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Transgenic hairy roots of Tetrastigma hemsleyanum: induction, propagation, genetic characteristics and medicinal components

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Abstract

Tetrastigma hemsleyanum Diels & Gilg ex Diels (T. hemsleyanum) is an important medicinal plant in China. Its bulbous root is the most valuable part for medicine. There is an increasing demand for T. hemsleyanum in the medicinal market. However, the natural slow growth rate of T. hemsleyanum hardly meets the demand of the medicinal market. There is the need of alternative way for rapid and large-scale production of T. hemsleyanum. In this study, we investigated the induction of hairy roots by using Agrobacterium rhizogenes (A. rhizogenes) as an alternative method of increasing production of the secondary metabolites in hairy roots of T. hemsleyanum. Herein, we reported our successful induction of hairy roots of T. hemsleyanum by cucumopine type A. rhizogenes K599 and their rapid propagation. Firstly, we selected the optimal mediums for induction of hairy roots and for subculture of induced hairy roots. We found that when A. rhizogenes K599 carrying both an endogenous plasmid pRi2659 and an exogenous plasmid pRI101-AN-GFP was used to infect the leaves of T. hemsleyanum seedlings, the optimal medium for inducting hairy roots was the medium of MS + 0.4 mg/L 1-naphthaleneacetic acid. The optimal medium for subculture of hairy roots was the combination of MS + 1.0 mg/L indole-3-butyric acid + 0.5 or 1.0 mg/L kinetin. During suspension culture of hairy roots, the rapid growth phase occurred in the period of 15–28 days. Secondly, we employed polymerase chain reaction (PCR) assays to examine the integration of T-DNA of A. rhizogenes. The PCR results revealed the existence of a phenomenon of lacking of integration of one or more of 11 genes within T-DNA of plasmid pRi2659 into the genomic DNA of hairy roots. Thirdly, we conducted histocytological analysis to examine the structural features of the induced hairy roots and observed that xylem structures of the hairy roots were simplified as compared to those of the bulbous roots and ordinary fine roots of non-transgenic plant. Finally, we applied high performance liquid chromatography to analyze the medicinal components in several organs of the T. hemsleyanum plants. The results showed that the contents of kaempferol in all hairy root lines were significantly higher than those in bulbous roots, fine roots, stems and leaves. These results suggest that insertion and integration of T-DNA from A. rhizogenes can significantly increase the kaempferol content in hairy roots of T. hemsleyanum.

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Abbreviations

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

6-BA:

6-Benzylaminopurine

AS:

Acetosyringone

Cef:

Cefotaxime sodium

cus:

Cucumopine synthase

FAA:

Formaldehyde-acetic acid-alcohol solution

FITC:

Fluorescein isothiocyanate

GFP:

Green fluorescent protein

HPLC:

High performance liquid chromatography

IBA:

3-Indolebutyric acid

kD:

kiloDalton

Km:

Kanamycin

KT:

Kinetin

MS:

Murashige and Skoog medium (1962)

NAA:

α-Naphthaleneacetic acid

OD:

Optical density

ORF:

Open reading frame

PCR:

Polymerase chain reaction

rol:

Root loci

Ri:

Root inducing plasmid

Str:

Streptomycin

T-DNA:

Transferred DNA

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Acknowledgments

The work was supported by the Project of Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants (Grant No. 201302); the Hangzhou Science and Technology Development Plan (Grant No. 20110232B10) and the New-shoot Talents Program of Zhejiang Province (Grant No. 2013R421008).

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Authors and Affiliations

  1. College of Life and Environment Sciences, Hangzhou Normal University, Hangzhou, 310036, China

    Surui Du, Taihe Xiang, Yaling Song, Lianxiang Huang, Yang Sun & Yixuan Han

  2. Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou, 310036, China

    Taihe Xiang

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  1. Surui Du
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Correspondence to Taihe Xiang.

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Du, S., Xiang, T., Song, Y. et al. Transgenic hairy roots of Tetrastigma hemsleyanum: induction, propagation, genetic characteristics and medicinal components. Plant Cell Tiss Organ Cult 122, 373–382 (2015). https://doi.org/10.1007/s11240-015-0775-6

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