Chin Bull Bot ›› 2018, Vol. 53 ›› Issue (3): 334-340.doi: 10.11983/CBB17207

Special Issue: Medicinal Plant

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Induction and Plant Regeneration of Protocorm-like Bodies in Dioscorea opposita

Li Ruixue1, Li Jiqiang1, Pu Tengfei1, Zhang Xiaoli1,2,3, Zhao Xiting1,2,3, Li Junhua1,2,3, Li Mingjun1,2,3,*()   

  1. 1College of Life Sciences, Henan Normal University, Xinxiang 453007, China
    2Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs in Henan Province, Xinxiang 453007, China
    3Henan Province Engineering Laboratory of Green Medicinal Plant Biotechnology, Xinxiang 453007, China
  • Received:2017-11-06 Accepted:2018-01-12 Online:2018-09-11 Published:2018-05-01
  • Contact: Li Mingjun E-mail:limingjun2002@263.net

Abstract:

To improve the speed of in vitro propagation of Dioscorea opposita and shorten the breeding cycle, we used nodal segments with axillary buds of D. opposita cv. ‘Tiegun’ as the starting material. The optimal conditions for protocorm-like body (PLB) induction, proliferation, differentiation and plantlet regeneration were analyzed. The optimal culture medium for PLB induction was MS+1.0 mg·L-1 TDZ+30 g·L-1 sucrose, the optimal culture medium for PLB proliferation was MS+9 mg·L-1 6-BA+30 g·L-1 sucrose, and the optimal culture medium for PLB differentiation was MS+2 mg·L-1 KT+0.02 mg·L-1 NAA+30 g·L-1 sucrose. In culture medium containing 1/4MS+0.05 mg·L-1 NAA+1.0 mg·L-1 PP333+15 g·L-1 sucrose, the rooting rate of regenerated plantlets from PLBs was 80%, and the survival rate of regenerated plantlets was up to 85% after being sown in soil. The induction of protocorm like bodies and the establishment of plant regeneration system provide a new way for rapid propagation of D. opposita seedlings.

Key words: Dioscorea opposita, induction, plant regeneration, proliferation, protocorm-like body

Figure 1

Induction and regeneration of Dioscorea opposita cv. ‘Tiegun’ protocorm-like bodies (PLBs)(A) Explants; (B) Induction of PLBs; (C) Proliferation of PLBs; (D) Differentiation of PLBs; (E) Plants regenerated from PLBs; (F) Plants produced from PLBs after being sown in soil"

Figure 2

Influence of TDZ concentration on the induction rate (A) and the induction number (B) of Dioscorea opposita cv. ‘Tiegun’ protocorm-like bodies (PLBs)Different lowercase letters indicate significant differences at P< 0.05 among different treatments."

Table 1

Influence of 6-BA concentration on the multiplication of Dioscorea opposita cv. ‘Tiegun' protocorm-like bodies (PLBs)"

Concentration of 6-BA
(mg·L-1)
Multiplication index Differentiation
rate (%)
The growth of PLBs
5 4.50±1.15 a 50.00 There was no PLB proliferation during the later stage. 2-3 buds were differentiated from the PLBs
7 4.67±0.95 a 33.33 There was no PLB proliferation during the later stage. 4-5 buds were differentiated from the PLBs
9 5.42±1.61 a 8.33 Light green PLBs with larger size and bud tips differentiated
12 4.92±0.14 a 8.33 Pale green PLBs with larger size and significantly differentiated bud tips
15 4.17±1.13 a 8.33 Green PLBs with smaller size and no differentiation

Table 2

Influence of culture mediums on the differentiation of Dioscorea opposita cv. ‘Tiegun’ protocorm-like bodies (PLBs) (means±SE)"

