We thank Lic. Jes&#250;s Fuentes Gonz&#225;lez and N&#233;stor Iv&#225;n Robles Olivares for their assistance in filming the video footage. We acknowledge the generous gifts by Dr. Antonia Gutierrez Mora of CIATEJ (Theobroma cacao plants). We also thank CIATEJ and Laboratorio Nacional PlanTECC, M&#233;xico, for facility support. H.E.H.D. (CVU: 1135375) conducted master studies with funding from the Consejo Nacional de Humanidades, Ciencia y Tecnolog&#237;a, M&#233;xico (CONAHCYT). R.U.L. acknowledges support from Consejo Estatal de Ciencia y Tecnolog&#237;a de Jalisco (COECYTJAL), and Secretar&#237;a de Innovaci&#243;n Ciencia y Tecnolog&#237;a (SICYT), Jalisco, M&#233;xico (Grant 7270-2018).

1. Agrobacterium tumefaciens culture
<ol>
	<li>Thaw electrocompetent cells of Agrobacterium tumefaciens strain LBA4404.</li>
	<li>Add 1 mL of Yeast Malt (YM; Table 1) broth to a 17 mm x 100 mm culture tube. Save this tube for later, and keep it at room temperature (RT).</li>
	<li>In a 1.5 mL microfuge tube, add 30 µL of the thawed Agrobacterium cells and 100-250 ng (up to 5 µL) of the DNA containing 35S:RUBY. Mix gently. 
	NOTE: The 35S:RUBY was a gift from Yunde Zhao. To avoid sample arcing, reduce the presence of ionic compounds as much as possible. These ionic compounds may be residual salts from the ethanol precipitation of DNA19.</li>
	<li>At this point, place a 1 mm electroporation cuvette on ice.</li>
	<li>Transfer the previous suspension mix to a chilled 1 mm electroporation cuvette. Keep everything on ice. Wipe the metallic electrodes of the cuvette.</li>
	<li>Set the electroporator to Agr (2.2 kV, ~5 ms, 1 pulse). Place the cuvette inside the electroporation chamber.</li>
	<li>Press the Pulse button. Register the pulse parameters. If the sample arced, the electroporation process failed. 
	NOTE: It is critical to quickly transfer the cells to the YM broth right after the pulse. Delaying this transfer can dramatically reduce the transformation efficiency20.</li>
	<li>Immediately, use the saved YM broth to transfer the cells from the cuvette to the 17 mm x 100 mm tube. Resuspend the cells gently.</li>
	<li>Incubate the transformed cells for 3 h at 28 °C and 250 rpm on an orbital incubator. 
	NOTE: This culture does not have antibiotics; be cautious about proper aseptic conditions.</li>
	<li>Streak this culture onto selective YM agar plates21. For the transformed LBA4404-RUBY strain, ensure these plates contain rifampicin (25 µg/mL), spectinomycin (50 µg/mL), and streptomycin (50 µg/mL). Incubate this plate overnight in a 28 °C standing incubator. 
	NOTE: In this protocol, the vector 35S:RUBY was used, which confers bacterial resistance to spectinomycin (50 µg/mL) and functions as a visual reporter on infiltrated plant tissue.</li>
	<li>Inoculate colonies from the overnight culture on 12.5 mL of a mixture of YM broth and Luria Bertani (LB) broth (in a 9:1 proportion, respectively), 10 mM of MES, pH 5.722. Ensure that this selective liquid medium contains the same antibiotics used in step 1.10. Refer to Table 1 to see the ingredients and concentrations for these mediums.
	<ol>
		<li>When incubating Agrobacterium, leave enough aeration space for the culture, about 4 to 5 times the liquid volume. Use YM broth for Agrobacterium strain LBA4404 to avoid cell clumping23.</li>
	</ol>
	</li>
	<li>Incubate the culture for 16 h at 250 rpm on a 28 °C orbital incubator.</li>
	<li>Scale the culture up to 10 times the initial volume with the same medium used in step 1.11.</li>
	<li>Incubate the culture for 16 h and 250 rpm on a 28 °C orbital incubator.</li>
	<li>Adjust the overnight culture to an optical density (OD600) of 0.4. Add 20 µM of acetosyringone (AS).</li>
	<li>Incubate at 250 rpm on a 28 °C orbital incubator until OD600 reaches about 1.0.</li>
	<li>Centrifuge the cells at 4 500 x g for 10 min at 20 °C.</li>
	<li>Resuspend the pellet with suspension solution (10 mM MES, 10 mM MgCl2, pH 5.7), adjusting OD600 to 0.6. Add 200 µM AS24. 
	NOTE: Pre-incubate the suspension solution at 28 °C. If the suspension solution is cold when added, the cells will precipitate.</li>
	<li>Leave the bacterial suspension for 2-24 h in dark conditions and 25 °C. Agitation is not required22.</li>
</ol>
2. Plant selection
<ol>
	<li>Choose a plant with a branch with leaves in the optimal stage for agroinfiltration. 
	NOTE: The plant may be full-grown or a mature tree. For cacao, young leaves of C stage are recommended. These leaves are bronze to light green colored; they are not fully expanded nor as rigid as stage D leaves25(Figure 1).
