scale up culture of transformed agrobacterium tumefaciens and agroinfiltration of large-sized woody plants

Agrobacterium tumefaciensを用いたカカオ植物のプランタ一過性形質転換

Plant genetic transformation methods are essential for testing the biological functions of genes and are especially useful today given the large number of uncharacterized genes predicted in the post-genomic era1. These methods can be used to obtain fully transformed lines or to express genes transiently. Stable transformation occurs when the foreign DNA the host has taken up becomes fully and irreversibly integrated into the host genome, and the genetic modifications are passed down to subsequent generations. Transient expression, known as transient transformation, occurs from the multiple copies of T-DNA transferred by Agrobacterium into the cell, which have not been integrated into the host genome, and peaks 2-4 days post infection2. 
It is worth noting that transient expression assays are often sufficient for the functional characterization of genes and can offer several advantages over stable transformation. For example, transient transformation does not require tissue culture-based regeneration procedures. Another advantage is that it is compatible with in planta functional analysis of genes, existing several successful examples of protocols well standardized for model plant species, such as Arabidopsis thaliana3 and Nicotiana benthamiana4, but still limited in non-model species5.
The development of transient assays relies on the availability of efficient gene transfer methods. For this, the most popular approaches are based on Agrobacterium infiltration, which takes advantage of Agrobacterium's unique ability to transfer DNA to plant cells6. Another useful tool for these analyses is the use of reporter genes, such as green fluorescent proteins (GFP), &#946;-glucuronidase (GUS), luciferase, or RUBY, all of which are employed to track transformation events. Among these reporter systems, RUBY is currently the easiest to visualize and relies on the conversion of tyrosine into betalains through three enzymatic step reactions. As opposed to other reporter systems, the resultant betalains can be readily observed as brightly colored pigments on transformed plant tissue without the need for sophisticated equipment or additional reactants7.
When infiltrating an Agrobacterium suspension into the intercellular space of the leaf mesophyll, the most critical step for successful agroinfection is overcoming the physical barrier imposed by the epidermal cuticle of the leaves8. While for some plants, a pressure gradient created with a needle-less syringe (syringe Agroinfiltration) is enough for an efficient agroinfiltration, as occurs in Nicotiana benthamiana9, other plant species may require a larger pressure gradient such as the one created with the help of vacuum pumps10. In vacuum-assisted processes, agroinfiltration occurs in two steps. In the first one, vacuum serves to subject the plant material to reduced pressure, forcing the release of gases from the mesophyll air spaces through stomata and wounds. Then, during a repressurization phase, the Agrobacterium suspension infiltrates the intercellular spaces via the stomata and wounds11. 
Compared to syringe infiltration, vacuum infiltration allows for higher usage frequency, repeatability, and the ability to control pressure and duration at every stage of the infiltration process10. In leaves of different plant species such as spinach (Spinacia oleracea)12, peony (a woody perennial) (Paeonia ostii)13, and Cowpea (Vigna unguiculata)14, vacuum agroinfiltration protocols achieved a deeper infiltration rate than syringe infiltration. Similarly, in tomato (Lycopersicon esculentum)15, and gerbera (Gerbera hybrida)16, vacuum agroinfiltration produced stronger and more uniform gene silencing than syringe infiltration. An additional advantage of vacuum infiltration is the lower dependence on genotype, compared to syringe infiltration, which was observed recently in three citrus varieties (Fortunella obovata, Citrus limon, and C. grandis)17. However, when trying to apply vacuum agroinfiltration to plants that are too large to fit into desiccators, the size of the vacuum chambers can be a limitation, as typically occurs with tropical woody plants.
Below, we describe a protocol that overcomes the spatial limitation of vacuum chambers, testing its utility for in planta transient transformation of cacao leaves. We present the first localized vacuum infiltration method for cacao, which does not require additional equipment and even allows the use of the same laboratory desiccators used for the infiltration of the whole plant, but with a simple adaptation that allows the access of a part of the plant inside the vacuum chamber, allowing its use at different stages of plant development. To test the usefulness of the localized vacuum infiltration method proposed, we selected cacao as a proxy of a large-leaved tropical plant species that is difficult to transform. Using this localized infiltration method, we recently reported the first in planta transient expression in avocado by Agrobacterium-mediated vacuum infiltration with conditions previously optimized for detached leaves18, and here we report the first in planta transient expression in cacao.