Treatments Differentiation rate (%) Average plant height (cm) The growth of plant
1 100 3.1±0.43 c Plants with fewer branches and slightly unfolded leaves
2 100 1.4±0.54 b Plants with more branches, slightly unfolded leaves and horizontal stems
3 100 5.0±1.13 a Plants with the most branches, slightly unfolded leaves and vertical stems
[1] 毕玲, 刘凤栾, 董爱香, 周晓阳, 马男, 赵梁军 (2012). 狗蔷薇类原球茎遗传转化体系的建立. 核农学报 26, 270-274.
[2] 蔡建荣 (2008). 山药组织培养褐化反应的研究. 中国农学通报 24(8), 118-120.
[3] 陈钢, 林兰英, 王建勤 (1994). 药用金线莲组培类原球茎研究初报. 福建中医药 25(4), 23-24.
[4] 陈晓旋, 李洪波, 叶春海, 丰锋, 卢淑仪, 李柳菁 (2010). 文心兰类原球茎的诱导与增殖技术的优化. 热带作物学报 31, 1464-1468.
[5] 崔广荣, 侯喜林, 张子学, 张从宇, 胡能兵, 刘跃成 (2007). 蝴蝶兰叶片离体培养胚状体的发生及组织学观察. 园艺学报 34, 431-436.
[6] 崔广荣, 张子学, 张从宇, 胡能兵 (2009). 文心兰、蝴蝶兰类原球茎薄切片高频诱导PLBs. 安徽科技学院学报 23(2), 21-27.
[7] 古碧珠, 刘柏涛, 何嘉碧, 梁红 (2008). 石斛兰和秋花独蒜兰种子的原球茎诱导及其生长分化. 仲恺农业技术学院学报 21(4), 17-21.
[8] 韩林林, 李俊华, 赵喜亭, 张晓丽, 宋志辉, 刘世宇, 李明军 (2016). 农杆菌介导的怀山药叶片瞬时表达方法的建立. 河南师范大学学报(自然科学版) 44(6), 135-139.
[9] 韩晓勇, 闫瑞霞, 殷剑美, 张培通, 郭文琦, 李春宏 (2013). 铁棍山药组织培养快繁及试管珠芽离体再生体系研究. 西北植物学报 33, 2120-2125.
[10] 洪森荣, 李明军 (2006). 玻璃化法超低温保存怀山药种质的技术研究. 中草药 37, 1715-1718.
[11] 姜福星, 刘凤栾, 赵梁军 (2012). 植物类原球茎的研究与应用. 核农学报 26, 478-483.
[12] 江艳华, 李艾莲, 陈彩霞, 崔思然, 赵锋 (2013). 半夏类原球茎的诱导及其植株再生研究. 中国农学通报 29(19), 153-158.
[13] 李海兵, 周娜, 赵姣, 李翔, 冯秋妍, 赵喜亭, 李明军 (2010). 怀山药种质资源的包埋玻璃化超低温保存与植株再生. 植物学报 45, 379-383.
[14] 李纪强 (2013). 铁棍山药类原球茎诱导增殖及植株再生的研究. 硕士论文. 新乡: 河南师范大学. pp. 1-43.
[15] 李明军 (1995). 多效唑——一种优良的植物生长调节剂. 植物学通报 12(2), 27-31.
[16] 李明军 (2004). 怀山药组织培养及其应用. 北京: 科学出版社. pp. 5-160.
[17] 李明军, 陈明霞, 郭君丽, 洪森荣, 张晓丽, 徐鑫, 刘萍 (2004a). 生长调节物质和糖对怀山药微型块茎诱导形成的影响. 华北农学报 19, 69-72.
[18] 李明军, 李金亭, 朱命炜, 张嘉宝 (1999). 怀山药的离体繁殖. 中草药 30, 296-298.
[19] 李明军, 刘纪华, 张嘉宝, 施介村, 郭仲琛 (1997a). 多效唑(MET)在玉米组培中的作用. 作物学报 23, 759-761.
[20] 李明军, 刘萍, 张嘉宝 (2000a). 怀山药微型块茎的离体诱导. 植物生理学通讯 36, 41-42.
[21] 李明军, 刘欣英, 李萍, 张晓丽, 赵喜亭, 柳俊, 谢从华 (2008). 山药微型块茎诱导形成的影响因子研究. 中草药 39, 905-910.
[22] 李明军, 薛建平, 陈明霞, 郭闽英, 邱卫花, 张嘉宝 (2000b). 不同因子对山药愈伤组织诱导的影响. 广西植物 20, 156-160.
[23] 李明军, 杨建伟, 张嘉宝 (1997b). 怀山药的茎段培养和快速繁殖. 植物生理学报 33, 275-276.
[24] 李明军, 张宝华, 王天亮, 张峰, 陈明霞, 张嘉宝 (2004b). 怀山药脱毒快繁技术研究. 见:陈振光. 全国植物组织培养、脱毒快繁及工厂化生产学术研讨会. 北京: 中国农业科技出版社. pp. 92-97.
[25] 李明军, 张峰, 陈明霞, 于相丽 (2003). 怀山药病毒病的研究. 中草药 34, 附3-附5.
