An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Summary

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Abstract

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Introduction

Agrobacterium rhizogenes is a gram-negative soil bacterium from the family Rhizobiaceae. A. rhizogenes can infect almost all dicotyledons, a few monocotyledons, and individual gymnosperms through wounds, producing hairy roots in infected plants. The bacterium carries the Ri (root-inducing) plasmid, and the T-DNA of the Ri plasmid carries the opine synthesis gene and rol genes (root locus genes). After the T-DNA of the Ri plasmid enters a plant cell and is integrated into a host chromosome, the expression of the rol genes induces production of hairy roots¹. A plant binary expression vector carrying a target gene is transformed into A. rhizogenes, and the transformed A. rhizogenes is used to infect a plant. Transgenic roots can be induced in infected plants, producing composite plants containing transgenic roots and nontransgenic stems and buds. Generally, a composite plant can be obtained within 14-20 days. A. rhizogenes-mediated hairy root transformation is generally not limited by genotype in dicotyledonous plants². The hairy roots produced by A. rhizogenes-infected plants are characterized by a fast growth rate, stable inheritance, and easy operation. Hairy root transformation mediated by A. rhizogenes is currently widely used to study root-related biology. Furthermore, the transformation of hairy roots can also be used to validate and optimize the target-editing efficiency of the CRISPR/Cas9 system^3,4,5 and protein subcellular localization. Therefore, hairy root transformation is an important tool in research on plant gene function, metabolic engineering, and interactions between roots and rhizosphere microorganisms^6,7,8.

Composite plants containing transgenic roots obtained through hairy root transformation have been widely produced in dicotyledonous plants, especially in legumes. The traditional method of injecting the hypocotyl with A. rhizogenes has been used to produce composite Lotus corniculatus⁹, soybeans¹⁰, tomato¹¹, sweet potatoes¹², and many other plants^5,8. The hypocotyl-injection method is inefficient and is likely to cause the death of young or tiny hypocotyl plants. Therefore, the method was improved by cutting off the embryonic roots, coating the seedling incision with A. rhizogenes, and then placing the hypocotyl on sterile culture medium for rooting cultivation¹³. However, those steps are performed in a sterile environment, and the operation steps are relatively cumbersome. In particular, the resulting composite plants need to be transplanted, which increases the amount of work. In previous work, one-step A. rhizogenes-mediated (ARM) hairy root transformation was established in cucumber, soybean, Lotus japonicus, Medicago truncatula, and tomato^{2,14,15,16,17}. The primary root and partial hypocotyl were removed, the incision site of the remaining hypocotyl was coated with transformed A. rhizogenes, and the seedling was then planted into moist sterile vermiculite. After 12 days of cultivation, hairy roots were produced at the incision site. The one-step ARM method is highly efficient and requires less time to produce hairy roots. Transplanting after forming hairy roots is also not necessary. Because microbial contamination can be avoided without transplantation, the one-step ARM method can be particularly useful when studying interactions between plants and microorganisms, such as symbiotic nitrogen fixation between leguminous plants and Rhizobia, and symbioses between plants and arbuscular mycorrhizal fungi. In this paper, a detailed one-step A. rhizogenes-mediated hairy root transformation protocol is provided with examples of composite plants produced in wild soybean, Solanum americanum, and pumpkin. With the protocol, researchers can smoothly perform the one-step ARM transformation.

Protocol

1. Plant growth conditions and A. rhizogenes culture

1. Seed sowing
   NOTE: Wild soybean seeds were collected in Yanggu County, Liaocheng, China; seeds of S. americanum and pumpkin local variety Yinsu were purchased from a market.
   1. Collect seeds of wild soybean, S. americanum, and the local variety of pumpkin, sow them in vermiculite at a depth of 1 cm, and thoroughly water them. Plant 20 seeds in 8 cm x 11 cm x 9 cm plastic boxes. Cultivate the plants in a growth chamber at 24 ± 2 °C with a 16 h light/8 h dark cycle at approximately 70% relative humidity.
      NOTE: Coats of wild soybean seeds must be broken before sowing. If the research question focuses on interactions between plants and microorganisms, seeds, vermiculite, plastic boxes, and water must be sterilized before use.
   2. Activation and culture of A. rhizogenes K599
      NOTE: The A. rhizogenes K599 strain had one binary vector pRed1305² carrying a red fluorescent reporter gene DsRed2.
      1. Remove the A. rhizogenes strain K599 from a -80 °C freezer, and activate the bacteria on solid LB medium (with 50 mg/L kanamycin and 50 mg/L streptomycin) at 28 °C for 48 h.
      2. Pick out a single clone of strain K599 and culture it in 1 mL of liquid antibiotic-containing LB medium for 12 h.
      3. Evenly spread 500 µL of the bacterial suspension on solid antibiotic-containing LB medium, followed by incubation at 28 °C for 24 h.

