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

根関連生物学の進歩のためのワンステップ毛根形質転換法

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.

著者スポットライト:Agrobacterium rhizogenesを介した複合植物生産の合理化-媒介された毛深い根の形質転換 

アグロバクテリウム・リゾゲネスに基づく毛状根形質転換による効率的かつ再現性の高い複合植物の生産法

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  

アグロバクテリウム根粒菌に基づく毛状根形質転換による複合植物の生産

野生の大豆、S.Americanum、および地元の種類のカボチャの種子を集めることから始めます。1センチメートルの深さでバーミキュライトに種をまき、徹底的にそれらに水をまきます。相対湿度約70%の成長チャンバーで栽培します。A.rhizogenes K599株を摂氏80度の冷凍庫から取り出し、摂氏28度の固体LB培地で48時間活性化します。次に、K599株の単一クローンを選択し、LB培地を含む1ミリリットルの液体抗生物質で12時間培養します。500マイクロリットルの細菌懸濁液をLB培地を含む固体抗生物質に均等に広げ、摂氏28度で24時間インキュベートします。7日後、苗の子葉が広げられたら、滅菌した鋭いメスを使って胚軸の約0.5〜1センチメートルを切ります。一次根と胚軸の一部を捨てます。胚軸切開部をK599細菌でコーティングします。湿ったバーミキュライトに苗を植え、A.rhizogenesを介した毛状根の形質転換を使用して、各種の30の植物に感染します。次に、4分の1強度のガンボーB5基本培地に5ミリリットルの再懸濁したK599細菌懸濁液で植物に水をやります。鉢を透明なビニール袋で覆い、成長室に入れます。接種後約12日間植物を育てます。この時点で、新しい毛状の根が切開部位に生成され、通常は2〜5センチメートルの長さに達するはずです。540ナノメートルの緑色励起光と600ナノメートルの発光を備えたCAMI発光イメージングシステムを使用して、レポーター遺伝子DsRed2の発現を検出します。野生ダイズ、S.americanum、カボチャの複合植物の根を自然励起光と緑色励起光の下で可視化します。

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Research

JoVE Journal

Biology

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

536 ビュー.  Liaocheng University. 野生の大豆、S.Americanum、および地元の種類のカボチャの種子を集めることから始めます。1センチメートルの深さでバーミキュライトに種をまき、徹底的にそれらに水をまきます。相対湿度約70%の成長チャンバーで栽培します。A.rhizogenes K599株を摂氏80度の冷凍庫から取り出し、摂氏28度の固体LB培地で48時間活性化します。次に、K599株の単一クローンを選択し、LB培?...

ビデオ: 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 ...

生物学

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.