Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea

doi:10.11983/CBB18257

Abstract

Abstract: The plant can produce hairy roots after infection by Agrobacterium rhizogenes harboring Ri plasmids. Spinach (Spinacia oleracea) is a common edible vegetable, and its hairy root system has yet to be reported. In this study, A. rhizogenes LBA9402 was screened to be suitable for hairy root induction of spinach. The highest induction efficiency, 16%, was reached when the spinach stem was infected by LBA9402. Hairy roots grew vigorously on SH solid medium without exogenous hormones. The hairy roots of spinach were white and had enormous root hairs. The regeneration plant obtained from the callus redifferentiated from spinach hair roots; the rate of regeneration was 8%. In addition, Ri T-DNA and Ti T-DNA carrying rolB genes and GFP gene, respectively, were co-transferred into cells of spinach and produced transgenic hairy roots. PCR analysis and fluorescence microscopy assays showed rolB gene and GFP gene in the hairy roots of spinach and expressed stably; the rate of co-transformation was 50%.

Key words: co-transformation, hairy root, plant regeneration system, Spinacia oleracea

Yue Xu,Yingping Cao,Yu Wang,Chunxiang Fu,Shaojun Dai. Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea[J]. Chinese Bulletin of Botany, 2019, 54(4): 515-521.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18257

https://www.chinbullbotany.com/EN/Y2019/V54/I4/515

Figures/Tables 6

Figure 1 Schematic map of T-DNAs of pCAMBIA139IZ-eGFP (A) and Ri plasmid (B)

Figure 2 Effects of different explants and Agrobacterium rhizogenes strains on the induction rate of spinach hairy roots

Figure 3 The induction and morphological characteristics of spinach hairy roots (A) After Agrobacterium rhizogenes LBA9402 infection, multiple hairy roots were produced at one site; (B) The induction of hairy roots around the vascular bundle of the petiole; (C) The induction of hairy roots in veins; (D) The induction of hairy roots around the vascular bundle of stem; (E) Genomic PCR analysis of rolB (up) and virG (down) genes in hairy roots (Lane 1: 2 kb DNA marker; Lane 2-11: Different hairy roots plantlets; Lane 12: LBA9402; Lane 13: Untransformed roots; Lane 14: Mili Q water); (F) The hairy root induced by LBA9402; (G) The wild type root. (A)-(D), (F), (G) Bars=20 μm

Figure 4 The morphological characterization of spinach hairy roots grown on solid medium and in liquid medium (A) Hairy roots grown on solid medium in 0 day after inoculation; (B), (F) Hairy roots grown on solid medium in 14 days after inoculation; (C) Hairy roots grown in liquid medium in 0 day after inoculation; (D), (G) Hairy roots grown in liquid medium in 14 days after inoculation; (E) Increase times of spinach hairy roots fresh weight on solid medium and in liquid medium, respectively. (A)-(D) Bars=8 cm; (F), (G) Bars=20 μm

Figure 5 Regeneration system of spinach hairy roots (A) Calli produced from hairy roots on callus induction medium; (B) Calli induced from hairy roots and produced adventitious buds on the regeneration medium; (C) The plantlets regenerated from the calli produced from hairy roots; (D) The wild type plantlets; (E) Genomic PCR analysis of rolB (up) and virG (down) genes in the plantlets regenerated from the calli produced from hairy roots (Lane 1: 2 kb DNA marker; Lane 2-4: Different hairy roots plantlets regenerated from the calli produced; Lane 5: LBA9402; Lane 6: The wild type plantlets; Lane 7: Mili Q water). (A)-(D) Bars=4 cm

Figure 6 PCR analysis and GFP green fluorescence assay of the rolB gene and GFP gene in spinach transgenic hairy roots (A) Transgenic hairy root under the natural light; (B) Transgenic hairy root under the blue exciting light; (C) Genomic PCR analysis of rolB (up) and virG (middle) and GFP (down) genes in transgenic hairy roots (Lane 1: 2 kb DNA marker; Lane 2-11: Different of transgenic hairy roots lines; Lane 12: LBA9402 (GFP); Lane 13: The wild type root; Lane 14: Milli Q water). (A), (B) Bars=20 μm

