养对蝴蝶兰类原球茎（PLB）状态的影响，以只添加3 mg/L的6-BA和不添加植物生长调节剂（对照）培养基分别继代培养PLB 24个月，培养过程中观测PLB活力指标，分析相关氧化、抗氧化和DNA生化指标。结果表明，第8个月2组PLB活力指标没有显著差异，第16个月6-BA组PLB分化率显著升高，第24个月增值倍数和分化率分别显著降低和极显著降低，且PLB的玻璃化率较高;6-BA组PLB氧化胁迫水平在第8、第16个月都较对照低，第24个月较高，培养期间·OH相对含量和POD活性与氧化胁迫水平分别相向和反向消长;6-BA组PLB的DNA甲基化程度始终较对照低，增色效应到第24个月才降低。较强的氧化胁迫，·OH相对含量、H2O2和MDA含量提高，POD和CAT活性降低，以及DNA甲基化程度降低，与6-BA诱导PLB玻璃化有关。综上分析，含6-BA培养基长期继代培养蝴蝶兰类原球茎，在培养中期可减缓类原球茎衰老，培养后期则加剧了衰老退化，所以在蝴蝶兰类原球茎的继代培养中，应适时调整细胞分裂素用量和控制继代次数。

关键词：蝴蝶兰类原球茎;长期继代;6-BA;活力;生理生化

Abstract： To understand the effects of 6-BA in media on the protocorm like body （PLB） of Phalaenopsis hybrida through long-term subculture， PLB was subcultured respectively on the media supplemented with only 6-BA （3 mg/L） and that without plant growth regulator （control） for 24 months. Some indexes of vitality， oxidation， antioxidant and DNA biochemistry of PLB were observed and analyzed during the subculture. The results showed that there was no significant difference in the PLB vitality indexes between the two groups at the 8th month. The differentiation rate of PLB in 6-BA group increased significantly at the 16th month. At the 24th month proliferation multiple and differentiation rate of PLB in 6-BA group decreased significantly and extremely significantly， respectively， meantime the PLB had a higher hyperhydricity rate. The oxidative stress level of PLB in 6-BA group reduced than that in control group at the 8th and 16th months， but increased at the 24th month. Throughout this subculture， changes of relative content of ·OH showed the similar trend with the oxidative stress level， and that of POD activity did conversely. The degree of DNA methylation in 6-BA group remained below that in the control， also the DNA hyperchromic effect was lower only at the 24 months. In addition to this， PLB hyperhydricity induced by 6-BA was connected with the factors including stronger oxidative stress， the increase of the relative content of ·OH， contents of H2O2 and MDA， the decrease of POD and CAT activities， as well as the decrease of degree of DNA methylation. Based on the above analysis， it could be concluded that during the long-term subculture of P. hybrida PLB， 6-BA in media slowed down PLB senescence in the middle stage of subculture， whereas it accelerated the senescence in the later stage. Therefore， the amount of cytokinin in media should be adjusted in time and the times of subculture should be limited in the subculture of P. hybrida PLB.

Keywords： Phalaenopsis hybrida PLB; long-term subculture; 6-BA; vitality; physiology and biochemistry

兰科植物组培类原球茎（PLB）在快速育苗、基因转化、生物反应器的应用、人工种子研制、种质保存等方面具有相当应用潜力，在类原球茎的生产应用中，不可避免地进行频繁继代培养，同其他植物一样，将导致无性系生理和遗传上的衰老变异，一般认为是培养基植物激素在植物体内积累等因素作用的结果[1]。蝴蝶兰或其他兰科植物的组培类原球茎在含高浓度植物激素的培养基上培养，尤其经长期继代培养无性系变异率很高是普遍现象，而且伴随某些生理生化、遗传指标的变化[2-5]。……