不同外植体对橡胶树原生质体分离和再生的影响  

Effects of Different Explants on Isolation and Regeneration of Protoplast in Rubber Tree

作　　者：戴雪梅[1] 黄天带[1] 李季[1] 杨先锋[1] 黄华孙[1] 

机构地区：[1]中国热带农业科学院橡胶研究所,农业部橡胶树生物学与遗传资源利用重点实验室,国家橡胶树育种中心,儋州571737

出　　处：《分子植物育种》2014年第6期1259-1264,共6页Molecular Plant Breeding

基　　金：中国热带农业科学院橡胶研究所基本科研业务费专项(1630022013002)资助

摘　　要：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。t To seek a better approach for plant regeneration from protoplast culture, in this research, anthers and inner integuments of rubber tree, a cultivar of Reyan 7-33-97, were respectively used as initial explants for inducing calli to establish stable embryogenic cell suspensions by suspension culture. Isolating and culturing of protoplast from these two kind of suspension cells were further studied. Protoplast yields and viabilities from different initial explants, protoplast division and growth during feeder layer culture, and subsequently somatic embryogenesis and plant regeneration were statistically analyzed. Results showed that embryogenic cell suspen- sions derived from anthers and inner integuments enabled to yield 7.6×106 protoplasts per mL PCV and 12×106 protoplasts per ml PCV, with mean viabilities of 75.2% and 83.9%, respectively, which was under the inoculation conditions in enzyme solution containing 1.5% cellulase R-10, 0.15% pectolyase y-23 and 0.5% macerozyme R-10 for 12 h. Sustained mitotic divisions were both observed when the two kind ofprotoplasts were cultured on feeder layer, and 247 and 480 microcalli with the size 2 mm above were respectively formed from 5 ~ 105 anther- derived and inner integument-derived protoplasts after 45 d nursing culture, from which 56 and 18 embryos were respectively obtained after 60 d culture on medium for somatic embryo induction. Finally 4.7% embryosdeveloped from anther-derived protoplast were regenerated to plantlets, whereas all embryos obtained from inner integument- derived protoplasts failed to plant regeneration. In conclusion, anther-derived embryonic cell suspension was the optimal material for isolating protoplast bearing regenerated capacity, providing basis and reference for further optimizing protoplast culture system and interspecific somatic hybridization in rubber tree.

关 键 词：橡胶树 胚性悬浮细胞系 原生质体 体胚发生 植株再生 

分 类 号：S794.1[农业科学—林木遗传育种]