Context Memo

To: Prof. Kimberly Freeman, COLWRIT 161 Class, College Writing Program
From: Claire Yerby
Date: 17 October 2023
Subject: The Status of and Improvements in Agrobacterium-mediated Transformation of Tomato
(Solanum lycopersicum)

This literature review covers the use of Agrobacterium infection to genetically modify tomato
tissues. This process involves transforming the target tissue by exposing it to the bacteria which
injects the plant with the genetic material designed to be inserted into the tissue. Agrobacterium
is a genus of bacteria that are naturally pathogenic to plants. In nature, they infect plants and
induce tumor growth. These infections cause crown gall disease which can be observed as
large knobby growths on trees. This is achieved by the delivery of Transfer-DNA (T-DNA) into
the host genome where it is then transcribed and translated to induce tumorigenesis. This
mechanism is exploited in vitro to insert a binary vector into the target tissue and induce an
intentional genetic modification.

The Agrobacterium genetic material (plasmid) contains DNA for its metabolic processes as well
as the T-DNA which is only functional when inserted into the host. By inserting a Cas9 gene, for
example, into the T-DNA portion of the plasmid, scientists can make edits at specific loci in the
plant genome. Likewise, inserting a gene encoding for a visual marker such as GFP (green
fluorescent protein) would result in mutations randomly across the plant genome with the
outcome being a plant that glows green. The increased specificity that Cas9 allows for has
opened up the field of genome editing to a new realm of possibility.

Another form of DNA delivery that is briefly mentioned in this literature review is gold-particle
bombardment which involves bombarding the target tissue (explant) with gold particles coated
in DNA. This is not used in tomato, but it is highly utilized in other crops such as wheat and rice.
It is more precise but causes more damage to the explant, so delicate tissues such as tomato
are less likely to regrow from this process.

If I were to submit this to a journal, I would submit it to Frontiers in Genome Editing. This is a
newer journal, but it tends to focus heavily on plant genome editing. This journal utilizes an APA-
like citation style which is used in this literature review.
Abstract

Since the beginning of European exploration of South America, tomato (Solanum lycopersicum)

has been a global agricultural staple. Its agricultural importance and versatility led it to become

the first genetically modified plant to become commercially grown and available. The discovery

and improvement of tools for genetic modification such as CRISPR-Cas9 has expanded the

specificity and precision of gene editing. This allows scientists to more efficiently achieve the

modifications they wish to induce. In this review, I discuss the protocol that is widely used for

genome editing in tomato as well as genes and how this protocol varies with different explants

and experimental outcomes in mind. Unlike many other crops, this protocol for genetic

modification has remained consistent since the advent of genetic modification. Since the

introduction of the use of Cas9 to edit genomes, scientists working with other crops have aimed

to optimize the endonuclease activity to improve protocol efficiency. The prospect of adding

additional treatments to this protocol as has been done in other crops may be appealing to

many researchers aiming to improve tomato editing efficiency.

Introduction

The rapid increase in global climate change has caused a profound impact on agricultural

systems worldwide, necessitating the urgent need for enhanced genome editing efficiency. As a

result, numerous staple crops have emerged as model species for intensive research and

genetic manipulation due to their role in global economies and food security. Among these key

crops, tomato (Solanum lycopersicum) has garnered significance owing to its importance in

global food systems and its ability to be genomically edited. Notably, tomato genome editing has

been a pivotal aspect of commercial crop transformation, marking a milestone by producing the

first commercially available transgenic plant product. Given the role of tomato in both

agricultural and scientific domains, continuous improvements in the efficiency of tomato

transformation have become paramount for advancing the fields of plant transformation and

genomics. By optimizing and refining the transformation methods for this crop, research can

continue to unveil new opportunities for improving crop productivity, enhancing resistance to

impending environmental changes, and ensuring a future of sustainable food production.

Compared to other crops, tomato genome editing has not been as continuously improved due to

its ease of transformation. In addition to heat treatments, other protocols have implemented the

use of hydrolytic enzymes to increase editing efficiency by damaging the target tissue and

allowing the naturally virulent Agrobacterium to attack the tissue (Clement et al., 2016). This

could be utilized in tomato to open up the number of explants that can be used in

transformations. Currently, most of the transformations being done target cotyledon or whole

fruit tissues, and enzymatic treatments may allow for more recalcitrant tissues to be edited.

