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-inflammatory as well as anticancer and antidiabetic properties, have been demonstrated³.

Agrobacterium rhizogenes is a soil bacterium that contributes to the development of hairy root disease in several higher plants, especially dicotyledons, by infecting wound sites^4,5. This process is initiated by the transfer of the T-DNA in the root-inducing (Ri) plasmid^5,6 and is commonly accompanied by the integration and expression of an exogenous gene from the Ri plasmid and the subsequent steps of generating the hairy root phenotype⁷. A. rhizogenes-mediated transgenic hairy roots, as a powerful tool in the field of plant biotechnology, have been most widely used owing to their stable and high productivity and easy obtainment in a short period. Moreover, hairy roots induced by A. rhizogenes are efficiently distinguished by their plagiotropic root development and highly branching growth in a hormone-free medium⁸. They can be used in several fields of research, including artificial seed production, root nodule research, and in studying the interactions with other organisms such as mycorrhizal fungi, nematodes, and root pathogens^7,9. In addition, hairy root transformation cultures have been extensively used as an experimental system to investigate the biochemical pathways and chemical signaling and to produce plant secondary metabolites that are used as pharmaceuticals, cosmetics, and food additives^8,10. The valuable secondary metabolites, including indole alkaloids, aconites, tropane alkaloids, terpenoids, and flavonoids, synthesized in wild-type hairy roots have been investigated for several decades in numerous species, such as ginsenoside in Panax ginseng¹¹, coumarine in Ammi majus¹², and phenolic compounds in TB^2,13.

Hairy roots have been produced using A. rhizogenes in 79 plant species from 27 families¹⁴. For instance, A. rhizogenes-mediated hairy root transformation has been reported in soybean^15,16, Salvia¹⁷, Plumbago indica¹⁸, Lotus japonicus¹⁹, and chicory (Cichorium intybus L.)²⁰. TB hairy root transformation has also been investigated². Few detailed protocols are available regarding the development of hairy roots mediated by A. rhizogenes either carrying a binary vector or not. For instance, Sandra et al.²¹ introduced a method of producing transgenic potato hairy roots sustained in wild-type shoots. The fully developed hairy roots could be visualized 5-6 weeks after the injection of A. rhizogenes carrying the gus reporter gene into the stem internodes of potato plants. Another study had also reported a transgenic hairy root system induced by A. rhizogenes harboring the gusA reporter gene in jute (Corchorus capsularis L.)²². Furthermore, Supaart et al.²³ obtained transgenic tobacco hairy roots using A. rhizogenes transformed with the expression vector pBI121 carrying the gene of Δ¹-tetrahydrocannabinolic acid (THCA) synthase to produce THCA.

However, a step-by-step process for an effective generation of hairy root transformation, especially in TB, has been relatively less demonstrated. In this study, we have described a detailed protocol using A. rhizogenes carrying the reporter gene (GFP), a selective marker (Kan), and a gene of interest (b4, an identified from our group but unpublished gene from basic helix-loop-helix (bHLH) family) to generate hairy root genetic transformation in TB. The experiment lasted for 5-6 weeks, from the inoculation of seeds to generation of hairy roots, involving the explant preparation, infection, coculturing, subculturing, and subsequent propagation. Furthermore, A. rhizogenes containing a binary plasmid carrying the TB transgene of myeloblastosis transcription factor 116 (FtMYB116) was used to determine whether FtMYB116 can promote accumulation of flavonoids, particularly rutin, in TB at the gene and metabolic level through the TB hairy root transformation. FtMYB116, which is a light-induced transcriptional factor, regulates the synthesis of rutin under different light conditions⁵. Chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), flavonoid-3'-hydroxylase (F3'H), and flavonol synthase (FLS)²⁴ are key enzymes involved in the metabolic pathway of rutin biosynthesis. Therefore, this study demonstrates the overexpression of FtMYB116 in TB hairy roots and the expression of key enzyme genes as well as the content of rutin and other flavonoids such as quercetin.

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 operations were performed under aseptic conditions in a laminar flow hood. All the media ingredients for A. rhizogenes and in vitro plant cultures are provided in Table 1. After adjusting the pH, all media were autoclaved at 120 °C for 20 min. Solidified media were prepared by filling 25 mL of medium into a Petri dish of 9-cm diameter and allowing it to solidify.

