Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber

doi:10.11983/CBB18118

Abstract

Abstract: Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun’. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin (Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin (Kan) resistence marker. The optimal concentration of timentin (Tim) for Agrobacterium counter selection was 500 mg·L ^-1. For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L ^-1 and 20 mg·L ^-1, respectively, and the optimal concentration of Kan were 120 mg·L ^-1 and 160 mg·L ^-1, respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun’, which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.

Key words: Agrobacterium tumefaciens, genetic transformation, microtuber, Dioscorea opposita cv. ‘Tiegun’

Junhua Li,Shiyu Liu,Chenglong Li,Linlin Han,Yahui Dong,Xiaoli Zhang,Xiting Zhao,Mingjun Li. Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber[J]. Chinese Bulletin of Botany, 2019, 54(1): 72-80.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB18118

https://www.chinbullbotany.com/EN/Y2019/V54/I1/72

Figures/Tables 8

Figure 1 Schematic maps of the overexpression vector and silencing vector used in this study (A) The overexpression vector pCAMBIA1301-DoSERK2; (B) The silencing vector pART27-DoSERK2

Table 1 Medium used for genetic transformation and plant regeneration of Dioscorea opposita cv. ‘Tiegun’

Media	Composition
MS₀	MS major salts, MS minor salts and MS vitamins +30 g·L^-1 sucrose+6 g·L^-1 fungible agar, pH5.8- 6.2
MS₀ 1	MS₀+1 mg·L^-1 6-BA+1 mg?L^-1 IAA
MS₀ 2	MS₀+2 mg?L^-1PP₃₃₃+0.05 mg?L^-1 NAA
MS₀ 1-A	MS₀1+100 μmol?L^-1 Acetosyringone (AS)
MS₀ 1-T	MS₀1+500 mg?L^-1 Timentin (Tim)
MS₀1-TH	MS₀1+500 mg?L^-1 Tim+15 mg?L^-1Hyg
MS₀ 1-TK	MS₀ 1+500 mg?L^-1 Tim+120 mg?L^-1 Kan
MS₀ 2-H	MS₀ 2+20 mg?L^-1Hyg
MS₀ 2-K	MS₀2+160 mg?L^-1 Kan

Table 2 Sequences of primers

Primers	Sequences (5'-3')
DoSERK2-OE-F	ATGACGGCTTGGGTTTTC
DoSERK2-OE-R	TCACCTCGGACCAGATAGC
DoSERK2-RNAi-F	CAGATGATACAGAAAAGCACCG
DoSERK2-RNAi-R	TAACTTTCGGTAGAGCGGAC
DoActin-F	CTCATTGATCGGCATGGAAGC
DoActin-R	GGGGAACATAGTTGAACCACCAC
DoSERK2-qRT-F	TATCTGGACCAGTTCCATCC
DoSERK2-qRT-R	CTTCAGCAGGCACATCATAG

Figure 2 Effects of different concentrations of Tim on the proliferation and differentiation of protocorm-like bodies (PLBs) in Dioscorea opposita (A) 0 mg·L-1; (B) 100 mg·L-1; (C) 200 mg·L-1; (D) 300 mg·L-1; (E) 400 mg·L-1; (F) 500 mg·L-1. Bars=1 cm

Figure 3 Effects of antibiotic on microtuber regeneration and rooting of regenerated seedlings in Dioscorea opposita cv. ‘Tiegun’ (A)-(F) Effects of different concentrations of Hyg on microtuber regeneration, the concentrations of Hyg are 0, 5, 10, 15, 20, 25 mg·L-1, respectively; (G)-(L) Effects of different concentrations of Kan on microtuber regeneration, the concentrations of Kan are 0, 80, 100, 120, 140, 160 mg·L-1, respectively; (M) Effects of different concentrations of Hyg on rooting of regenerated plantlets; (N) Effects of different concentrations of Kan on rooting of regenerated plantlets. (A)-(L) Bars=0.5 cm; (M), (N) Bars=2 cm

Table 3 Effects of antibiotic concentrations on regeneration of microtuber slices in Dioscorea opposita cv. ‘Tiegun’

Concentration of Hyg (mg·L^-1)	Regeneration (%)	Concentration of Kan (mg·L^-1)	Regeneration (%)	Growth of seedlings
0	60.00±2.00	0	65.56±2.52	Strong growth vigor
5	55.56±1.15	80	48.89±1.53	Well
10	33.33±2.00	100	34.44±1.53	Slow, albefaction in some seedlings
15	25.56±0.58	120	8.89±3.06	Slow, albefaction in many seedlings
20	2.22±1.15	140	1.11±0.58	Growth stopped, albefaction seriously
25	0	160	0	All dead

Table 4 Effects of antibiotic concentrations on rooting of regenerated seedlings in Dioscorea opposita cv. ‘Tiegun’

Concentration of Hyg (mg·L^-1)	Rooting (%)	Concentration of Kan (mg·L^-1)	Rooting (%)	Growth of roots
0	100	0	100	Strong and flourishing
5	100	120	100	Well
15	3±2.00	140	1±1.20	Seldom rooting
20	0	160	0	Hardly rooting
25	0	180	0	Hardly rooting, albefaction