	<ol>
		<li>As a control, simultaneously perform agroinfiltration on other plants (e.g., Nicotiana tabacum) with a high agroinfiltration efficiency that is reported for the strain and vector used. 
		NOTE: If no positive results are obtained in this control, it is possible that the negative results are due to the strain or the vector used.</li>
	</ol>
	</li>
</ol>
3. Vacuum chamber setup
<ol>
	<li>As a vacuum chamber, use a desiccator that has a vacuum gauge to measure the vacuum pressure inside.</li>
	<li>Add 250 µM of Jasmonic acid (JA)18,26 to the Agrobacterium suspension from step 1.19.</li>
	<li>Transfer the Agrobacterium culture to a wide-mouth beaker to submerge the selected branch and leaves. Then, place the beaker with Agrobacterium culture inside the desiccator.</li>
	<li>Place the branch between the desiccator and its lid. Make sure to submerge the desired leaves inside the Agrobacterium culture. Next, use a jump ring, which is a round ring with a cutout that allows the plant branch to enter the desiccator. The Jump Ring also acts as a spacer between the bottom and top of the Desiccator.</li>
	<li>Ensure that the gasket is structurally stable enough to avoid being squished with the lid, flexible enough to bend and adjust to the circumference of the desiccator, and not made of a porous material. 
	NOTE: This study used a stranded metal wire consisting of several smaller wires twisted together, coated with a nonporous plastic material resembling a gasket (Figure 2).</li>
	<li>To fix the branch onto the desiccator, use silicone impression material. Ensure that the material is sticky, nonporous, chemically inert to the desiccator and the plant, and easy to apply and fill small gaps between the branch, the gasket, and the desiccator.</li>
	<li>Once the silicone impression material polymerizes (this takes about 1 min) and fixes up the branch in place, close the desiccator. Make sure not to leave any gaps.</li>
	<li>Connect the desiccator to the vacuum pump (Figure 3).</li>
</ol>
4. Vacuum infiltration
<ol>
	<li>Start the vacuum pump until it gets to -0.07 MPa.</li>
	<li>Once it reaches this pressure, close the pressure valve and turn off the vacuum pump. Maintain this pressure for 5 min.</li>
	<li>Open the pressure valve to restore the chamber pressure. 
	NOTE: This is a critical step. Restore the chamber pressure gradually and steadily. It may take up to 3 min to fully repressurize the desiccator. Extended re-pressurization increases the number of bacteria infiltrated inside the tissue10.</li>
	<li>Repeat this process two more times.</li>
	<li>Take the branch off the cell suspension and the desiccator.</li>
	<li>Clean the infiltrated leaves with distilled water.</li>
</ol>
5. Incubation of infiltrated leaves
<ol>
	<li>Let the infiltrated leaves stay in dark conditions at 25 °C for 48 h.</li>
	<li>Then, expose the infiltrated tissue to a 16/8-h light/dark photoperiod.</li>
	<li>Evaluate the transient leaf transformation 3-7 days post-infection (DPI).</li>
</ol>
<img alt="Leaf growth stages diagram; leaf A-E variation; scale 10 cm; botany research; plant development." class="xfigimg" src="/files/ftp_upload/66024/66024fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Cacao leaves developmental stages. (A-E) Developmental stages25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Vacuum desiccator diagram; pressure gauge, lid, seal, valve, chamber setup; moisture removal process." class="xfigimg" src="/files/ftp_upload/66024/66024fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Vacuum chamber configuration and its components. The vacuum chamber is a desiccator connected to a vacuum gauge. The gasket/ O-ring is cut so it has an opening where the branch will be placed. (A) Vacuum gauge, (B) Lid, (C) Gasket/ O-ring, (D) Pressure valve, (E) Desiccator, (F) Hose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plant transpiration experiment diagram with vacuum pump setup for water loss measurement." class="xfigimg" src="/files/ftp_upload/66024/66024fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: In planta vacuum agroinfiltration system. To avoid vacuum losses during the infiltration process, it is critical to secure the branch to the desiccator and the gasket/O-ring with silicone impression material. (A) Cacao plant, (B) Vacuum chamber, (C) Silicone impression material, (D) Leaves submerged on Agrobacterium suspension, (E) Vacuum pump. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

