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为了探寻橡胶树原生质体培养再生植株的较佳途径,本研究以橡胶树热研7-33-97花药和内珠被为起始外植体分别诱导愈伤组织,进行悬浮培养建立稳定的胚性悬浮细胞系,并分别对其进行原生质体的分离和培养研究,分析比较不同起始外植体来源原生质体的产量、活力及其在看护培养过程中的分裂生长、体胚发生及植株再生情况。结果显示,在含1.5%纤维素酶、0.15%果胶酶和0.5%离析酶的酶液中酶解处理12 h后,花药和内珠被来源的悬浮细胞原生质体产量分别为7.6×106个/m L PCV和12×106个/m L PCV,平均活力分别为75.2%和83.9%;在看护培养基上这两种来源的原生质体均能发生持续分裂,45 d后从5×105个原生质体形成2 mm以上的小愈伤组织数分别为247个和480个;经体胚诱导培养60 d后获得的体胚数分别为56个和18个,最终花药来源原生质体发育的体胚约有4.7%转化成完整植株,而内珠被来源的原生质体没能得到正常的植株。研究结果表明,经花药培养建立的胚性悬浮细胞系是获得具有植株再生能力原生质体的最佳材料,可为进一步优化橡胶树原生质体培养体系及进行橡胶树品种间体细胞杂交提供依据和参考。
In order to explore a better way to cultivate rubber plant protoplasts, the embryogenic callus was induced by rubbery embryo 7-33-97 and the explants were cultured in suspension to establish a stable embryogenic suspension Cell lines. The protoplasts were isolated and cultured separately. The yield and vigor of the protoplasts from different explants and their division, growth and somatic embryogenesis were analyzed. The results showed that the protoplast production of suspension cells derived from anthers and inner beads were 7.6 × 10 6 after enzymolysis for 12 h in enzyme solution containing 1.5% cellulase, 0.15% pectinase and 0.5% isozyme, respectively / m L PCV and 12 × 106 cells / m L PCV respectively, the average viability was 75.2% and 83.9%, respectively. Protoplasts from these two sources could continue to divide on the medium of care, and from 45 × 105 The numbers of small calluses with protoplasts forming more than 2 mm were 247 and 480, respectively. The number of somatic embryos obtained after somatic embryo induction culture for 60 d were 56 and 18, respectively. Finally, the somatic embryo derived from protoplasts Approximately 4.7% were transformed into intact plants, while the inner bead-derived protoplasts failed to obtain normal plants. The results showed that the embryogenic suspension cell line established by anther culture is the best material for obtaining protoplasts with plant regeneration ability and can provide the basis and references for further optimization of the protoplast culture system of rubber tree and somatic hybridization among rubber tree species.