[26] 李青 (2016). 绿色食品铁棍山药栽培技术. 河南农业 11, 31-32.
[27] 林江波, 王伟英, 李海明, 吴水金, 邹晖, 李跃森, 戴艺民 (2016). 铁皮石斛茎段原球茎的诱导、分化与植株再生. 福建农业学报 31, 1075-1079.
[28] 刘雯 (2014). 铁棍山药微型块茎形成的形态解剖及生理生化变化研究. 硕士论文. 新乡: 河南师范大学. pp. 1-34.
[29] 刘晓芳, 黄闽敏, 姜敏, 牛俊丽 (2009). 离体培养春兰原球茎增殖的影响因子研究. 西北林学院学报 24(3), 88-90.
[30] 罗剑飘, 吴坤林, 翁殊斐 (2015). 麒麟火焰兰叶片及根类原球茎的诱导研究. 浙江农业学报 27, 1173-1177.
[31] 马林, 张玲, 李卫锋 (2004). 黄山药丛生芽诱导与植株快速繁殖. 生物技术 14(2), 53-54.
[32] 彭晓英, 周朴华, 张良波, 彭尽晖, 周双德 (2010). 盾叶薯蓣类原球茎的离体诱导及快繁体系的建立. 植物遗传资源学报 11, 629-634.
[33] 乔永旭, 张永平, 陈超, 韩兴辉, 王旭, 刘桂萍 (2010). 蝴蝶兰组培苗类原球茎的诱导与分化. 北方园艺 (4), 138-141.
[34] 王丽萍, 梁淑云 (2010). 铁皮石斛原球茎诱导与增殖研究. 中国农学通报 26, 265-268.
[35] 许云, 高艳强, 高洪昌, 夏赟, 吴文嫱, 黄东益 (2014). 大薯类原球茎的离体诱导及再生体系的建立. 植物生理学报 50, 1027-1032.
[36] 杨美纯, 周歧伟, 许鸿源, 卢美英 (2000). 外部因子对蝴蝶兰叶片原球茎状体发生的影响. 广西植物 20, 42-46.
[37] 于倩, 李明军 (2004). 怀山药微型块茎愈伤组织的诱导形成及高频率再生. 生态学报 24, 1022-1026.
[38] 詹忠根, 徐程, 张铭, 罗紫娟 (2005). 铁皮石斛离体根尖经体细胞胚再生植株研究. 浙江大学学报(农业与生命科学版) 31, 579-580.
[39] 张超, 荣松, 张茜茜, 楼楠男 (2010). 基本培养基、碳源及植物生长调节剂对文心兰原球茎增殖的影响. 浙江农业科学 1, 55-59.
[40] 张瑞姿 (2012). 植物生长调节剂在观赏植物组织培养中的应用. 园林绿化 (5), 39-40.
[41] 张晓丽, 王医鹏, 刘雯, 郭晓博, 李书杰, 李明军 (2013). 铁棍山药试管苗快繁培养基的优化. 河南师范大学学报(自然科学版) 41, 123-126.
[42] 张玉, 李艳敏, 张和臣, 王利民, 王慧娟, 张鸽香 (2012). 蝴蝶兰叶片诱导类原球茎初探. 河南农业科学 41, 126-128.
[43] 张振霞, 叶静鹏, 郑莉, 陈贵豪, 郑玉忠 (2016). 广山药组织培养技术的研究. 江苏农业科学 44(2), 76-80.
[44] 赵喜亭, 王苗, 邵换娟, 李翔, 李海兵, 李明军 (2012). 山药种质包埋玻璃化超低温保存再生植株的稳定性分析. 华北农学报 27, 234-238.
[45] Li MJ, Li JH, Liu W, Liu LY, Lu J, Niu J, Liu XY, Yang QX (2014). A protocol for in vitro production of microtubers in Chinese yam ( Dioscorea opposita). Biosci Biotechnol Bio- chem 78, 1005-1009.
[46] Li MJ, Zhao XT, Hong SR, Zhang XL, Li P, Liu J, Xie CH (2009). Cryopreservation of plantlet nodes of Dioscorea opposita Thunb. using a vitrification method. CryoLetters 30, 19-28.
[47] Tian CW, Chen Y, Zhao XL, Zhao LJ (2008). Plant regene- ration through protocorm-like bodies induced from rhi- zoids using leaf explants of Rosa spp. Plant Cell Rep 27, 823-831.
[1] Zhenxing Wang 艳华 齐. Optimization of Tissue Culture and Plant Regeneration System of Mature Embryo of Leymus chinensis [J]. Chin Bull Bot, 2020, 55(2): 0-0.
