巴西橡胶树体胚胚性愈伤组织悬浮系的建立和植株再生
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摘要
以巴西橡胶树‘热研7-33-97’品种花药体胚胚性愈伤组织的新鲜增殖组织为培养材料,建立和优化适合橡胶树胚性愈伤组织增殖的悬浮培养体系,并对悬浮组织进行体细胞胚的成熟诱导和植株再生。结果表明:橡胶树体细胞胚诱导的易碎胚性愈伤组织是建立稳定、均一的悬浮细胞系非常适合的材料;改良MS+1.0 mg/L 2,4-D+1.5 mg/L KT+0.5 mg/L NAA有利于细胞分裂,最适初始接种量为1.2 g/50 mL,旧培养液比例为1/3;悬浮培养物易分散、细胞大小均一、形态一致,细胞团由4～15个细胞聚合而成,细胞颜色淡黄色,培养基清澈透亮;胚性愈伤悬浮细胞的生长曲线呈S形,第3～第7天是指数增长期,最适继代周期为7～8 d;在一个生长周期内,培养液的pH先降后升,最后逐渐趋于稳定,而电导率则持续下降;三次继代后,将悬浮培养物接种到固体分化培养基上分化可得到成熟体细胞胚状体,将其接种到植株再生培养基上,45 d后可成功获得再生植株。橡胶树花药体胚胚性愈伤组织悬浮系的建立,为橡胶树胚性愈伤组织的快速增殖及体细胞胚的规模化繁育提供帮助。
The fresh proliferative tissue of the anther embryogenic calli from H. brasiliensis 'Reyan 7-33-97' was used as the culture material. A suspension culture system suitable for embryogenic callus development was established and optimized to explore the main factors affecting the tissue proliferation in Hevea brasiliensis. On the basis of these, somatic embryo maturation induction and plant regeneration of suspension tissue were carried out.The results showed the fragile embryogenic callus induced by anther somatic embryos of Hevea brasiliensis was a suitable material for establishing stable and homogeneous suspension cell lines. The optimum culture medium for suspension was modified MS medium supplemented with 1.0 mg/L 2,4-D, 1.5 mg/L KT and 0.5 mg/L NAA, with the initial inoculation amount of 1.2 g/50 mL and the ratio of the old culture medium was 1/3. The medium was suitable for cells proliferation and could produce uniform cells and small cell aggregates composed of4～15 cells, which were uniform in sizes and consistent in shapes. The cell aggregates looked yellowish and were easy to disperse in the liquid medium, which was clear and transparent. The growth curve of suspension cells was typical of "S". From 3 to 7 days, the cells proliferated exponentially. The optimum subculture period was within 7 to 8 days to sustain the strong cell division competence and cell viability. During a growth cycle, the pH of the culture medium first decreased, then increased and gradually stabilized, while the electrical conductivity showed a downward trend. After three times of subcultures, the small cell aggregates were moved into the solid differentiation medium to obtain the mature embryos. The mature embryos were transferred to the plant regeneration medium, and the regenerated plants were successfully obtained after 45 days. The establishment of suspension system of embryogenic callus from somatic embryos in Hevea brasiliensis set up a basis for rapid proliferation of embryogenic callus and large-scale propagation of somatic embryos.
The fresh proliferative tissue of the anther embryogenic calli from H. brasiliensis 'Reyan 7-33-97' was used as the culture material. A suspension culture system suitable for embryogenic callus development was established and optimized to explore the main factors affecting the tissue proliferation in Hevea brasiliensis. On the basis of these, somatic embryo maturation induction and plant regeneration of suspension tissue were carried out.The results showed the fragile embryogenic callus induced by anther somatic embryos of Hevea brasiliensis was a suitable material for establishing stable and homogeneous suspension cell lines. The optimum culture medium for suspension was modified MS medium supplemented with 1.0 mg/L 2,4-D, 1.5 mg/L KT and 0.5 mg/L NAA, with the initial inoculation amount of 1.2 g/50 mL and the ratio of the old culture medium was 1/3. The medium was suitable for cells proliferation and could produce uniform cells and small cell aggregates composed of4～15 cells, which were uniform in sizes and consistent in shapes. The cell aggregates looked yellowish and were easy to disperse in the liquid medium, which was clear and transparent. The growth curve of suspension cells was typical of "S". From 3 to 7 days, the cells proliferated exponentially. The optimum subculture period was within 7 to 8 days to sustain the strong cell division competence and cell viability. During a growth cycle, the pH of the culture medium first decreased, then increased and gradually stabilized, while the electrical conductivity showed a downward trend. After three times of subcultures, the small cell aggregates were moved into the solid differentiation medium to obtain the mature embryos. The mature embryos were transferred to the plant regeneration medium, and the regenerated plants were successfully obtained after 45 days. The establishment of suspension system of embryogenic callus from somatic embryos in Hevea brasiliensis set up a basis for rapid proliferation of embryogenic callus and large-scale propagation of somatic embryos.
