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

Transformação transitória de plantas de cacau em plantas de cacau usando Agrobacterium tumefaciens

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.

Autor em destaque: Transformação mediada por agrobacterium livre de cultura de tecidos em grandes plantas lenhosas

Agroinfiltração Forçada a Vácuo para Transformação In planta de Plantas Recalcitrantes: Cacau como Estudo de Caso

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.

Ampliação da Cultura de Agrobacterium tumefaciens Transformado e Agroinfiltração de Plantas Lenhosas de Grande Porte

Para começar, a cultura de células de Agrobacterium tumefaciens em placas de ágar de levedura manitol suplementadas com antibióticos seletivos. Incube as placas durante a noite a 28 graus Celsius em uma incubadora em pé. No dia seguinte, inocular colônias da cultura em 12,5 mililitros de mistura de caldo de levedura Mannitol Luria-Bertani e incubar em um agitador orbital a 28 graus Celsius por 16 horas a 250 RPM.Inocular 12,5 mililitros da cepa Agrobacterium LBA4404 em 112,5 mililitros de caldo de Levedura Manitol com antibióticos. Incubar a cultura em um agitador orbital a 28 graus Celsius por 16 horas a 250 RPM. Ajustar a cultura noturna para uma densidade óptica de 0,4 com o caldo de cultura.Em seguida, adicione 20 micromolares de acetoseringona antes da incubação. Uma vez que a cultura incubada atinja uma densidade óptica de um, centrifugar para pellet as células. Ressuspender o pellet em solução de suspensão até uma densidade óptica de 0,6 e adicionar 200 acetosiringonas micromolares antes da incubação.Para a agroinfiltração, colher plantas lenhosas de cacau que tenham galhos com folhas. Adicionar ácido jasmônico 250 micromolares à suspensão de Agrobacterium. Em seguida, configure um exsicador com um medidor de vácuo como uma câmara de vácuo para medir a pressão interna.Coloque um anel de salto para permitir a passagem do galho da planta. Coloque o copo com cultura de Agrobacterium dentro do dessecador, e coloque o galho entre o exsicador e sua tampa, com as folhas desejadas submersas na cultura de Agrobacterium. Em seguida, use material de impressão de silicone para selar as lacunas entre o anel de salto, o ramo da planta e o dessecador.Uma vez que o material de silicone tenha polimerizado, feche o dessecador, não deixando lacunas, e conecte-o à bomba de vácuo. Ligue a bomba de vácuo até atingir menos 0,07 megapascals. Quando a pressão desejada for atingida, feche a válvula de pressão e desligue a bomba para manter a pressão por cinco minutos.Abra a válvula de pressão de forma gradual e constante para restaurar a pressão da câmara. Depois de repetir a infiltração mais duas vezes, retire o ramo da suspensão celular. Limpe as folhas infiltradas com água destilada e, em seguida, coloque as plantas a 25 graus Celsius no escuro por 48 horas.Após a incubação, expor o tecido infiltrado a um período de fotos claras de 16 horas e oito horas de escuro. As folhas infiltradas das plantas aparecem mais escuras em certas partes, indicando a penetração de Agrobacterium. O sistema repórter usado produziu acúmulo de betalaína nas folhas, que pode ser facilmente observada como pigmentação vermelha brilhante a olho nu.Observou-se que o acúmulo de betalaína varia com a viabilidade tecidual.

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 ...

Biologia