Chin Bull Bot ›› 2020, Vol. 55 ›› Issue (2): 192-198.doi: 10.11983/CBB19223

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Optimization of Tissue Culture and Plant Regeneration System of Mature Embryo of Leymus chinensis

Xiao Yan1,Wang Zhenxing1,Li Dongming2,Qi Yanhua2, Enhebayaer1()   

  1. 1 College of Life Science and Technology, Inner Mongolia Normal University, Huhehot 010022, China
    2 Key Laboratory of Herbage and Endemic Crop Biotechnology, Ministry of Education, School of Life Sciences, Inner Mongolia University, Huhehot 010000, China
  • Received:2019-11-17 Accepted:2020-02-26 Online:2020-02-26 Published:2020-03-01
  • Contact: Enhebayaer E-mail:nmsdenhe@imnu.edu.cn

Abstract:

Leymus chinensis is a heterotetraploid grass. It is hard to use genetic transformation method for improving breeding since the efficiency of regeneration using callus induced from mature embryo is very low. In this study, we optimized the protocol of culturing mature embryo with L. chinensis as explants by screening optimum plant hormone concentrations, light conditions and culturing temperatures at different culturing stages including callus induction, differentiation, rooting and transplanting. Our results showed at the callus induction stage, the induction rate could reach up to 74.1% under 2.0 mg·L -12,4-D, variable temperature and dark culture. The differentiation rate could reach up to 57.1% with optimum condition of 1.0 mg·L -16-BA and 1.0 mg·L -1NAA. In the rooting stage, the survival rate was 100% when cultured with 0.25 mg·L -1NAA.

Key words: mature embryo, Leymus chinensis, callus, plant hormone, plant regeneration

Figure 1

Induction efficiency of different 2,4-D concentra- tions in mature embryos of Leymus chinensis under light/dark conditions for 20 days * P<0.05; ** P<0.01; ****P<0.0001"

Table 1

Differentiation efficiency of Leymus chinensis callus under different concentrations of 6-BA and NAA (means±SE)"

6-BA/NAA (mg·L-1) Callus number The number of adventitious buds in proliferation The number of roots in reproduction Differentiation
efficiency (%)
0.5/0.25 21 0 0 0
1.0/0.25 21 1A 4+ 4.8±4.8 ab
2.0/0.25 21 0 0 0
3.0/0.25 21 3AB 0 14.3±8.3 abcd
4.0/0.25 21 8AB 0 38.1±4.8 efgh
5.0/0.25 21 4A 0 19.1±4.8 abcde
0.5/0.5 21 0 0 0
1.0/0.5 21 7BC 6+ 33.3±9.5 defg
2.0/0.5 21 10BC 5+ 47.6±9.5 gh
3.0/0.5 21 7AB 4 33.3±4.8 defg
4.0/0.5 21 10AB 0 47.6±9.5 gh
5.0/0.5 21 3BC 0 14.3±0 abcd
0.5/1.0 21 2A 8+ 9.5±4.8 abc
1.0/1.0 21 12AB 4 57.1±4.8 h
2.0/1.0 21 9A 0 42.9±8.3 fgh
3.0/1.0 21 5AC 3 23.8±8.3 bcdef
4.0/1.0 21 4A 1+ 19.1±4.8 abcde
5.0/1.0 21 5A 0 23.8±12.6 bcdef
0.5/2.0 21 5ABC 9+ 23.8±4.8 bcdef
1.0/2.0 21 1A 5 4.8±4.8 ab
2.0/2.0 21 0 0 0
3.0/2.0 21 2A 1+ 9.5±4.8 abc
4.0/2.0 21 2AB 4 9.5±4.8 abc
5.0/2.0 21 0 0 0
0.5/3.0 21 1A 3 4.8±4.8 ab
1.0/3.0 21 4A 3 19.1±4.8 abcde
2.0/3.0 21 7AB 3 33.3±4.8 defg
3.0/3.0 21 10ABC 1 47.6±9.5 gh
4.0/3.0 21 0 0 0
5.0/3.0 21 0 0 0
0.5/4.0 21 6A 3 28.6±8.2 cdefg
1.0/4.0 21 0 0 0
2.0/4.0 21 8AB 2 38.1±4.8 efgh
3.0/4.0 21 0 0 0
4.0/4.0 21 3AB 5 14.3±8.2 abcd
5.0/4.0 21 6AB 0 28.6±8.2 cdefg

Figure 2

Effect of different NAA concentrations on rooting of tissue culture seedlings in Leymus chinensis (A) Rooting of 2.0-3.0 cm of seedlings; (B) Rooting of 3.1-5.0 cm of seedlings; (C) Rooting of 5.1-7.0 cm of seedlings. All seedlings with (0.5±0.1) cm of primary root and 3-5 of adventitious roots were used to the related experiments."