Figure 4 Genetic transformation and regeneration of Dioscorea opposita cv. ‘Tiegun’ microtuber slices and molecular verification of trans- genic plants (A) Microtuber slices infected by Agrobacterium; (B) Microtuber slices after co-cultivation with Agrobacterium for 3 days; (C) Tissues cultured on regeneration medium for 40 days; (D) Tissues cultured on regeneration medium for 70 days; (E) Antibiotic-resistant regenerated seedlings that had been transferred to rooting medium; (F) Regenerated seedlings that had been cultured on rooting medium for 30 days; (G) PCR assay of genome DNA from transgenic plants (M: DNA marker; P: Plasmid pCAMBIA1301-DoSERK2 (left panel) and pART27-DoSERK2 (right panel) as template; WT: Genome DNA of wild-type plants as template; OE: Genome DNA from overexpression transgenic plants as template; RNAi-1-6: Genome DNA from line 1-6 of transgenic plants of the silencing vector as template); (H) GUS activity assay in the leaf of an overexpression transformant, nodal or leaf segments of a wild-type plant (WT) or transgenic plant (OE) were stained with GUS staining solution; (I) RT-qPCR assay of the endogenous expression levels of DoSERK2 (WT: cDNA of wild-type plants as template; RNAi-1-6: cDNA from line 1-6 of transgenic plants of the silencing vector as template). (A)-(F), (H) Bars=1 cm

References 24

1	范俊强 ( 2008). 根癌农杆菌介导的盾叶薯蓣转化体系的建立. 硕士论文. 武汉: 华中农业大学. pp. 1-5. DOI URL
2	付洪冰, 崔崇士, 赵曦, 刘琦 ( 2010). 农杆菌介导南瓜遗传转化体系的建立. 植物学报 45, 472-478. DOI URL
3	郭利军, 曾炳山, 刘英 ( 2013). 农杆菌介导巨桉Eg5高效遗传转化. 植物学报 48, 87-93. DOI URL
4	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, 135-139. DOI URL
5	李明军 (2013).提高山药商品性栽培技术问答. 北京: 金盾出版社. pp. 36-50.
6	李明军, 陈明霞, 洪森荣, 徐鑫, 张晓丽 ( 2004). NAA、IBA和PP₃₃₃对怀山药试管苗生长发育的影响. 广西植物 24, 376-379. DOI URL
7	李明军, 刘世宇, 刘雯, 李俊华, 张晓丽, 赵喜亭 ( 2017). 怀山药微型块茎形成过程中的生理生化变化. 植物生理学报 53, 807-814.
8	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, U003-U005. DOI URL
9	李瑞雪, 李纪强, 蒲腾飞, 张晓丽, 赵喜亭, 李俊华, 李明军 ( 2018). 怀山药类原球茎的诱导形成与植株再生. 植物学报 53, 334-340.
10	李卫, 郭光沁, 郑国锠 ( 2000). 根癌农杆菌介导遗传转化研究的若干新进展. 科学通报 45, 798-807. DOI URL
11	廖华兰, 黎秀琼, 李可, 李伯凌, 熊茜, 苏童, 李春霞, 陈银华, 罗丽娟 ( 2016). 农杆菌介导的木薯遗传转化体系的优化. 热带生物学报 7, 427-434. DOI URL
12	王善平, 许智宏, 卫志明 ( 1990). 毛白杨叶外植体的遗传转化. 植物学报 32, 172-177.
13	王运英 ( 2016). 怀山药脱毒微型块茎管内管外萌发条件及生理生化机制研究. 硕士论文. 新乡: 河南师范大学. pp. 9-19.
14	许云 ( 2014). 大薯遗传多样性的AFLP分析和类原球茎遗传转化体系的研究. 硕士论文. 海口: 海南大学. pp. 61-66.
15	杨亚萍, 李永兰, 梁月荣, 陆建良, 郑新强 ( 2015). 发根农杆菌抑菌剂的抑菌效果及对茶组培苗丛生芽的影响. 茶叶科学 35, 437-442.
16	张宁, 司怀军, 李学才, 王蒂 ( 2004). 根癌农杆菌介导的马铃薯高效遗传转化体系的研究. 中国马铃薯 18, 132-135. DOI URL
17	赵喜亭, 蒋丽薇, 王苗, 朱玉婷, 张文芳, 李明军 ( 2016). 怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化. 植物学报 51, 525-532. DOI URL
18	Edwards K, Johnstone C, Thompson C ( 1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19, 1349. DOI URL PMID
19	Li MJ, Li JH, Wang YP, Liu W, Guo XB, Li SJ, Han LL, Song ZH, Zhao XT, Yang QX ( 2015). A simple method for microtuber production in Dioscorea opposita using single nodal segments . Pakistan J Bot 47, 665-668.
20	Mignouna HD, Abang MM, Asiedu R (2008). Genomics of yams, a common source of food and medicine in the tropics. In: Moore PH, Ming R, eds. Genomics of Tropical Crop Plants. Plant Genetics and Genomics: Crops and Models, Vol. 1. Berlin: Springer. pp. 549-570.
21	Nyaboga E, Tripathi JN, Manoharan R, Tripathi L ( 2014). Agrobacterium-mediated genetic transformation of yam ( Dioscorea rotundata): an important tool for functional study of genes and crop improvement.Front Plant Sci 5, 463.
22	Tör M, Ainsworth C, Mantell SH ( 1993). Stable transfor-mation of the food yam Dioscorea alata L.by particle bombardment. Plant Cell Rep 12, 468-473.
23	Tör M, Twyford CT, Funes I, Boccon-Gibod J, Ainsworth CC, Mantell SH ( 1998). Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies.Plant Cell Tissue Organ Cult 53, 113-126. DOI URL
24	Zhao XT, Zhang XL, Guo XB, Li SJ, Han LL, Song ZH, Wang YY, Li JH, Li MJ ( 2016). Identification and validation of reference genes for qRT-PCR studies of gene expression in Dioscorea opposita.Biomed Res Int 2016, 3089584. DOI URL PMID