[2] Zhang Wenting,He Yanhong,Shu Ning,Xing Jingjing,Liu Baojun,Bao Manzhu,Liu Guofeng. Plant Regeneration and Rapid Propagation System of Lilium bakerianum var. aureum [J]. Chin Bull Bot, 2019, 54(6): 773-778.
[3] Feng Ying, Qian Lianwen, Lin Qingliang. The Effect of Different Hormones on Explant Browning and Callus Browning in Cyclocarya paliurus [J]. Chin Bull Bot, 2019, 54(5): 634-641.
[4] Guo Jia, Li Yansu, He Chaoxing, Yan Yan, Yu Xianchang. Establishing a High-efficiency Regeneration System in Pumpkin (Cucurbita moschata) [J]. Chin Bull Bot, 2019, 54(4): 539-546.
[5] Xu Yue, Cao Yingping, Wang Yu, Fu Chunxiang, Dai Shaojun. Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea [J]. Chin Bull Bot, 2019, 54(4): 515-521.
[6] Li Junhua, Liu Shiyu, Li Chenglong, Han Linlin, Dong Yahui, Zhang Xiaoli, Zhao Xiting, Li Mingjun. Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber [J]. Chin Bull Bot, 2019, 54(1): 72-80.
[7] Zhang Xuhong, Wang Di, Liang Zhenxu, Sun Meiyu, Zhang Jinzheng, Shi Lei. Callus Induction and Establishment of a Plant Regeneration System with Lilium martagon [J]. Chin Bull Bot, 2018, 53(6): 840-847.
[8] Zheng Yunfeng, Zhang Xiaoman, Liu Xiao. Plant Regeneration by Inducing Axillary Buds of Sterile Seedlings of Primula denticulata [J]. Chin Bull Bot, 2018, 53(5): 686-692.
[9] Qianqian Zhang, Tong Zheng, Qian Yu, Lei Ge. Auxin and the Maintenance of Root Stem Cell Niches in Plants [J]. Chin Bull Bot, 2018, 53(1): 126-138.
[10] Sun Yingkun, Hu Shaoqing, Pang Jiliang, Gao Kai, Liu Huahong, Chen Huanwei, Yao Tao, Chen Linjing, Shen Bochun. Establishment of a Tissue Culture and Propagation System for Ardisia violacea, a Rare and Endangered Species [J]. Chin Bull Bot, 2017, 52(6): 764-773.
[11] Ren Ruyi, Xue Jukun, Guo Huiyan, Wei Jicheng. Induction of Hairy Roots of Scrophularia buergeriana and Its Plant Regeneration [J]. Chin Bull Bot, 2017, 52(6): 783-787.
[12] Liping Yan, Li Li, Cuilan Liu, Dejun Wu, Yinhua Wang, Fei Ren, Liangjun Zhao. Somatic Embryo Induction and Plantlet Regeneration of Fraxinus velutina [J]. Chin Bull Bot, 2016, 51(6): 807-816.