In Planta Transient Transformation of Cacao Plants Using Agrobacterium tumefaciens

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Authors have no conflict of interest to declare.

In this work, we presented an efficient, low-cost agroinfiltration protocol for the in planta transient transformation of woody plants, using cacao plants as an example. Given the well-known constraint that the cuticle of leaves represents for the transformation of plant tissues, we concentrated on developing a strategy to facilitate agroinfiltration by vacuum in woody plants, which are usually recalcitrant to this procedure.
The achieved vacuum pressure inside the vacuum chamber was only possible due to the efficient sealing and filling of the small gaps created between the desiccator, the tree branch, the gasket/O-ring, and the desiccator&#39;s lid, which was the main challenge of the protocol. For this reason, it is critical to use silicone impression material to fix the branch onto the desiccator. This material is nonporous, proves to be sticky enough to adhere the branch to the desiccator and the gasket, and is chemically inert, so neither the infiltrated branch nor the desiccator is damaged.
In addition, it is necessary to use a vacuum pump to achieve a pressure gradient inside the leaf section, causing air to be sucked out of the stomatal cavity and mesophyll regions, then a large pressure gradient during repressurization pushes the Agrobacterium suspension into the leaf through stomata and wounds10. We used a freeze dryer as a vacuum pump. This is an example of the versatility and adaptability of this protocol to every laboratory&#39;s needs. Using the freeze dryer as a vacuum pump, it took about 3 min to reach -0.07 MPa. This is effective for cacao since previous authors have also vacuum infiltrated cacao detached leaves with the same vacuum pressure25. This is the preferred infiltration method since it is more repeatable than non-assisted or needleless syringe infiltration (Supplementary Figure 1). When using a vacuum pump for vacuum infiltration, the user can control the pressure and time that the tissues are exposed to the vacuum10. 
In other plants, like poplar, it has been studied that leaf structure may affect infiltration efficiency due to the arrangement of the mesophyll cells. The different and variable arrangements of these cells may result in an uneven infiltration by the Agrobacterium suspension8,27 as the interior structure of the leaf has intercellular air gaps8. Although spontaneous infiltration through the stomata of the leaves may happen when submerging the leaves in an Agrobacterium suspension, atmospheric pressure is not effective enough to overcome the epidermal cuticle and displace these air spaces with the bacterial suspension; this has been tried with other recalcitrant species such as Persea americana18. For this reason, forced vacuum-mediated agroinfiltration proves to be an efficient method to facilitate substance penetration into the leaves through the stomata or wounds10. Inoculation of young cacao seedlings with the badnavirus cacao swollen shoot Ghana M virus infectious clone by biolistic inoculation and agroinoculation has been recently reported28. In cacao, most transient transformation assays are performed on detached leaves25,29, resulting in reduced transient expression upkeep due to the plant material degradation. To our knowledge, there are no reports of in planta transient genetic transformation on cacao plants. Here, we achieved the first transient transformation of cacao, and previously, our same study group achieved this in Persea americana18, making this a viable methodology for possible localized in vivo analysis of metabolites or molecules that are only expressed on leaves.
Transient transformation with the 35S:RUBY vector was confirmed by the transient expression of betalains on the cacao leaves. This vector reconstitutes the betalain biosynthetic pathway on plants that do not naturally produce these pigments. Only some Caryophyllales can naturally biosynthesize betalains from tyrosine with their own metabolic pathways30. To replicate this metabolism, 35S:RUBY contains the genetic information for the heterologous expression of three enzymes that convert the commonly present amino acid tyrosine into betalains7.
In planta transient transformation protocols have many advantages over stable transformation protocols, including faster, cost-effective, and enhanced transformation efficiency31. Moreover, in planta transient transformation may be applied for many functional genomics analysis32. Functional genomics studies on cacao aim to solve the disease susceptibility of this species to many pathogens, such as Moniliophthora perniciosa and Phytophthora spp33, increase their drought resistance34, and improve its organoleptic qualities35. This protocol has the potential to improve and deepen cacao functional genomics studies since it makes in planta transient transformation possible on large-sized cacao trees or even in other large recalcitrant and perennial plant species.