Figure 3

Induction, subculture, differentiation and transplanting of mature embryos of Leymus chinensis (A) Mature embryo induction for 5 days; (B) Mature embryo induction for 20 days; (C) Subculture of callus; (D) Callus differentiation; (E) The differentiated 40-day seedlings; (F) Rooting culture for 14 days; (G) Plants transplanted for 14 days; (H) Plants transplanted for 2 months. Bars=1 cm"

[1] 崔秋华, 张玉珍, 朴铁夫, 顾德峰, 张为群, 许耀奎, 孙振雷, 刘海学 ( 1990). 羊草胚性愈伤组织的形成及植株再生. 吉林农业大学学报 12(3), 1-5.
[2] 韩德复 ( 1996). 羊草组织培养的研究. 吉林农业大学学报 18(S1), 140-141.
[3] 孔祥军, 梁正伟 ( 2007). 羊草分子生物学研究进展. 生命科学研究 11, 289-294.
[4] 孔祥军, 梁正伟, 马红媛, 刘淼 ( 2008). 变温培养对羊草胚性愈伤组织诱导率的影响. 生物技术 10(5), 60-62.
[5] 李艳波, 李凤芹 ( 1998). 松嫩盐碱草地羊草群落的产量动态. 黑龙江大学自然科学学报 15(2), 103-106.
[6] 刘滨硕, 康春莉, 王鑫, 包国章 ( 2014). 羊草对盐碱胁迫的生理生化响应特征. 农业工程学报 30(23), 166-173.
[7] 刘公社, 汪恩华, 刘杰, 齐冬梅, 李芳芳 ( 2002). 羊草幼穗离体培养诱导植株再生的研究. 草地学报 10, 198-202.
[8] 马红媛, 梁正伟 ( 2007). 不同pH值土壤及其浸提液对羊草种子萌发和幼苗生长的影响. 植物学通报 24, 181-188.
[9] 孟宪宝 ( 2010). 优质饲草羊草栽培技术. 黑龙江畜牧兽医 13, 97-98.
[10] 曲同宝 ( 2004). 羊草遗传转化受体系统的建立及转BADH基因的研究. 硕士论文. 吉林: 吉林农业大学. pp.12-25.
[11] 曲同宝, 孟繁勇, 张友民, 王丕武 ( 2010). 影响羊草愈伤组织分化因素的研究. 安徽农业科学 38, 6125-6127, 6130.
[12] 曲同宝, 王丕武, 关淑艳, 刘玲芝 ( 2004). 羊草组织培养及再生系统的建立. 草业学报 13(5), 91-94.
[13] 汪恩华 ( 2002). 羊草繁殖生物学特性的研究.硕士论文. 北京: 中国科学院植物研究所. pp.39-45.
[14] 魏琪, 胡国富, 李凤兰, 胡宝忠 ( 2005). 羊草种子愈伤组织的诱导及植株再生. 东北农业大学学报 36, 41-44.
[15] 张莹, 李晓峰, 刘公社, 陈耀锋 ( 2007). 羊草愈伤组织状态的调控. 西北农林科技大学学报(自然科学版) 35(5), 111-114.
[16] 张玉芬, 周道玮 ( 2002). 羊草分化及育种研究进展. 中国草地 24(2), 54-58, 74.
[17] 周道玮, 李强, 宋彦涛, 王学志 ( 2011). 松嫩平原羊草草地盐碱化过程. 应用生态学报 22, 1423-1430.
[18] 邹吉祥 ( 2012). 羊草高频再生体系建立及转CodA基因的初步探索. 硕士论文. 吉林: 延边大学. pp.3-4.
[19] Hiei Y, Ohta S, Komari T, Kumashiro T ( 1994). Efficient transformation of rice ( Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6, 271-282.
[20] Liu BS, Kang CL, Wang X, Bao GZ ( 2015). Tolerance mechanisms of Leymus chinensis to salt-alkaline stress. Acta Agric Scand Sect B-Soil Plant Sci 65, 723-734.
[21] Mathur S, Tomar RS, Jajoo A ( 2019). Arbuscularmycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress. Photosyn Res 139, 227-238.
[22] Olson RE, Rudney H ( 1983). Biosynthesis of ubiquinone. Vitam Horm 40, 1-43.
[23] Zhu TC, Li JD, Yang DC ( 1981). A study of the ecology of Yang-cao (Leymus chinensis) grassland in Northern China. In: Proceedings of the 14th International Grassland Congress. Lexington: Westview. pp. 429-431.
[1] Lai Xianjun,Zhang Yizheng,Gu Yinghong,Yan Lang. Transformation of Insect Derived Antifreeze Gene into Sweet Potato (Ipomoea batatas) and Enhanced Its Freeze-tolerance [J]. Chin Bull Bot, 2020, 55(1): 9-20.
[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] 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.
[5] 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.