References 21

[1]	付春祥 ( 2006). 雪莲细胞培养物中黄酮类物质代谢调控及其生物活性成分分析. 博士论文. 北京: 中国科学院植物研究所. pp. 28-45.
[2]	付春祥, 金治平, 杨睿, 吴风燕, 赵德修 ( 2004). 新疆雪莲毛状根的诱导及其植株再生体系的建立. 生物工程学报 20, 366-371.
[3]	耿晓霞 ( 2009). 菠菜叶绿体表达载体构建及遗传转化. 硕士论文. 济南: 山东大学. pp. 57-59.
[4]	胡凤, 杨万年 ( 2013). 大豆组培苗水培生根与培养基生根比较研究. 大豆科学 32, 333-335.
[5]	晋四清 ( 2013). 菠菜露地越冬杂交制种技术. 乡村科技 ( 10), 20.
[6]	钱伟, 张合龙, 刘伟, 徐兆生 ( 2014). 菠菜遗传育种研究进展. 中国蔬菜 1(3), 5-13.
[7]	任如意, 薛巨坤, 国会艳, 魏继承 ( 2017). 北玄参毛状根诱导及其植株再生. 植物学报 52, 783-787.
[8]	施和平, 王蓓, 杨树楠, 郭亚鹏 ( 2016). 五寸石竹毛状根诱导及其植株再生. 植物学报 51, 363-368.
[9]	孙晶, 徐洁森, 赵立子, 魏建和, 杨洪一, 隋春 ( 2013). 北柴胡毛状根诱导及其植株再生体系的建立. 药学学报 48, 1491-1497.
[10]	孙敏, 汪洪, 王颖, 伍春莲 ( 2002). 长春花转化毛状根诱导及培养条件的优化. 西南师范大学学报(自然科学版) 27, 549-552.
[11]	王晓武, 杜永臣 ( 2007). 蔬菜作物分子育种研究现状与趋势. 中国农业科技导报 9(2), 14-18.
[12]	闻玉莉, 杨世海 ( 2010). 罗勒毛状根的诱导及培养. 安徽农业科学 38, 1727-1730.
[13]	Akhgari A, Yrjönen T, Laakso I, Vuorela H, Oksman- Caldentey KM, Rischer H ( 2015). Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production. Plant Cell Rep 34, 1939-1952.
[14]	Hu Z, Du M ( 2006). Hairy root and its application in plant genetic engineering. J Integr Plant Biol 48, 121-127.
[15]	Porter JR, Flores H ( 1991). Host range and implication of plant infection by Agrobacterium rhizogenes. CRC Crit Rev Plant Sci 10, 387-421.
[16]	Ron M, Kajala K, Pauluzzi G ( 2014). Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166, 455-469.
[17]	Sharafi A, Hashemi Sohi H, Mousavi A, Azadi P, Razavi K, Ntui VO ( 2013). A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tissue Organ Cult 113, 1-9.
[18]	Veena V, Taylor GG ( 2007). Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant 43, 384-403.
[19]	Xu C, Jiao C, Sun H, Cai X, Wang X, Ge C, Zheng Y, Liu W, Sun X, Xu Y, Deng J, Zhang Z, Huang S, Dai S, Mou B, Wang Q, Fei Z, Wang Q ( 2017). Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions. Nat Commun 8, 15275.
[20]	Zhang HX, Zeevaart JAD ( 1999). An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach( Spinacia oleracea L.). Plant Cell Rep 18, 640-645.
[21]	Zhao Q, Chen W, Bian J, Xie H, Li Y, Xu C, Ma J, Guo S, Chen J, Cai X, Wang X, Wang Q, She Y, Chen S ( 2018). Proteomics and phosphoproteomics of heat stress-responsive mechanisms in spinach. Front Plant Sci 9, 800.