CAUTION: Deposit all the genetically modified bacteria and plants into the appropriate waste container. Operate all hazardous chemicals in a fume cupboard and deposit them in the hazardous waste container.

1. Preparation of TB explants

1. Preparation of TB seeds
   1. Select plump and undamaged TB seeds (Figure 1A1) that have been preserved at a temperature less than−20 °C for no more than 2 years.
   2. Soak the seeds in water at 28 °C for approximately 20 min so that the seed coat can be easily peeled off (Figure 1A2). If necessary, use scissors to cut a slot on the seeds to facilitate the peeling.
2. Sterilization of TB seeds
   1. Place 100–200 peeled seeds into a 100 mL sterilized conical flask.
   2. Disinfect the seeds using 75% ethanol for 30 s.
   3. Replace the ethanol with 5% sodium hypochlorite and disinfect for 15 min.
      NOTE: Disinfection using mercury bichloride at a concentration of 1 g/L for 8 min may be used as an alternative sterilizer to replace sodium hypochlorite in any case of inadequate sterilization.
      CAUTION: Mercury bichloride is hazardous and not an environment-friendly material. Operate it in the fuming cupboard and deposit it into the hazardous waste container, if mercury bichloride is used in any case.
   4. Pour out the sodium hypochlorite.
   5. Wash the seeds using sterile deionized water 5 times.
   6. Blot the seeds dry with a sterile bibulous paper.
3. Preparation of TB seedlings
   1. Prepare 50 mL Murashige and Skoog (MS) basal medium (1962) supplemented with 30 g/L sucrose and 7 g/L agar powder (MSSA) (Table 1) in a 300 mL plant tissue culture bottle.
   2. Adjust the pH to 5.8 before autoclaving.
   3. Distribute 10 seeds evenly per bottle of MSSA medium.
   4. Germinate the seeds in a culture room at 25 °C ± 1 °C under the light condition for 7–10 days.
4. Preparation of sterile explants
   1. Select robust seedlings of TB (Figure 1C), when the 2 pieces of cotyledons are unfolded.
   2. Cut off the seedlings from the roots (Figure 1C red-dash arrowheads), avoiding contact with the medium.
   3. Place them in a sterile Petri dish.
   4. Cut the hypocotyls into 0.8–1 cm segments, and shear the cotyledons into approximately 0.5 cm pieces.
   5. Preculture these explants on MSSA medium under the light condition for 24 h.
   6. Transfer them into a 100 mL sterilized conical flask, which are now ready for infection.

2. Preparation of A. rhizogenes for transformation

NOTE: The A. rhizogenes strain ACCC10060 was kindly provided by the Institute of Medicinal Plant Development and preserved at −80 °C. A. rhizogenes was transformed with the binary vector pK7GWIWG2D (II) that harbors a T-DNA carrying the b4 gene accompanying a GFP as an indicator gene and the Kan resistance gene as a selectable marker. The gene b4 is a member of the transcription factor bHLH family, which has not yet been published. To evaluate the potential of TB hairy roots, A. rhizogenes was transformed with the binary vector pK7WG2D containing the MYB116 gene to investigate its effect on the production of secondary metabolites such as flavonoids at the level of gene expression and by metabolic analyses. Activated A. rhizogenes should be well prepared at the same time with the explants.

1. Activation of A. rhizogenes
   1. Thaw A. rhizogenes on ice
   2. Dip the bacteria and line them evenly onto yeast mannitol medium (YEB) supplemented with 15 g/L agar powder, 50 mg/L rifampicin, and 50 mg/L spectinomycin (YEBARS, pH 7.0).
   3. Incubate the bacteria at 28 °C for 12–16 h.
   4. Pick a monoclonal colony and culture it in another Petri dish in the same above-described manner.
   5. Select monoclonal colonies and culture them in a 100 mL sterilized conical flask containing 20 mL of YEB medium supplemented with 50 mg/L rifampicin and 50 mg/L spectinomycin (YEBRS, pH 7.0) at 28 °C and 200 rpm for 16–18 h until the OD₆₀₀ value reaches 2.0.
   6. Incubate 2%–4% of the abovementioned culture in another 100-mL conical flask containing 20 mL of YEBRS medium at 28 °C and 200 rpm for 4-5 h until the OD₆₀₀ value reaches approximately 0.5 (Figure 1D).

