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

In Planta Transient Transformation of Cacao Plants Using &lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;

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.

Author Spotlight: Tissue Culture-Free &lt;em&gt;Agrobacterium&lt;/em&gt;-Mediated Transformation in Large Woody Plants

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

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.

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

To begin, streak transform Agrobacterium tumefaciens cell culture onto selective antibiotic supplemented Yeast Mannitol Agar plates. Incubate the plates overnight at 28 degrees Celsius in a standing incubator. The next day, inoculate colonies from the culture on 12.5 milliliters of Yeast Mannitol Luria-Bertani broth mix, and incubate on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM.Inoculate 12.5 milliliters of the Agrobacterium strain LBA4404 in 112.5 milliliters of Yeast Mannitol broth with antibiotics. Incubate the culture on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM. Adjust the overnight culture to an optical density of 0.4 with the culture broth.Then add 20 micromolar of acetosyringone before incubation. Once the incubated culture reaches an optical density of one, centrifuge to pellet the cells. Resuspend the pellet in suspension solution to an optical density of 0.6, and add 200 micromolar acetosyringone before incubation.For agroinfiltration, pick woody cacao plants that have branches with leaves. Add 250 micromolar jasmonic acid to the Agrobacterium suspension. Next, set up a desiccator with a vacuum gauge as a vacuum chamber to measure the pressure inside.Place a jump ring to allow the plant branch to pass through. Place the beaker with Agrobacterium culture inside the desiccator, and place the branch between the desiccator and its lid, with the desired leaves submerged in the Agrobacterium culture. Next, use silicone impression material to seal the gaps between the jump ring, the plant branch, and the desiccator.Once the silicone material has polymerized, close the desiccator, leaving no gaps, then connect it to the vacuum pump. Start the vacuum pump until it reaches minus 0.07 megapascals. When the desired pressure is reached, close the pressure valve, and turn off the pump to maintain the pressure for five minutes.Open the pressure valve gradually and steadily to restore the chamber pressure. After repeating infiltration two more times, take the branch off the cell suspension. Clean the infiltrated leaves with distilled water, then place the plants at 25 degrees Celsius in the dark for 48 hours.After incubation, expose the infiltrated tissue to a 16 hour light and eight hour dark photo period. Infiltrated leaves of the plants appear darker in certain parts, indicative of Agrobacterium penetration. The reporter system used produced betalain accumulation on the leaves, which can be easily observed as bright red pigmentation with the naked eye.Betalain accumulation was observed to vary with the tissue viability.

scale-up-culture-transformed-agrobacterium-tumefaciens

Research

JoVE Journal

Biology

91 Görüntüleme.  Centro de Investigación Y Asistencia en Tecnología Y Diseño del Estado de Jalisco (CIATEJ). Başlamak için, çizgi Agrobacterium tumefaciens hücre kültürünü seçici antibiyotik takviyeli Maya Mannitol Agar plakalarına dönüştürür. Plakaları gece boyunca 28 santigrat derecede ayakta duran bir inkübatörde inkübe edin. Ertesi gün, kültürden kolonileri 12.5 mililitre Maya Mannitol Luria-Bertani et suyu karışımı üzerine aşılayın ve 250 RPM'de 16 saat boyunca 28 santigrat derecede bir orbital çalkalayıcı üzerinde inkübe edin.112.5 mililitre Maya Manni...