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.

<table><tbody><tr><td>&lt;em&gt;35S:RUBY&lt;/em&gt; plasmid</td><td>Addgene</td><td>160908</td><td>http://n2t.net/addgene:160908 ; RRID:Addgene_160908</td></tr><tr><td>1 mm electroporation cuvette</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>FB101</td><td>Fisherbrand&amp;nbsp;Electroporation Cuvettes Plus</td></tr><tr><td>Desiccator</td><td>Bel-Art SP SCIENCEWARWE</td><td>F42400-2121</td><td></td></tr><tr><td>Freeze dryer</td><td>LABCONCO</td><td>700402040</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>P8281-500G</td><td>For YM medium add 0.38 g/L</td></tr><tr><td>LBA4404 ElectroCompetent Agrobacterium</td><td>Intact Genomics USA</td><td>1285-12</td><td>https://intactgenomics.com/product/lba4404-electrocompetent-agrobacterium/</td></tr><tr><td>Mannitol</td><td>Sigma Aldrich</td><td>63560-250G-F</td><td>For YM medium add 10 g/L</td></tr><tr><td>MES</td><td>Sigma Aldrich</td><td>PHG0003</td><td>(For LB, YM and resuspension medium) add 1.95 g/L (10mM)</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266</td><td>For resuspension medium add 0.952 g/L (10 mM)</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Sigma Aldrich</td><td>63138-1KG</td><td>For YM medium add 0.204 g/L</td></tr><tr><td>MicroPulser Electroporation Apparatus</td><td>Biorad&amp;nbsp;</td><td>165-2100</td><td></td></tr><tr><td>NaCl</td><td>Karal</td><td>60552</td><td>For LB medium add 5 g/L; For YM medium add 0.1 g/L</td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>13-400-518</td><td></td></tr><tr><td>President Silicone Impression material</td><td>COLTENE</td><td>60019938</td><td></td></tr><tr><td>Rifampicin&amp;nbsp;</td><td>Gold-Bio</td><td>R-120-1</td><td>(100 mg/mL)</td></tr><tr><td>Silicone Impression material gun</td><td>Andent</td><td>TBT06</td><td></td></tr><tr><td>Spectinomycin</td><td>Gold-Bio</td><td>S-140-SL10</td><td>(100 mg/mL)</td></tr><tr><td>Streptomycin</td><td>Gold-Bio</td><td>S-150-SL10</td><td>(100 mg/mL)</td></tr><tr><td>Tryptone enzymatic digest from casein</td><td>Sigma Aldrich</td><td>95039-1KG-F</td><td>For LB medium add 10 g/L</td></tr><tr><td>Yeast extract</td><td>MCD LAB</td><td>9031</td><td>For LB medium add 5 g/L; For YM medium add 0.4 g/L</td></tr></tbody></table>

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 rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