3. Infection and screening of TB explants

NOTE: The objective of this protocol is to obtain genetically transformed hairy roots. The wild-type roots were used as the negative control to assess the transgenic expression. In this protocol, A. rhizogenes was transformed with binary vector either pK7WG2D carrying the gene of FtMYB116 or pK7GWIWG2D (II) carrying the gene of b4 in advance.

1. Resuspension of A. rhizogenes
   1. Transfer the culture obtained in step 2.1.6 into a 50-mL sterilized centrifugal tube.
   2. Spin at 4,000 x g for 10 min at 20 °C.
   3. Remove the supernatant and resuspend the bacterial pellet with MS medium supplemented with 30 g/L sucrose and 300 μM acetosyringone (AS) (MSSAS, pH 5.8) to OD₆₀₀ ≈ 0.2.
2. Infection of explants
   1. Infuse the bacterial suspension obtained in step 3.1.3 into a conical flask containing the explants prepared in step 1.4.6 for 10 min (Figure 1E).
   2. Take out the explants, and blot them dry using a sterile bibulous paper.

4. Coculture of explants with A. rhizogenes

1. Place a sterile 9 cm diameter filter paper on the MS medium, which is solidified using 7 g/L agar powder supplemented with 30 g/L sucrose and 100 μM AS (MSSAAS medium, pH 5.2).
2. Overlay the explants on the filter paper at 25 °C for 3 days in the dark (Figure 1F).

5. Induction and selective culture

1. Place approximately 20 infected explants on the MSSA medium supplemented with 500 mg/L cefotaxime and 50 mg/L kanamycin (Kan) (MSSACK, pH 5.8) (Figure 1G).
2. Incubate them vertically under the light condition at 25 °C ± 1 °C. The hairy roots occur approximately 1 week after incubation (Figure 1H black-dash arrowheads indicate occurrence of hairy roots).
   NOTE: Replace MSSACK medium every 15 days if necessary.

6. Subculturing TB hairy roots

NOTE: This procedure aims to harvest vigorous hairy roots. Regularly observe the growth of hairy roots during propagation, and remove the contaminated and inactivated ones in a timely manner. If necessary, repeat the following steps to propagate more hairy roots. It takes approximately 10–14 days from subculturing to harvest.

1. Select the hairy roots showing white appearance and rapid growth.
2. Cut them into pieces of 2-3 cm.
3. Clearly number them on a clean bench.
4. Subculture them in a 100 mL sterilized conical flask containing 5 mL of MS medium supplemented with 30 g/L sucrose and 50 mg/L Kan (MSSK, pH 5.8) at a rotary speed of 80 rpm at 25 °C in the dark until they overspread to the bottom of the flask (Figure 1I).

7. Identification of transformed hairy roots and conservation

NOTE: Transformed hairy roots can be identified based on the aspects of morphology and gene level. Identification can also be conducted according to the hairy root genome and resistance, which are not covered in this protocol. This procedure primarily focuses on reporter gene and target gene identification.

1. Remove tawny and contaminated hairy roots and select those with white appearance.
2. Evaluate if there is green fluorescence under a blue/light dual ultraviolet transilluminator.
3. Select the hairy roots exhibiting a strong fluorescence signal in the numbered tubes or wrapped using a marked tinfoil after drying them out with an absorbent paper.
4. Lyophilize them in liquid nitrogen, followed by storing all the harvest at −80 °C for further investigation.
5. Gene identification
   1. Triturate 0.1 g of the hairy roots into fine powder in liquid nitrogen.
   2. Prepare the genomic DNA of independent transgenic lines of TB using the modified cetyltrimethylammonium bromide (CTAB) method²⁵ according to the instruction of the manufacturer of the plant genomic DNA kit.
   3. Perform polymerase chain reaction (PCR) using 100 ng of genomic DNA template and primers listed in Table 2.
   4. Perform the amplification cycle as follows: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, and primer extension at 72 °C for 30 s. After 36 cycles and a final extension step at 72 °C for 10 min, analyze the amplification products on 1% agarose gels.
   5. Stain the gels with nucleic acid staining and visualize them under UV light.

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

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