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

Summary

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.

Abstract

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.

Introduction

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), β-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.

Protocol

1. Agrobacterium tumefaciens culture

1. Thaw electrocompetent cells of Agrobacterium tumefaciens strain LBA4404.
2. 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).
3. 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.
4. At this point, place a 1 mm electroporation cuvette on ice.
5. Transfer the previous suspension mix to a chilled 1 mm electroporation cuvette. Keep everything on ice. Wipe the metallic electrodes of the cuvette.
6. Set the electroporator to Agr (2.2 kV, ~5 ms, 1 pulse). Place the cuvette inside the electroporation chamber.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
    1. 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.
12. Incubate the culture for 16 h at 250 rpm on a 28 °C orbital incubator.
13. Scale the culture up to 10 times the initial volume with the same medium used in step 1.11.
14. Incubate the culture for 16 h and 250 rpm on a 28 °C orbital incubator.
15. Adjust the overnight culture to an optical density (OD₆₀₀) of 0.4. Add 20 µM of acetosyringone (AS).
16. Incubate at 250 rpm on a 28 °C orbital incubator until OD₆₀₀ reaches about 1.0.
17. Centrifuge the cells at 4 500 x g for 10 min at 20 °C.
18. 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.
19. Leave the bacterial suspension for 2-24 h in dark conditions and 25 °C. Agitation is not required²².

2. Plant selection

1. 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).
   1. 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.

3. Vacuum chamber setup

1. As a vacuum chamber, use a desiccator that has a vacuum gauge to measure the vacuum pressure inside.
2. Add 250 µM of Jasmonic acid (JA)^18,26 to the Agrobacterium suspension from step 1.19.
3. 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.
4. 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.
5. 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).
6. 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.
7. 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.
8. Connect the desiccator to the vacuum pump (Figure 3).

4. Vacuum infiltration

1. Start the vacuum pump until it gets to -0.07 MPa.
2. Once it reaches this pressure, close the pressure valve and turn off the vacuum pump. Maintain this pressure for 5 min.
3. 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.
4. Repeat this process two more times.
5. Take the branch off the cell suspension and the desiccator.
6. Clean the infiltrated leaves with distilled water.

5. Incubation of infiltrated leaves

1. Let the infiltrated leaves stay in dark conditions at 25 °C for 48 h.
2. Then, expose the infiltrated tissue to a 16/8-h light/dark photoperiod.
3. Evaluate the transient leaf transformation 3-7 days post-infection (DPI).

Figure 1: Cacao leaves developmental stages. (A-E) Developmental stages²⁵. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Representative Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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.

Figure 6: Abaxial view of adult cacao leaves after completion of vacuum agroinfiltration. Please click here to view a larger version of this figure.

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

	LB Medium	YM Medium
Yeast extract	5 g/L	0.4 g/L
Tryptone enzymatic digest from casein	10 g/L
NaCl	5 g/L	0.1 g/L
Mannitol		10 g/L
MgSO4·7h20		0.204 g/L
K2HPO4		0.38 g/L
MES	1.95 g/L (10 mM)	1.95 g/L (10 mM)
MgCl2

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. Please click here to download this File.

Discussion

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'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'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 suspension^8,27 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 leaves^25,29, 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.

Materials

Name	Company	Catalog Number	Comments
35S:RUBY plasmid	Addgene	160908	http://n2t.net/addgene:160908 ; RRID:Addgene_160908
1 mm electroporation cuvette	Thermo Fisher Scientific	FB101	Fisherbrand Electroporation Cuvettes Plus
Desiccator	Bel-Art SP SCIENCEWARWE	F42400-2121
Freeze dryer	LABCONCO	700402040
K2HPO4	Sigma Aldrich	P8281-500G	For YM medium add 0.38 g/L
LBA4404 ElectroCompetent Agrobacterium	Intact Genomics USA	1285-12	https://intactgenomics.com/product/lba4404-electrocompetent-agrobacterium/
Mannitol	Sigma Aldrich	63560-250G-F	For YM medium add 10 g/L
MES	Sigma Aldrich	PHG0003	(For LB, YM and resuspension medium) add 1.95 g/L (10mM)
MgCl2	Sigma Aldrich	M8266	For resuspension medium add 0.952 g/L (10 mM)
MgSO4·7H20	Sigma Aldrich	63138-1KG	For YM medium add 0.204 g/L
MicroPulser Electroporation Apparatus	Biorad	165-2100
NaCl	Karal	60552	For LB medium add 5 g/L; For YM medium add 0.1 g/L
NanoDrop One Microvolume UV-Vis Spectrophotometer	Thermo Fisher Scientific	13-400-518
President Silicone Impression material	COLTENE	60019938
Rifampicin	Gold-Bio	R-120-1	(100 mg/mL)
Silicone Impression material gun	Andent	TBT06
Spectinomycin	Gold-Bio	S-140-SL10	(100 mg/mL)
Streptomycin	Gold-Bio	S-150-SL10	(100 mg/mL)
Tryptone enzymatic digest from casein	Sigma Aldrich	95039-1KG-F	For LB medium add 10 g/L
Yeast extract	MCD LAB	9031	For LB medium add 5 g/L; For YM medium add 0.4 g/L