Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Summary

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

Abstract

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7–10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p < 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Introduction

Tartary buckwheat (TB) (Fagopyrum tataricum (L.) Gaertn) is a type of dicotyledon belonging to the genus Fagopyrum and the family Polygonaceae¹. As a type of Chinese medicine homologous food, TB has been receiving considerable interest owing to its distinctive chemical composition and diverse bioactivities against diseases. TB is primarily rich in carbohydrates, proteins, vitamins, and carotenoids as well as in polyphenols such as phenolic acids and flavonoids¹. Various biological and pharmacological activities of flavonoids, including antioxidative, antihypertensive², and anti-i....

Protocol

The TB used in this study was named as BT18, which originated from the breed of "JinQiao No.2" cultivated by the Research Center of Small Miscellaneous Grain of Shanxi Academy of Agricultural Science. The primary steps of this protocol are illustrated in Figure 1.

NOTE: Operate explants-related manipulation rapidly, and when possible, keep the Petri dishes closed to avoid wilting and contamination. Unless otherwise stated, all the explant incubations were conducted under the condition of a 14-h light and a 10-h dark photoperiod at 25 °C. Unless otherwise stated, all explants- or bacteria-related op....

Results

Agrobacterium rhizogenes-mediated TB hairy root transformation
This study describes the step-by-step protocol that was established to obtain genetically transformed hairy roots using A. rhizogenes. It took approximately 5-6 weeks from the inoculation of TB seeds to the harvesting of the identified hairy roots, and some key steps are depicted in Figure 1 (A-H). Briefly, sterilized shelled seeds were inoculated (Figur.......

Discussion

TB has been used in several studies related to secondary metabolites at genetic and metabolic levels^1,2,5,27,28. Hairy root culture, as a unique source for metabolite production, plays a pivotal role in metabolic engineering²⁹ and can be used to alter metabolic pathways by inserting the related genes. Kim et al.².......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2*Taq PCR MasterMix	Aidlab, China	PC0901
Agar powder	Solarbio Life Science, Beijing, China	A8190
Applied Biosystems 2720 thermo cycler	ThermoFisher Scientific, US	A37834
AS	Solarbio Life Science, Beijing, China	A8110	Diluted in DMSO, 100 mM
binary vectors	ThermoFisher Scientific (invitrogen), US	/	pK7WG2D/pK7GWIWG2D (II)
Cefotaxime,sodium	Solarbio Life Science, Beijing, China	C8240	Diluted in Water, 200 mg/mL
CF15RXII high-speed micro	Hitachi, Japan	No. 90560201
Diposable Petri-dish	Guanghua medical instrument factory, Yangzhou, China	/
DYY-6C electrophoresis apparatus	Bjliuyi, Beijing China	ECS002301
EASYspin Plus Plant RNA Kit	Aidlab, China	RN38
ELGA purelab untra bioscience	ELGA LabWater, UK	82665JK1819
Epoch Microplate Spectrophotometer	biotek, US	/
Gateway BP/LR reaction enzyme	ThermoFisher Scientific (invitrogen), US	11789100/11791110
HYG-C multiple-function shaker	Suzhou Peiying Experimental Equipment Co., Ltd. China	/
Kan	Solarbio Life Science, Beijing, China	K8020	Diluted in Water, 100 mg/mL
MLS-3750 Autoclave sterilizer	Sanyo, Japan	/
MS salts with vitamins	Solarbio Life Science, Beijing, China	M8521
NaCl	Solarbio Life Science, Beijing, China	S8210
Other chemicals unstated	Beijing Chemical Works, China		ethanol, mercury bichloride, etc.
PHS-3C pH meter	Shanghai INESA Scientific Instrument Co., Ltd, China	a008
Plant Genomic DNA Kit	TIANGEN BIOTECH (BEIJING) CO., LTD	DP305
Rifampin	Solarbio Life Science, Beijing, China	R8010	Diluted in DMSO, 50 mg/mL
Spectinomycin	Solarbio Life Science, Beijing, China	S8040	Diluted in Water, 100 mg/mL
Sucrose	Solarbio Life Science, Beijing, China	S8270
Trans2K DNA Marker	TransGen Biotech, Beijing, China	BM101-01
Tryptone	Solarbio Life Science, Beijing, China	LP0042
Whatman diameter 9 cm Filter paper	Hangzhou wohua Filter Paper Co., Ltd	/
Yeast Extract powder	Solarbio Life Science, Beijing, China	LP0021