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

Agrobacterium tumefaciens를 사용한 카카오 식물의 Plan 일시적 변형

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.

저자 스포트라이트: 대형 목본 식물의 조직 배양이 필요 없는 아그로박테리움 매개 형질전환

난치성 식물의 식물 변형 을 위한 진공 강제 농업 여과: 사례 연구로서의 카카오

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.

형질전환된 Agrobacterium tumefaciens 의 스케일업 배양 및 대형 목본 식물의 아그로인 침투

먼저, 줄무늬는 Agrobacterium tumefaciens 세포 배양을 선택적 항생제 보충 효모 만니톨 한천 플레이트로 변환합니다. 스탠딩 인큐베이터에서 섭씨 28도에서 밤새 플레이트를 배양합니다. 다음날 배양액에서 콜로니를 12.5ml의 효모 만니톨 Luria-Bertani 육수 혼합물에 접종하고 섭씨 28도의 궤도 셰이커에서 250RPM으로 16시간 동안 배양합니다.112.5ml의 효모 만니톨 국물에 12.5ml의 Agrobacterium 균주 LBA4404를 항생제와 함께 접종합니다. 섭씨 28도의 궤도 셰이커에서 250RPM으로 16시간 동안 배양액을 배양합니다. 배양액을 사용하여 야간 배양액을 광학 밀도 0.4로 조정합니다.그런 다음 배양 전에 20 마이크로 몰의 아세토시린곤을 첨가하십시오. 배양된 배양액이 광학 밀도 1에 도달하면 원심분리하여 세포를 펠릿화합니다. 현탁액에 펠릿을 0.6의 광학 밀도로 재현탁시키고 배양 전에 200마이크로몰 아세토시링곤을 첨가합니다.아그로인스탈리즘의 경우, 잎이 있는 가지가 있는 목본 카카오 식물을 선택하십시오. 250 마이크로몰 자스몬산을 아그로박테리움 현탁액에 첨가합니다. 다음으로 진공 게이지가 있는 데시케이터를 진공 챔버로 설정하여 내부 압력을 측정합니다.식물 가지가 통과할 수 있도록 점프 링을 놓습니다. 아그로박테리움 배양액이 담긴 비커를 데시케이터 안에 넣고 원하는 잎이 아그로박테리움 배양액에 잠기도록 데시케이터와 뚜껑 사이에 가지를 놓습니다. 다음으로 실리콘 인상재를 사용하여 점프 링, 식물 가지 및 데시케이터 사이의 틈을 밀봉합니다.실리콘 재료가 중합되면 데시케이터를 닫고 틈을 남기지 않은 다음 진공 펌프에 연결합니다. 마이너스 0.07메가파스칼에 도달할 때까지 진공 펌프를 시작합니다. 원하는 압력에 도달하면 압력 밸브를 닫고 펌프를 꺼서 5분 동안 압력을 유지합니다.압력 밸브를 점차적으로 꾸준히 열어 챔버 압력을 복원합니다. 침투를 두 번 더 반복한 후 세포 현탁액에서 가지를 떼어냅니다. 침투한 잎을 증류수로 세척한 다음 식물을 섭씨 25도의 어두운 곳에 48시간 동안 둡니다.배양 후 침윤된 조직을 16시간의 밝은 사진 기간과 8시간의 어두운 사진 기간에 노출시킵니다. 식물의 침투 된 잎은 특정 부분에서 더 어둡게 나타나며 Agrobacterium 침투를 나타냅니다. 사용된 리포터 시스템은 잎에 베타린 축적을 생성했으며, 이는 육안으로 밝은 적색 색소 침착으로 쉽게 관찰할 수 있습니다.베타린 축적은 조직 생존력에 따라 달라지는 것으로 관찰되었습니다.

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 ...

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