文心兰类原球茎的愈伤组织诱导及其植株再生

Callus induction and plant regeneration of Oncidium ‘Gower Ramsey' by means of protocorm-like body culture

关键词:  文心兰, 组织培养, 愈伤组织增殖, TDZ, 再分化

Key words:  Oncidium ‘Gower Ramsey', tissue culture, callus proliferation, TDZ, redifferentiation

摘要:

该研究首次以文心兰的类原球茎(protocorm-like bodies, PLBs)为外植体进行愈伤组织诱导及其植株再生培养,并分析了不同浓度的TDZ和2,4-D配比对愈伤组织增殖的影响。结果表明:以1/2MS为基本培养基,添加1 mg·L^-1 TDZ与3 mg·L^-1 2,4-D,从接种的 PLBs上可以诱导出乳白色的、较疏松的愈伤组织,诱导频率达到100%。愈伤组织继代培养时,在2,4-D浓度为0.5～2.0 mg·L^-1 的范围内,其增殖主要受TDZ浓度的影响,TDZ浓度从1.0 mg·L^-1降低到0.5 mg·L^-1,愈伤组织鲜重增殖倍数显著增加,由最低的4.50倍增加到最高的6.04倍。愈伤组织增殖的最适培养基为1/2MS + 0.5 mg·L^-1 TDZ + 1.0 mg·L^-1 2,4-D。将在最适愈伤组织增殖培养基上继代培养约1个月的愈伤组织转移到T2培养基(3.5 g·L^-1 花宝1号 + 20 g·L^-1红薯 + 25 g·L^-1香蕉 + 1 g·L^-1 tryptone + 20 g·L^-1蔗糖 + 3.5 g·L^-1 phytagel)上,黑暗培养1个月后,每克鲜重的愈伤组织约诱导出1 328.67个PLBs。将诱导出的PLBs转移到新鲜的T2 培养基上光照培养1个月,萌发率为90.12%。而将小植株转移到添加1 g·L^-1活性炭的1/2MS培养基上,成苗率达到100%。该研究结果成功建立了文心兰的高频愈伤组织诱导及其植株再生体系,为文心兰基因工程育种提供了一个高效、稳定的转化受体系统。

Abstract:

Oncidium ‘Gower Ramsey' is a species of orchid with high ornamental value and strong market competitiveness. Gene engineering breeding technology provides an effective way for the genetic improvement of ornamental plants. And callus is an ideal transformation receptor. Up to now, callus induction and plant regeneration of Onciudium have been successfully achieved using stem segment, root tip and shoot tip. But there are some shortcomings for these in vitro regeneration systems, such as low frequency and long period of callus induction, difficulty in maintaining embryogenic state of callus, etc. In this research, for the first time protocorm-like bodies of Oncidium ‘Gower Ramsey' were used as explants for callus induction and plant regeneration. The effects of different concentrations and combinations of TDZ and 2,4-D on callus proliferation were investigated. The results showed that 1/2MS medium supplemented with 1 mg·L^-1 TDZ and 3 mg·L^-1 2,4-D could promote the induction of whitish and friable callus, the induction rate was 100%. The proliferation rate of callus was mainly affected by the concentration of TDZ, while the callus subculture medium consisted of TDZ in combination with 2,4-D at the concentrations of 0.5 to 2.0 mg ·L^-1. When the concentration of TDZ decreased from 1.0 to 0.5 mg·L^-1, proliferation rate of the callus fresh weigh increased significantly. And the optimal medium for callus proliferation contained 1/2MS basal medium, 0.5 mg·L^-1 TDZ and 1.0 mg·L^-1 2,4-D. After four weeks of subculture in the dark, the highest proliferation rate of the callus fresh weigh, which was 6.04 folds, appeared on the optimal medium, while the lowest proliferation rate of the callus fresh weigh, which was 4.50 folds, appeared on the medium containing 1/2MS basal medium, 1.0 mg·L^-1 TDZ and 2.0 mg·L^-1 2,4-D. The callus subcultured on the optimal proliferation medium for about one month were transferred to T2 medium(3.5 g·L^-1 Hyponex 1 + 20 g·L^-1 sweet potato + 25 g·L^-1 banana + 1 g·L^-1 tryptone + 20 g·L^-1 sucrose + 3.5 g·L^-1 phytagel)and cultured in the dark for PLBs induction. Approximately, 1 328.67 protocorm-like bodies could be generated in one month from an initial culture of 1 g callus fresh weight. Subsequently, the cultures transferred onto the fresh T2 medium. The germination rate of protocorm-like bodies was as high as 90.12% after one month of culture under the light. When transplanted on 1/2MS medium supplemented with 1 g·L^-1active carbon, all shoots became healthy plants after two months. In this study, the callus induction and plant regeneration system of Oncidium ‘Gower Ramsey' with high frequency was successfully developed to provide an efficient and stable transformation receptor system for the genetic transformation of Oncidium.