引文
Blanc G.,Baptiste C.,Oliver G.,Martin F.,and Montoro P.,2006,Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Müll Arg.plants,Plant Cell Rep.,24(12):724-733
Cai Z.Y.,and Huang G.X.,2011,Research advances in anthracnose of Hevea brasiliensis,Xinan Linye Daxue Xuebao(Journal of Southwest Forestry College),31(1):89-93(蔡志英,黄贵修,2011,巴西橡胶树炭疽病研究进展,西南林业大学学报,31(1):89-93)
Chen T.,Chang J.,Huang T.D.,Huang H.S.,and Hua Y.W.,2015,HLPC analysis of anthocyanin in somatic embryo of rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2774-2778(陈涛,畅姣,黄天带,黄华孙,华玉伟,2015,橡胶树体细胞胚花青素HPLC分析,基因组学与应用生物学,34(12):2774-2778)
Guo P.,Liang P.,He Q.G.,Liu W.B.,Lin C.H.,Miao W.G.,and Zheng F.C.,2016,Clone and expression analysis of MAT-related gene(STE2 gene)from Oidium heveae B.A.Steinm,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):879-885(郭鹏,梁鹏,何其光,刘文波,林春花,缪卫国,郑服丛,2016,橡胶树白粉菌MAT相关基因(STE2基因)的克隆及表达分析,基因组学与应用生物学,35(4):879-885)
He Y.,Tian Z.H.,and Zheng Y.L.,2005,Establishment and optimization of suspension cell system in creeping dichondra(Dichondra repens Forst.),Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),24(4):401-405(何勇,田志宏,郑用琏,2005,马蹄金悬浮细胞系的建立与优化,华中农业大学学报,24(4):401-405)
Jiang X.C.,Liu C.,Ming J.,Mu D.,and Yuan S.X.,2011,Establishment of cell suspension culture and plantlet regeneration of Lilium regale,Yuanyi Xuebao(Acta Horticulturae Sinica)38(2):327-334(姜新超,刘春,明军,穆鼎,袁素霞,2011,岷江百合悬浮细胞系的建立及植株再生,园艺学报,38(2):327-334)
Lai Z.X.,Huang Q.,Lin X.L.,Lin Y.L.,Chen Y.T.,Lai C.C.,and Cai Y.Q.,2007,Rapid establishment of embryogenic cell suspensions and plant regeneration via somatic embryogenesis in litchi,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),23(1):28-32(赖钟雄,黄浅,林秀莲,林玉玲,陈义挺,赖呈纯,蔡英卿,2007,荔枝胚性悬浮细胞系的快速建立及其体胚植株再生,中国农学通报,23(1):28-32)
Ni Y.M.,2010,Primary studies on friable embryogenic calli induetion,long-term subculture and plant regeneration in Hevea brasiliensis,Thesis for M.S.,Hainan University,Supervisor:Li Z.,pp.1-23(倪燕妹,2010,橡胶树易碎胚性愈伤组织诱导、长期继代培养及其植株再生研究,硕士学位论文,海南大学,导师:李哲,pp.1-23)
Tian L.,2010,A review on application of biotechnology in genetic improvement of Hevea brasiliensis,Shaanxi Linye Ke ji(Shaanxi Forest Science and Technology),(6):105-118(田郎,2010,生物技术在巴西橡胶树遗传改良中的应用综述,陕西林业科技,(6):105-118)
Wu C.T.,Li W.G.,and Huang H.S.,2013,Research progresses on Hevea germplasm resources and breeding methods,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),28(2):118-124(吴春太,李维国,黄华孙,2013,近年来国内外橡胶树种质资源与育种方法研究新进展,西北林学院学报,28(2):118-124)
Xu S.Q.,Li X.D.,and Zhang J.G.,2011,Callus induction and establishment of cell suspension culture system for Thymus vulgaris L.,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),20(10):112-119(徐世千,李晓东,张建国,2011,百里香愈伤组织的诱导及细胞悬浮培养体系的建立,西北农业学报,20(10):112-119)
Yang Y.Y.,He Y.Q.,and Zheng C.X.,2005,Induction of callus from the ovule of Pinus tabulaeformis Carr and establishment of suspension cell line,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(5):591-594(杨远媛,贺窑青,郑彩霞,2005,油松胚珠愈伤组织诱导和悬浮细胞系的建立,植物生理学通讯,41(5):591-594)
Zhao M.Q.,Gao Y.Z.F.,and Liu C.K.,2012,Analysis of protein subcellular localization by using plant suspension cultured cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(4):389-393(赵蒙晴,高野哲夫,柳参奎,2012,利用植物悬浮培养细胞进行蛋白质快速定位分析的方法,基因组学与应用生物学,31(4):389-393)
Zhou X.G.,Ding Y.M.,Sun M.L.,Chen L.,Su Y.,Zhang S.S.,and Li C.Y.,2008,The establishment of cell suspension culture of 24 rice monogenic lines of IRRI-Japan,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),21(3):642-647(周晓罡,丁玉梅,孙茂林,陈亮,苏源,张绍松,李成云,2008,24个水稻抗稻瘟病单基因鉴别品系细胞悬浮系的建立,西南农业学报,21(3):642-647)
