Name: Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation
Uploaded: 2023-06-30T14:20:00
Description: Watch this Scientific Journal Video about Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation at JoVE.com

production of composite plants by agrobacterium rhizogenes-based hairy root transformation

뿌리 관련 생물학 발전을 위한 One-Step Hairy Root Transformation 방법

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 system3,4,5 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 microorganisms6,7,8.
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,&#160;and many other plants5,8. 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,&#160;and tomato2,14,15,16,17. 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&#160;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.

저자 스포트라이트: Agrobacterium rhizogenes-Mediated Hairy Root Transformation을 통한 복합 식물 생산 간소화 

Agrobacterium rhizogenes 기반 털이 많은 뿌리 형질전환에 의한 복합 식물 생산을 위한 효율적이고 재현 가능한 방법

Our team focuses on Agrobacterium rhizogenes-based hairy root transformation. Hairy root transformation is crucial in studying plant gene function, metabolic engineering, and interactions between roots and Rhizosphere microorganisms. This article provides a one-step transformation method, mediated by Agrobacterium rhizogenes.Hairy root transformation is established in various dicotyledons and monocotyledons species, and is almost independent with the genotype. The one-step method mediated by Agrobacterium rhizogenes for inducing hairy roots is simpler and more efficient for creating composite plants compared to the hypocotyl injection method. The hypocotyl injection method is inefficient and is likely to cause the death of young or tiny hypocotyl plants.The one-step Agrobacterium rhizogenes-mediated method is highly efficient and requires less time to produce hairy roots. Moreover, there is no need for transplanting past the formation of hairy roots. Alpha-sign regulation genes and beta-sign synthesis genes were used as viral screen markers in hairy root transformation mediated by Agrobacterium rhizogenes.

Watch this Scientific Journal Video about Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation at JoVE.com

Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation  

Agrobacterium rhizogenes 기반 털이 많은 뿌리 형질전환에 의한 복합 식물 생산

야생 콩, S.Americanum 및 현지 품종의 호박의 씨앗을 수집하는 것으로 시작하십시오. 1 센티미터의 깊이에 질석에 씨앗을 뿌리고 완전히 물을주십시오. 약 70 %의 상대 습도에서 성장 챔버에서 재배하십시오.섭씨 80도 냉동고에서 A.rhizogenes 균주 K599를 꺼내 섭씨 28도의 고체 LB 배지에서 48시간 동안 활성화합니다. 그런 다음 균주 K599의 단일 클론을 선택하고 LB 배지를 함유한 1밀리리터의 액체 항생제에서 12시간 동안 배양합니다. 500 마이크로 리터의 박테리아 현탁액을 LB 배지를 함유 한 고체 항생제에 골고루 뿌리고 섭씨 28도에서 24 시간 동안 배양합니다.7 일 후, 묘목 자엽이 펼쳐지면 멸균 된 날카로운 메스를 사용하여 배축의 약 0.5-1 센티미터를 자릅니다. 기본 뿌리와 배축의 일부를 버리십시오. 배축 절개 부위를 K599 박테리아로 코팅합니다.축축한 질석에 묘목을 심고 A.rhizogenes 매개 털이 많은 뿌리 변형을 사용하여 각 종의 30개 식물을 감염시킵니다. 그런 다음 5 밀리리터의 K599 박테리아 현탁액을 4 분의 1 강도의 Gamborg B5 기본 배지에 재현 탁하여 식물에 물을줍니다. 냄비를 투명한 비닐 봉지로 덮고 성장 챔버에 넣으십시오.접종 후 약 12 일 동안 식물을 재배하십시오. 이때 절개 부위에 새로운 털이 많은 뿌리가 생성되어야 하며 일반적으로 길이가 2-5cm에 이릅니다. 540 나노 미터에서 녹색 여기 빛과 600 나노 미터에서 방출되는 CAMI 발광 이미징 시스템을 사용하여 리포터 유전자 DsRed2의 발현을 검출합니다.야생 콩, S.Americanum 및 호박의 복합 식물의 뿌리가 자연 및 녹색 여기광 아래에서 시각화됩니다.

production-composite-plants-agrobacterium-rhizogenes-based-hairy-root

Research

JoVE Journal

Biology

Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

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

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&#160;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.

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

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

연구

생물학

Begin by collecting seeds of wild soybean, S.Americanum, and the local variety of pumpkin. Sow the seeds in vermiculite at a depth of one centimeter and water them thoroughly. Cultivate them in a growth chamber at approximately 70%relative humidity.Remove the A.rhizogenes strain K599 from the 80 degrees Celsius freezer and activate it on a solid LB medium at 28 degrees Celsius for 48 hours. Then select a single clone of strain K599 and culture it in one milliliter of liquid antibiotic containing LB medium for 12 hours. Spread 500 microliters of the bacterial suspension evenly on a solid antibiotic containing LB medium and incubate at 28 degrees Celsius for 24 hours.After seven days, when seedling cotyledons are unfolded, use a sterilized sharp scalpel to cut approximately 0.5 to one centimeter of the hypocotyl. Discard the primary root and a portion of the hypocotyl. Coat the hypocotyl incision with K599 bacteria.Plant seedlings in moist vermiculite and infect 30 plants of each species using A.rhizogenes-mediated hairy root transformation. Then water the plants with five milliliters of resuspended K599 bacterial suspension in quarter-strength Gamborg B5 basic medium. Cover the pots with a transparent plastic bag and place them in a growth chamber.Grow the plants for approximately 12 days post-inoculation. At this time, new hairy roots should be generated at the incision site, typically reaching lengths of two to five centimeters. Using a CAMI luminescence imaging system with green excitation light at 540 nanometers and emission at 600 nanometers, detect the expression of the reporter gene DsRed2.Roots of composite plants of wild soybean, S.Americanum, and pumpkin under natural and green excitation light are visualized.