[13] Heping Shi, Bei Wang, Shunan Yang, Yapeng Guo. Induction of Hairy Roots of Dianthus chinensis and Its Plant Regeneration [J]. Chin Bull Bot, 2016, 51(3): 363-368.
[14] Fang Liu, Yinghong Tang, Youmei Yuan, Qingquan Guo, Fan Shen, Jianrong Chen. Tissue Culture of the Succulent Plant Sedum clavatum [J]. Chin Bull Bot, 2016, 51(2): 251-256.
[15] Lili Liu, Shubo Lu, Jiaping Xu, Qingtian Zhang, Changyu Li. Establishment of Plant Regeneration System from Hairy Roots of Aconitum coreanum [J]. Chin Bull Bot, 2015, 50(5): 623-627.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Yang Li-rui and Cheng Mu-chu. Relationship between Plant Stress Resistance and Photorespiration[J]. Chin Bull Bot, 1991, 8(01): 43 -47 .
[2] He Ping. Investigation of Pest Species and the Control of the Main Insect Pests in the Exhibition Green House of Beijing Botanical Garden[J]. Chin Bull Bot, 1996, 13(02): 44 -47 .
[3] Cui Kai-rong;Chen Ke-ming;Wang Xiao-zhe and Wang Ya-fu. Current Reseach on Plant Somatic Embryogenesis[J]. Chin Bull Bot, 1993, 10(03): 14 -20 .
[4] Huang Yao Li Chao-luan Ma Cheng Wu Nai-hu. Chloroplast DNA and Its Application to Plant Systematic Studies[J]. Chin Bull Bot, 1994, 11(02): 11 -25 .
[5] WANG Pu ZHAO Xiu-Qin. The Effect of Extracting Condition on the Analysis Result of Allelochemicals in Wheat Straw[J]. Chin Bull Bot, 2001, 18(06): 735 -738 .
[6] Yun Zihou;Liang Mingxia;Zhang Cunjie and Tan Zhiyi. The Determination of Trace Cytokinin in a Small Plant Sample by Gas Chromatography[J]. Chin Bull Bot, 1988, 5(01): 60 -63 .
[7] Yanxia He;Zicheng Wang*. Variation of DNA Methylation in Arabidopsis thaliana Seedlings After the Cryopreservation[J]. Chin Bull Bot, 2009, 44(03): 317 -322 .
[8] Yiting Shi, ShuhuaYang. Chinese Scientists Made Breakthrough in Study on Ethylene Signaling Transduction in Plants[J]. Chin Bull Bot, 2016, 51(3): 287 -289 .
[9] Rongpei Yu, Yang Li, Dong Li, Xuanhuai Zhan, Lei Shi. Radiosensitivity of Green Globular Bodies of Matteuccia struthiopteris Exposed to 60Coγ Radiation[J]. Chin Bull Bot, 2015, 50(5): 565 -572 .
[10] L Chao-Qun, SUN Shu-Cun. A REVIEW ON THE DISTRIBUTION PATTERNS OF CARBON DENSITY IN TERRESTRIAL ECOSYSTEMS[J]. Chin J Plan Ecolo, 2004, 28(5): 692 -703 .