Cai Z.Y.,and Huang G.X.,2011,Research advances in anthracnose of Hevea brasiliensis,Xinan Linye Daxue Xuebao(Journal of Southwest Forestry College),31(1):89-93(蔡志英,黄贵修,2011,巴西橡胶树炭疽病研究进展,西南林业大学学报,31(1):89-93)
Chen T.,Chang J.,Huang T.D.,Huang H.S.,and Hua Y.W.,2015,HLPC analysis of anthocyanin in somatic embryo of rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2774-2778(陈涛,畅姣,黄天带,黄华孙,华玉伟,2015,橡胶树体细胞胚花青素HPLC分析,基因组学与应用生物学,34(12):2774-2778)
Guo P.,Liang P.,He Q.G.,Liu W.B.,Lin C.H.,Miao W.G.,and Zheng F.C.,2016,Clone and expression analysis of MAT-related gene(STE2 gene)from Oidium heveae B.A.Steinm,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):879-885(郭鹏,梁鹏,何其光,刘文波,林春花,缪卫国,郑服丛,2016,橡胶树白粉菌MAT相关基因(STE2基因)的克隆及表达分析,基因组学与应用生物学,35(4):879-885)
He Y.,Tian Z.H.,and Zheng Y.L.,2005,Establishment and optimization of suspension cell system in creeping dichondra(Dichondra repens Forst.),Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),24(4):401-405(何勇,田志宏,郑用琏,2005,马蹄金悬浮细胞系的建立与优化,华中农业大学学报,24(4):401-405)
Jiang X.C.,Liu C.,Ming J.,Mu D.,and Yuan S.X.,2011,Establishment of cell suspension culture and plantlet regeneration of Lilium regale,Yuanyi Xuebao(Acta Horticulturae Sinica)38(2):327-334(姜新超,刘春,明军,穆鼎,袁素霞,2011,岷江百合悬浮细胞系的建立及植株再生,园艺学报,38(2):327-334)
Lai Z.X.,Huang Q.,Lin X.L.,Lin Y.L.,Chen Y.T.,Lai C.C.,and Cai Y.Q.,2007,Rapid establishment of embryogenic cell suspensions and plant regeneration via somatic embryogenesis in litchi,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),23(1):28-32(赖钟雄,黄浅,林秀莲,林玉玲,陈义挺,赖呈纯,蔡英卿,2007,荔枝胚性悬浮细胞系的快速建立及其体胚植株再生,中国农学通报,23(1):28-32)
Ni Y.M.,2010,Primary studies on friable embryogenic calli induetion,long-term subculture and plant regeneration in Hevea brasiliensis,Thesis for M.S.,Hainan University,Supervisor:Li Z.,pp.1-23(倪燕妹,2010,橡胶树易碎胚性愈伤组织诱导、长期继代培养及其植株再生研究,硕士学位论文,海南大学,导师:李哲,pp.1-23)
Tian L.,2010,A review on application of biotechnology in genetic improvement of Hevea brasiliensis,Shaanxi Linye Ke ji(Shaanxi Forest Science and Technology),(6):105-118(田郎,2010,生物技术在巴西橡胶树遗传改良中的应用综述,陕西林业科技,(6):105-118)
Wu C.T.,Li W.G.,and Huang H.S.,2013,Research progresses on Hevea germplasm resources and breeding methods,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),28(2):118-124(吴春太,李维国,黄华孙,2013,近年来国内外橡胶树种质资源与育种方法研究新进展,西北林学院学报,28(2):118-124)
Xu S.Q.,Li X.D.,and Zhang J.G.,2011,Callus induction and establishment of cell suspension culture system for Thymus vulgaris L.,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),20(10):112-119(徐世千,李晓东,张建国,2011,百里香愈伤组织的诱导及细胞悬浮培养体系的建立,西北农业学报,20(10):112-119)
Yang Y.Y.,He Y.Q.,and Zheng C.X.,2005,Induction of callus from the ovule of Pinus tabulaeformis Carr and establishment of suspension cell line,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(5):591-594(杨远媛,贺窑青,郑彩霞,2005,油松胚珠愈伤组织诱导和悬浮细胞系的建立,植物生理学通讯,41(5):591-594)
Zhao M.Q.,Gao Y.Z.F.,and Liu C.K.,2012,Analysis of protein subcellular localization by using plant suspension cultured cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(4):389-393(赵蒙晴,高野哲夫,柳参奎,2012,利用植物悬浮培养细胞进行蛋白质快速定位分析的方法,基因组学与应用生物学,31(4):389-393)
Zhou X.G.,Ding Y.M.,Sun M.L.,Chen L.,Su Y.,Zhang S.S.,and Li C.Y.,2008,The establishment of cell suspension culture of 24 rice monogenic lines of IRRI-Japan,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),21(3):642-647(周晓罡,丁玉梅,孙茂林,陈亮,苏源,张绍松,李成云,2008,24个水稻抗稻瘟病单基因鉴别品系细胞悬浮系的建立,西南农业学报,21(3):642-647)