This protocol presents an effective agroinfiltration method for large-sized woody plants. With this protocol, we were able to achieve a vacuum pressure of -0.07 MPa, resulting in the effective, localized infiltration of cacao leaves. In Figure 4, we observe the infiltration system setting up process, and in Figure 5, the final configuration.
<img alt="Leaf gas exchange analysis using infrared gas analyzer; plant physiology experiment setup." class="xfigimg" src="/files/ftp_upload/66024/66024fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Vacuum chamber setup. (A) Notice the branch placement between the two ends of the gasket. This gasket is large enough to allow the branch to pass through between the desiccator and its lid. Observe that there are gaps formed due to the irregular surface of the branch. (B) The branch is fixed onto the desiccator, and the formed gaps are filled with the silicone impression material. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plant transpiration experiment setup with desiccator and pot, analyzing water vapor pressure." class="xfigimg" src="/files/ftp_upload/66024/66024fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Vacuum infiltration system setup. Example of the setup for the vacuum infiltration system for a large-sized cacao plant using the proposed protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
During the first stage of agroinfiltration, the tissue is subjected to reduced pressure, which causes gases to be released from the stomatal cavities and adjacent mesophyll air spaces through the stomata and through the wounds. Small bubbles on the surface of the leaves immersed in the Agrobacterium suspension can be seen with the naked eye as the vacuum increases. The next step is to repressurize the tissues and force the Agrobacterium suspension inside to fill the void left by the air11. Infiltrated leaves appear darker in color in some areas (Figure 6). This indicates that the Agrobacterium suspension has penetrated the tissue and spread throughout, filling the intercellular spaces of the leaf8.
<img alt="Plant disease identification, leaf chlorosis, diagram, botanical research, disease diagnosis." class="xfigimg" src="/files/ftp_upload/66024/66024fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Abaxial view of adult cacao leaves after completion of vacuum agroinfiltration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol achieved agroinfiltration on localized leaves of large-sized cacao plants. By doing so, we transiently transformed large cacao plants with a vector carrying 35S:RUBY as a reporter system. We found this reporter system a useful indicator of successful transient transformation in cacao because the accumulation of betalains in the leaves indicates the efficiency of the transformation. Betalains are bright red pigments, producing an unmistakable signal on the green leaves that can be observed with the naked eye (Figure 7). Since the leaves are attached to the plant, betalains may accumulate if the infiltrated tissue maintains its viability. Figure 8 shows a series of results, varying from a high to a low betalain accumulation on transformed cacao leaves of stage C. It is worth mentioning that this is the first in planta transient transformation reported on this species.
<img alt="Leaf disease analysis; close-up diagram highlighting veins and lesions; plant pathology study." class="xfigimg" src="/files/ftp_upload/66024/66024fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Cacao leaves transiently transformed to overexpress 35S: RUBY. Red spots on cacao leaves resulting from betalain accumulation are a simple and quick way to evaluate the proposed protocol of localized vacuum-based agroinfiltration in planta. Image at 6 DPI, scale bar: 1 cm, optical zoom: 8x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Leaf microscopy showing disease spots; sequential close-ups; plant pathology analysis." class="xfigimg" src="/files/ftp_upload/66024/66024fig08.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Betalain accumulation on cacao leaves. Different levels and patterns of betalain accumulation on cacao leaves at 6 DPI by Agrobacterium-mediated transient transformation with the RUBY reporter. Scale bar: 1 cm, Optical zoom: 8x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/66024fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>LB Medium</td>
			<td>YM Medium</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>5 g/L</td>
			<td>0.4 g/L</td>
		</tr>
		<tr>
			<td>Tryptone enzymatic digest from casein</td>
			<td>10 g/L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 g/L</td>
			<td>0.1 g/L</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td></td>
			<td>10 g/L</td>
		</tr>
		<tr>
			<td>MgSO4·7h20</td>
			<td></td>
			<td> 0.204 g/L</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td></td>
			<td>0.38 g/L</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>1.95 g/L (10 mM)</td>
			<td>1.95 g/L (10 mM)</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Composition of YM and LB mediums.
Supplementary Figure 1: Representative image of forced agroinfiltration on cacao leaves using a needle-less syringe. Syringe infiltration did not result in RUBY expression in the leaves visible to the naked eye but did result in damage at the point where pressure was applied with the needleless syringe. Image at 6 DPI, scale bar: 5 mm, optical zoom: 25x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66024/Supplemental%20Figure%201.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study at JoVE.com

Author Spotlight: Tissue Culture-Free Agrobacterium-Mediated Transformation in Large Woody Plants

Here, we present the first protocol for localized vacuum infiltration for in vivo studies of the genetic transformation of large-sized plants. Using this methodology, we achieved for the first time the Agrobacterium-mediated in planta transient transformation of cacao.

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 ...

vacuum-forced-agroinfiltration-for-planta-transformation-recalcitrant

Nanyang Technological University

Research

JoVE Journal

Biology

2.3K Views.  Centro de Investigación Y Asistencia en Tecnología Y Diseño del Estado de Jalisco (CIATEJ). In vivo genetische methoden zijn nodig om de biologische functie van genen in tropische vaste planten en tuinbouwgewassen te bestuderen. Veel van deze soorten zijn echter in elke generatie recalcitrant voor transformatie. Het doel van dit onderzoek is het ontwikkelen van een eenvoudige en efficiënte genetische transformatiemethode die toepasbaar is op grote planten.Ik zal de dingen illustreren. Agrobacterium-gemedieerde genetische transformatie van planten met moeilijk te infiltreren ...

Video: Auteur in de schijnwerpers: Weefselkweekvrije agrobacterium-gemedieerde transformatie in grote houtachtige planten

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

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

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