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.
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.
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.
1. Agrobacterium tumefaciens culture
2. Plant selection
3. Vacuum chamber setup
4. Vacuum infiltration
5. Incubation of infiltrated leaves
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.
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.
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 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.
The authors have nothing to disclose.
We thank Lic. Jesús Fuentes González and Néstor Iván 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, México, for facility support. H.E.H.D. (CVU: 1135375) conducted master studies with funding from the Consejo Nacional de Humanidades, Ciencia y Tecnología, México (CONAHCYT). R.U.L. acknowledges support from Consejo Estatal de Ciencia y Tecnología de Jalisco (COECYTJAL), and Secretaría de Innovación Ciencia y Tecnología (SICYT), Jalisco, México (Grant 7270-2018).
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 |