[6] Liu Xiaomei, Sun Lili, Fu Xiangdong, Liao Hong. An Effective Method for the Rooting of Tea Cuttings [J]. Chin Bull Bot, 2019, 54(4): 531-538.
[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] Zhang Tianpeng, Yang Xinghong. Advances in the Molecular and Physiological Mechanisms of Early Development of Tomato Fruit [J]. Chin Bull Bot, 2018, 53(6): 856-866.
[9] 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.
[10] Li Ruixue, Li Jiqiang, Pu Tengfei, Zhang Xiaoli, Zhao Xiting, Li Junhua, Li Mingjun. Induction and Plant Regeneration of Protocorm-like Bodies in Dioscorea opposita [J]. Chin Bull Bot, 2018, 53(3): 334-340.
[11] Hongliang Wang, Siyi Guo, Pengtao Wang, Chunpeng Song. Research Progress in Stomatal Development Mechanism [J]. Chin Bull Bot, 2018, 53(2): 164-174.
[12] 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.
[13] Lingyu Li, Haomeng Yang, Weibo Ren, Xinhong Wu, Fang Huang. Assessment of Phosphate and Nitrogen Deficiency in Sheepgrass by Chlorophyll Fluorescence Spectroscopy [J]. Chin Bull Bot, 2017, 52(3): 271-276.
[14] Yan Chun, Xueyong Li. Research Progress in Genetic Regulation of Rice Panicle Architecture [J]. Chin Bull Bot, 2017, 52(1): 19-29.
[15] 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.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Cui Gao;Yuxia Chen;Ying Bao;Min Feng;Anming Lu. Studies on Sexual Organs and Embryological Development Morphology of Speirantha gardenii (Convallariaceae)[J]. Chin Bull Bot, 2010, 45(06): 705 -712 .
[2] ZHANG Yi-Shun HUANG Shang-Zhi FU Jia-Rui. Progress in Study on Raffinose Family Oligosaccharides in Seeds[J]. Chin Bull Bot, 2001, 18(01): 16 -22 .
[3] Jiang Gao-ming. The Impact of Globae Increasing of CO2 on Plants[J]. Chin Bull Bot, 1995, 12(04): 1 -7 .
[4] YU Bing-Jun LIU You-Liang. Chlorine, Chloride Channel and Chlorine Tolerance in Plants[J]. Chin Bull Bot, 2004, 21(04): 402 -410 .
[5] Tang Yan-cheng. A Short Guide to the International Code of Botanical Nomenclature V.[J]. Chin Bull Bot, 1984, 2(04): 51 -57 .
[6] Xu Ji. The Protective Protein of Nitrogenase Against Oxygen Damage-Fe-S Protein[J]. Chin Bull Bot, 1986, 4(12): 1 -4 .
[7] . [J]. Chin Bull Bot, 2001, 18(05): 633 .
[8] Huang Zhao-xiang;Zheng Zhen-gui and Zhu Du. Ecological Effect of Taxodium ascendens-Oryza sativa Ecosystem(I) The Growing Characteristic of Taxodium Ascendens in the Ecosystem[J]. Chin Bull Bot, 1996, 13(02): 48 -51 .
[9] Zhihong Xu;Jiayang Li. Plant Hormones Research in China: Past, Present and Future[J]. Chin Bull Bot, 2006, 23(5): 433 -442 .
[10] GU Rui-Sheng;LIU Qun-Lu;CHEN Xue-Mei and JIANG Xiang-Ning. Comparison and Optimization of the Methods on Protein Extraction and SDS-PAGE in Woody Plants[J]. Chin Bull Bot, 1999, 16(02): 171 -177 .