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

In Planta transiente Transformation von Kakaopflanzen mit 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 im Rampenlicht: Gewebekulturfreie Agrobakterien-vermittelte Transformation in großen Gehölzen

Vakuumforcierte Agroinfiltration zur In-planta-Transformation widerspenstiger Pflanzen: Kakao als Fallstudie

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-Kultur von transformiertem Agrobacterium tumefaciens und Agroinfiltration von großformatigen Gehölzen

Zu Beginn transformieren Sie die Zellkultur von Agrobacterium tumefaciens auf selektive, mit Antibiotika ergänzte Hefe-Mannitol-Agar-Platten. Inkubieren Sie die Platten über Nacht bei 28 Grad Celsius in einem stehenden Inkubator. Am nächsten Tag die Kolonien aus der Kultur mit 12,5 Millilitern Hefe-Mannitol-Luria-Bertani-Brühe impfen und 16 Stunden lang bei 250 U/min auf einem Orbitalschüttler bei 28 Grad Celsius inkubieren.12,5 Milliliter des Agrobacterium-Stammes LBA4404 in 112,5 Milliliter Hefe-Mannitol-Brühe mit Antibiotika impfen. Inkubieren Sie die Kultur auf einem Orbitalschüttler bei 28 Grad Celsius für 16 Stunden bei 250 U/min. Stellen Sie die Übernachtkultur mit der Kulturbrühe auf eine optische Dichte von 0,4 ein.Fügen Sie dann vor der Inkubation 20 Mikromolaren Acetosyringon hinzu. Sobald die inkubierte Kultur eine optische Dichte von eins erreicht hat, zentrifugieren Sie, um die Zellen zu pelletieren. Resuspendieren Sie das Pellet in Suspensionslösung auf eine optische Dichte von 0,6 und fügen Sie vor der Inkubation 200 mikromolares Acetosyringon hinzu.Für die Agroinfiltration pflücken Sie holzige Kakaopflanzen mit Zweigen mit Blättern. Geben Sie 250 mikromolare Jasmonsäure in die Agrobacterium-Suspension. Als nächstes richten Sie einen Exsikkator mit einem Vakuummeter als Vakuumkammer ein, um den Druck im Inneren zu messen.Legen Sie einen Biegering an, damit der Pflanzenzweig hindurchpasst. Legen Sie das Becherglas mit der Agrobacterium-Kultur in den Exsikkator und legen Sie den Zweig zwischen den Exsikkator und seinen Deckel, wobei die gewünschten Blätter in die Agrobacterium-Kultur getaucht werden. Verwenden Sie als Nächstes Silikonabformmaterial, um die Lücken zwischen Biegering, Pflanzenzweig und Exsikkator abzudichten.Sobald das Silikonmaterial polymerisiert ist, schließen Sie den Exsikkator lückenlos und schließen Sie ihn dann an die Vakuumpumpe an. Starten Sie die Vakuumpumpe, bis sie minus 0,07 Megapascal erreicht. Wenn der gewünschte Druck erreicht ist, schließen Sie das Druckventil und schalten Sie die Pumpe aus, um den Druck fünf Minuten lang aufrechtzuerhalten.Öffnen Sie das Druckventil allmählich und gleichmäßig, um den Kammerdruck wiederherzustellen. Nachdem Sie die Infiltration noch zweimal wiederholt haben, nehmen Sie den Zweig von der Zellsuspension. Reinigen Sie die infiltrierten Blätter mit destilliertem Wasser und stellen Sie die Pflanzen dann 48 Stunden lang bei 25 Grad Celsius in die Dunkelheit.Setzen Sie das infiltrierte Gewebe nach der Inkubation einer 16-stündigen hellen und acht Stunden dunklen Fotoperiode aus. Infiltrierte Blätter der Pflanzen erscheinen an bestimmten Stellen dunkler, was auf das Eindringen von Agrobacterium hinweist. Das verwendete Reportersystem erzeugte eine Betalain-Anreicherung auf den Blättern, die mit bloßem Auge leicht als leuchtend rote Pigmentierung beobachtet werden kann.Es wurde beobachtet, dass die Betalain-Akkumulation mit der Lebensfähigkeit des Gewebes variiert.

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 ...

Forschung

Biologie