橡胶树热垦525胚性悬浮细胞系的建立及其胚性能力的维持  

Establishment of Embryogenic Cell Suspensions of Hevea brasiliensis Clone Reken 525 and Maintenance of Its Embryogenic Competence

作　　者：戴雪梅[1] 彭素娜[1] 成镜[1] 顾晓川[1] 黄天带[1] DAI Xuemei;PENG Suna;CHENG Jing;GU Xiaochuan;HUANG Tiandai(Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs/State Key Laboratory Incubation Base for Cultivation&Physiology of Tropical Crops/Engineering Center for Rapid Propagation of Tropical Woody Plants/Haikou Key Laboratory of Tropical Plant Seedling Innovation,Haikou,Hainan 571101,China)

机构地区：[1]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室/省部共建国家重点实验室培育基地‒海南省热带作物栽培生理学重点实验室/热带林木种子种苗工程中心/海口市热带植物种苗创新重点实验室,海南海口571101

出　　处：《热带作物学报》2024年第7期1385-1392,共8页Chinese Journal of Tropical Crops

基　　金：海南省重点研发计划国际科技合作研发项目(No.GHYF2022013);海南省重点研发计划项目(No.ZDYF2022XDNY-204);海南省国际科技合作人才与交流项目(No.G20241024008E)。

摘　　要：易碎胚性愈伤组织和胚性悬浮细胞系都是基因工程和细胞工程的理想受体材料。然而橡胶树(Hevea brasil-iensis)易碎胚性愈伤组织诱导频率低、耗时长,且其胚性能力通常随继代次数的增加而下降甚至完全丧失,限制了相关研究的持续开展。因此,快速获得易碎胚性愈伤组织,建立具有高效体胚发生能力的胚性悬浮细胞系,并尽可能较长时间维持其胚性能力是当前橡胶树基因工程和细胞工程研究的重要内容。本研究以橡胶树热垦525未成熟花药为外植体进行愈伤组织的诱导和胚性悬浮细胞系的建立,分析比较固液2种长期继代方式对维持其胚性能力的影响。结果表明:花药外植体在愈伤诱导培养基中获得的黄色致密初代愈伤组织体胚发生能力低,分散性差,不适合进行悬浮培养。将初代愈伤组织转移至胚性愈伤组织诱导培养基中培养70~80 d后,可观察到有胚性结构的形成和早期体细胞胚发生,同时周围长出鲜黄色小颗粒状易碎胚性愈伤组织。通过常规方法建立的Ⅰ型胚性悬浮细胞系具备胚性细胞的典型特征,体胚发生能力显著高于胚性愈伤组织,但在体胚发生过程中容易重新愈伤化;而通过筛选特定形态的胚性愈伤组织低密度启动悬浮培养建立的Ⅱ型胚性悬浮细胞系,在显微镜下可观察到细胞处在有序的胚性结构中,其体胚发生频率可达100%。在含2 mg/L2,4-D的液体培养中持续继代2 a后细胞明显老化且增殖缓慢,体胚发生能力几近丧失;而在去除2,4-D并添加低浓度脱落酸(0.1 mg/L)和水解酪蛋白(0.5 g/L)的固体培养基上持续继代2 a后,体胚发生能力仍能维持在较高水平。所建立的Ⅱ型胚性悬浮细胞系可为橡胶树遗传转化及原生质体培养等相关研究长期提供优质充足且状态相对稳定的材料来源。Both friable embryogenic callus and suspension cells are ideal materials for genetic and cell engineering.However,the induction of friable embryogenic callus of Hevea brasiliensis is low frequency and time-consuming,and its embryogenic competence usually decreases gradually or even completely loses with the increase of subculture times.Therefore,to quickly obtain friable embryogenic callus,establish embryogenic cell suspensions with high efficiency of somatic embryogenesis,and maintain the embryogenic competence as long as possible,are important research contents for genetic and cell engineering of H.brasiliensis.In this study,the immature anthers of H.brasiliensis clone Reken 525 was used as the explants to induce callus and establish embryogenic cell suspensions,and the effects of two long-term subculture methods-solid and liquid on the maintenance of embryogenic competence were compared and analyzed.The results showed that the yellow and compact primary callus tissue induced from anther explants on callus induction me-dium had a low ability for somatic embryogenesis,and was not suitable for suspension culture due to poor dispersion.After 70-80 d of culture on the medium for embryogenic callus induction,the formation of embryogenic structures and early somatic embryogenesis were observed,and bright yellow small granular friable embryogenic calli grew around them.The typeⅠembryogenic suspension cell line established through routine methods possessed typical characteri tics of embryogenic cells,and the frequency of somatic embryogenesis was significantly higher than that of embryo-s-genic callus tissue,but proned to callus formation during somatic embryogenesis;the typeⅡembryogenic suspension cell line established by screening specific embryogenic tissue and initiating with low density suspension culture was in an orderly embryonic structure observed by microscope,and its frequency of somatic embryogenesis could reach up to 100%.After two years of continuous subculture in liquid culture medium containing 2 mg

关 键 词：橡胶树 易碎胚性愈伤组织 胚性悬浮细胞系 体胚发生 胚性维持 

分 类 号：S794.1[农业科学—林木遗传育种]