[1]	Liyuchen YuchenLI Hai-Xia ZHAO Xi-Ping JIANG. Establishment of stable genetic transformation system of Agropyron mongolicum mediated by Agrobacterium tumefaciens [J]. Chinese Bulletin of Botany, 2024, 59(4): 0-0.
[2]	Yu Xiaomin, Wang Yaqin, Liu Yuhan, Yi Qingping, Cheng Wenhan, Zhu Yu, Duan Feng, Zhang Lixue, He Yanhong. Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold (Tagetes erecta) [J]. Chinese Bulletin of Botany, 2023, 58(5): 760-769.
[3]	Lan Yang, Ya Liu, Yang Xiang, Xiujuan Sun, Jingwei Yan, Aying Zhang. Establishment and Optimization of a Shoot Tip-based Genetic Transformation System for Foxtail Millet [J]. Chinese Bulletin of Botany, 2021, 56(1): 71-79.
[4]	Lijun Guo, Bingshan Zeng, Ying Liu. Agrobacterium-mediated High-efficient Transformation of Eucalyptus grandis Clone Eg5 [J]. Chinese Bulletin of Botany, 2013, 48(1): 87-93.
[5]	Guimei Cui, Yi Sun, Yaoshan Hao, Jianzhong Du, Yixue Wang. The Improvement of Maize Pollen In Vitro Germination Method and Its Role in Pollen-mediated Plant Genetic Transformation [J]. Chinese Bulletin of Botany, 2012, 47(2): 155-161.
[6]	Xuanyu Liu, Qingyun Wang, Shujun Liu, Songquan Song. Advances in the Genetic Transformation of Sorghum bicolor [J]. Chinese Bulletin of Botany, 2011, 46(2): 216-223.
[7]	Hongbing Fu;Chongshi Cui;Xi Zhao;Qi Liu. Establishment of Cucurbita moschata Genetic Transformation System by Agrobacterium tumefaciens Transfection [J]. Chinese Bulletin of Botany, 2010, 45(04): 472-478.
[8]	Daojie Wang, Cuiling Yang, Ming Lu. Transformation of Brassica napus by Vacuum Infiltration [J]. Chinese Bulletin of Botany, 2009, 44(02): 216-222.
[9]	Kaifa Wei*;Yiping Liu;Ziying Lin;Yafang Yang;Zehong Zhang;Wensuo Jia. Problems and Solutions in Agrobacterium tumefaciens-mediated Genetic Transformation of Monocotyledons [J]. Chinese Bulletin of Botany, 2008, 25(04): 491-496.
[10]	Tingbo Jiang*;Xinhua Tang;Fengjuan Li;Baojian Ding;Hong Chen. Effects of Ferritin Gene Expression on Transgenic Tobacco for Low Iron Tolerance [J]. Chinese Bulletin of Botany, 2008, 25(02): 167-175.
[11]	Jianbin Hu*;Jun Liu. Progress in Tissue Culture and Genetic Transformation of Amorphophallus Blume [J]. Chinese Bulletin of Botany, 2008, 25(01): 14-19.
[12]	JI Feng_Yuan, WANG Ge_Liang, XU Yi_Nong. THE EFFECTS OF ANTIOXIDANTS ON THE TRANSIENT EXPRESSION OF GUS GENE IN SOYBEAN HYPOCOTYLS MEDIATED BY AGROBACTERIUM TUMEFACIENS [J]. Chin J Plant Ecol, 2006, 30(2): 330-334.
[13]	GAO Li-Ping BAO Man-Zhu. Advances in Plant Regeneration and Genetic Transformation of Roses [J]. Chinese Bulletin of Botany, 2005, 22(02): 231-237.
[14]	ZHU Gen-Fa GUO Zhen-Fei. Progress on Molecular Biology of Main Ornamental Orchidaceae [J]. Chinese Bulletin of Botany, 2004, 21(04): 471-477.
[15]	SONG Li-Ying TAN Zheng GAO Feng DENG Shu-Yan. Advances in in vitro Culture of Cucurbitaceae in China [J]. Chinese Bulletin of Botany, 2004, 21(03): 360-366.