著者スポットライト:大型木本植物における組織培養フリー のアグロバクテリウム媒介形質転換

難治性植物の プランタ内 変態のための真空強制農業浸透:ケーススタディとしてのカカオ

In vivo genetic methods are required to study the biological function of genes in tropical perennial species and horticultural tree crops. However, many of these species are recalcitrant to transformation in every generation. The scope of this research is to develop a simple and efficient genetic transformation method applicable to large-sized plants.I will illustrate the things. Agrobacterium-mediated genetic transformation of plants with difficult to infiltrate tissues has been made possible by forced infiltration using a vacuum pump. However, the protocols currently in use are limited to small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum chamber.The size of the vacuum chambers, which are usually laboratory dessicators, prevents the vacuum infiltration of plants that are too large to fit into dessicators, as is the case of tropical woody plants. We have developed a protocol for efficient localized vacuum infiltration of tropical woody plants, making in vivo genetic transformation studies of large plant species possible. This protocol allowed us to overcome plant size limitations for Agrobacterium-mediated vacuum infiltration in horticultural tree crops such as cacao and avocado.Tissue culture-free Agrobacterium-mediated transformation of attached leaves by localized vacuum infiltration can be performed in a rapid, straightforward, and cost-effective manner by laboratories that already use vacuum infiltration, since the same pump and vacuum vessel can be used with no need to add costly equipment.

形質転換 アグロバクテリウム・ツメファシエンスの スケールアップ培養と大型木本植物のアグロインフィルトレーション

まず、Agrobacterium tumefaciens細胞培養液を選択的抗生物質添加酵母マンニトール寒天培地にストリーク形質転換します。プレートを常設インキュベーターで摂氏28度で一晩インキュベートします。翌日、12.5ミリリットルの酵母マンニトールルリア-ベルタニブロスミックスの培養物からコロニーを接種し、28°Cのオービタルシェーカー上で250RPMで16時間インキュベートします。112.5ミリリットルの酵母マンニトールブロスに12.5ミリリットルのアグロバクテリウム株LBA4404抗生物質を接種します。培養液を摂氏28度のオービタルシェーカーで250RPMで16時間インキュベートします。培養液で一晩培養液を光学濃度0.4に調整します。次に、インキュベーション前に20マイクロモルのアセトシリンゴンを加えます。インキュベートした培養物が光学濃度1に達したら、遠心分離して細胞をペレット化します。ペレットを懸濁液に光学濃度0.6に再懸濁し、インキュベーション前に200マイクロモルのアセトシリンゴンを添加します。農業浸透のために、葉のある枝を持つ木質カカオ植物を選びます。アグロバクテリウム懸濁液に250マイクロモルのジャスモン酸を加えます。次に、真空チャンバーとして真空計付きのデシケーターを設置し、内部の圧力を測定します。植物の枝が通るようにジャンプリングを配置します。アグロバクテリウム培養液の入ったビーカーをデシケーター内に置き、デシケーターとその蓋の間に枝を置き、目的の葉をアグロバクテリウム培養液に浸します。次に、シリコーン印象材を使用して、ジャンプリング、植物枝、およびデシケーターの間の隙間を塞ぎます。シリコーン材料が重合したら、隙間を残さずにデシケーターを閉じ、真空ポンプに接続します。マイナス0.07メガパスカルに達するまで真空ポンプを始動します。希望の圧力に達したら、圧力バルブを閉じ、ポンプをオフにして圧力を5分間維持します。圧力バルブを徐々に着実に開いて、チャンバーの圧力を回復します。さらに2回浸潤を繰り返した後、細胞懸濁液から枝を取り除きます。浸潤した葉を蒸留水できれいにし、植物を摂氏25度で暗所に48時間置きます。インキュベーション後、浸潤した組織を16時間の明色および8時間の暗色写真期間に曝露します。植物の浸透した葉は、特定の部分で暗く見え、アグロバクテリウムの浸透を示しています。レポーターシステムは、葉にベタレインを蓄積させ、肉眼で真っ赤な色素沈着として容易に観察することができます。ベタレインの蓄積は、組織の生存率によって異なることが観察されました。.

Research

JoVE Journal

Biology

Scale Up Culture of Transformed Agrobacterium tumefaciens and Agroinfiltration of Large-Sized Woody Plants

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation ...

生物学

形質転換 アグロバクテリウム・ツメファシエンスの スケールアップ培養と大型木本植物のアグロインフィルトレーション

形質転換アグロバクテリウム・ツメファシエンスのスケールアップ培養と大型木本植物のアグロインフィルトレーション