2. One-step A. rhizogenes-mediated hairy root transformation method

1. Hypocotyl incision
   1. After 7 days (define the sowing of the seeds as day 0), seedling cotyledons have just unfolded (Figure 1A), use a sterilized, sharp scalpel to cut approximately 0.5-1 cm of the hypocotyl (Figure 1B). Discard the primary root and some of the partial hypocotyl.
      NOTE: Use the scalpel with care.
2. K599 inoculation
   1. Coat the hypocotyl incision with K599 bacteria (Figure 1C).
   2. Plant the seedlings in moist vermiculite (Figure 1D).
   3. Infect 30 plants of each species via A. rhizogenes-mediated hairy root transformation. Water each plant with 5 mL of resuspended K599 bacterial suspension (OD₆₀₀ = 0.5-0.6) in a quarter strength (0.25x) Gamborg B-5 basic medium (Figure 1E).
   4. Cover the pots with a highly transparent plastic bag (Figure 1F) and place them in a growth chamber.

3. Hairy root production

1. Grow the plants for ~12 days post inoculation, new hairy roots will be induced and generated at the incision site. After 15 days, the hairy root lengths typically reach 2-5 cm (Figure 2).
2. To determine whether the hairy roots produced are transgenic, examine the expression of reporter genes dependent on the transformed vector in K599.
   1. Detect the expression of the reporter gene DsRed2 using a chemiluminescence imaging system with green excitation light at 540 nm and emission at 600 nm (Figure 2B, Figure 2D, and Figure 2F).

Representative Results

Highly efficient one-step A. rhizogenes-mediated hairy root transformation
Hairy roots were produced at the hypocotyl incision site 12 days after inoculation with engineered K599. Transgenic hairy roots were determined based on the expression of the reporter gene contained in the binary vector. Transgenic roots transformed with the reporter gene DsRed2 of composite wild soybean, S. americanum, and pumpkin were observed under natural (Figure 2A, Figure 2C, and Figure 2E) and green excitation light (Figure 2B, Figure 2D, and Figure 2F).

When a composite plant contained at least one transgenic root, it was designated a transgenic composite plant. Among the 30 inoculated plants of each of the three species, 28 wild soybean, 18 S. Americanum, and 30 pumpkin plants were transgenic composites. Thus, the hairy root transformation efficiency was 93.3% (soybean), 60% (S. americanum), and 100% (pumpkin). A comparison of the three types of plants indicated that plants with thick hypocotyls produced more transgenic hairy roots than those with thin hypocotyls.

Figure 1: One-step A. rhizogenes-mediated hairy root transformation. (A) Seven-day-old pumpkin seedlings. (B) Apical portion of hypocotyl cut in K599 bacterial solution. (C) K599 bacterial mass coating the hypocotyl incision. (D) Explant planted in wet, sterile vermiculite. (E) Watering with 5 mL of resuspended K599 bacterial solution in quarter strength (0.25x) Gamborg B-5 basic medium. (F) Highly transparent plastic bag covering. Scale bars = 1 cm. Please click here to view a larger version of this figure.

Figure 2: Composite plants obtained from one-step A. rhizogenes-mediated hairy root transformation. Roots of composite plants of (A,B) wild soybean, (C,D) Solanum americanum, and (E,F) pumpkin under (A,C,E) natural and (B,D,F) green excitation light. White arrows indicate transgenic hairy roots; black arrows indicate nontransgenic hairy roots. Scale bars = 1 cm. Please click here to view a larger version of this figure.

Discussion

The one-step A. rhizogenes-mediated hairy root method is a simpler and more efficient method for producing composite plants than the hypocotyl-injection method. The one-step ARM method significantly improves the efficiency of hairy root transformation, shortens the time to produce hairy roots, increases the number of hairy roots, and reduces the amount of work involved. The improved transformation protocol is optimal for studies on symbioses between leguminous plants and rhizobia and between plants and arbuscular mycorrhizal fungi. This can be attributed to the fact that transplantation of composite plants after the production of hairy roots is not required, which avoids the contamination with noninoculated strains that occurs during transplanting. Furthermore, the transformation efficiency was 100% in one plant species (pumpkin).

The following reasons might explain why the one-step ARM method was more efficient than the hypocotyl-injection method in hairy root transformation. First, although the primary root was removed, seedling transpiration was maintained. Thus, transpiration pull facilitated A. rhizogenes invasion of hypocotyl cells in the incision. Second, the wound area caused by the hypocotyl incision was larger than that caused by the hypocotyl-injection method, and therefore, more plant cells were infected by A. rhizogenes. Last, after removing the primary root, the hypocotyl incision was buried in dark and moist vermiculite, which is a favorable environment in which to produce roots¹⁸.

Materials

kanamycin	Sangon Biotech (Shanghai) Co., Ltd.	A506636
LB medium	Sangon Biotech (Shanghai) Co., Ltd.	B540113
plastic box	LiaoSu		8 cm x 11 cm x 9 cm
pumpkin			local variety Yinsu
streptomycin	Sangon Biotech (Shanghai) Co., Ltd.	A610494
Tanon-5200Multi machine	Tanon Co., Ltd., China	5200Multi	chemiluminescence imaging system
tomato			local variety Zhongshu4
wild soybean			collected in Yanggu County, Liaocheng, China