Bolistic DNA delivery is commonly used to transform recalcitrant tissues, but it is too harsh for

most tomato tissues which are generally more delicate. Scientists achieve organogenesis in

tomato via Agrobacterium-mediated transformation. This method decreases the incidence of

chimeric events (Frary, A. and Van Eck, J., 2005) compared to biolistic DNA delivery.

Agrobacterium-mediated transformation allows for the transfer of large DNA fragments into the

target genome with great accuracy and efficiency (Tanaka et al., 2022). This method was first

outlined by Horsch et al. in 1985 and has proven to be a more efficient method of genome

editing than particle bombardment in non-recalcitrant plant species. The first documentation of

this method being used to transform tomato followed shortly after in 1986 (McCormick et al.,

1986). This method of DNA delivery has been continually improved upon since its initial

introduction to transform a variety of genotypes and increase editing efficiency (Chyi and

Phillips, 1987; Fillatti et al.,1987; Frary and Earle, 1996; Park et al., 2003; Sun et al., 2006; Van

Eck et al., 2006; Gupta and Van Eck, 2016). The advent of CRISPR/Cas9 technology has
allowed for precise genome editing, further increasing the editing capabilities of

Agrobacterium-mediated transformation.

The use of RNA-guided Cas9 has become a largely popular method for editing plant genomes

and has been used in all major crop species. Poddar et al. utilizes RNA-guided PDS knockout in

wheat as a visual marker for successful editing events. Optimizing the temperature at which

tissues are infected and cultured is one way to increase editing efficiency greatly. Plant tissues

and endonucleases involved in transformation have greatly different optimal temperatures (24°C

and 37°C respectively). Temperature treatments have been previously studied in wheat (Tanaka

et al., 2022; Poddar et al., 2023) and rice (Illa-Berenguer et al., 2023) which both show an

intermediate temperature to have the greatest increase in the number of editing events. These

previous studies have shown that finding a temperature at which the endonucleases can reach

their full functional potential and infected tissues can continue proliferating and regenerating

new transgenic crops greatly increases genomic editing efficiency.

This review serves to highlight the current state of tomato transformation, and improvement

methods that have been used in other plants. These methods for improvement have the

potential to similarly improve the outcome of tomato transformations. The need for improved

transformation and genome editing protocols is only increasing as climate change becomes an

increasing threat to our planet. This review discusses current transformation methods used in

tomato, explants commonly used in tomato transformation, the value of using PDS as a target

gene, and two potential treatments for improving transformation and genome editing

frequencies.

Transformation Methods

Tomato transformation via Agrobacterium co-cultivation has remained the dominant method of

transformation for tomatoes (Gerszberg et al., 2015) since it was first published in 1985 (Horsch
et al., 1985). This method involves first transforming the bacteria to contain the gene of interest

in the pathogenic portion of its genome. This half of the bacteria’s genome will be injected into

the plant using the bacteria’s natural pathogenic mechanism. Plant tissue is then exposed to the

Agrobacterium in a bacterial solution, allowing the naturally virulent bacteria to infect the target

tissues. Typically, the bacterial construct will contain a selective gene to weed out tissues that

have been successfully transformed and those that have not (Frary and Van Eck, 2004; Horsch

et al., 1985; McCormick et al.,1986).

Protocols have since been improved upon to improve the efficiency of the regeneration process.

This includes altering growth mediums slightly by adding hormones to the rate of tissue growth

(Gupta and Van Eck, 2016). These mediums are made to optimize plant growth so that whole

transgenic plants can be regenerated from transformed plantlets. Regenerative transformations

are more common in crops such as tomato because whole transgenic plants are essential for

developing crop lines that can be used commercially (Gerszberg et al., 2015). This is why it

remains the primary method of transformation in tomato. Rather than exposing the explant to a

solution of bacteria, another method is to inject a mature fruit with the Agrobacterium (Yang et

al., 2022; Bao et al., 2022, Naing et al., 2019), but this does not allow for regeneration. Although

transformation methods have been improved upon since the advent of genome editing, it has

not been necessary to improve tomato transformation in the same way due to tomato’s intrinsic

ability to take genetic modifications. This does not mean that the standard tomato

transformation protocol should not be improved upon by experimenting with various

experimental methods including various explants, target genes, and post-transformational

treatments.
Explants

The two most commonly used explants for tomato transformation are cotyledon and mature fruit.

Some studies (McCormick et al. 1985) use mature leaf tissue. The reason for using one explant

over another varies from study to study, but transforming whole fruit allows scientists to observe

the expression of edited genes in the whole fruit without waiting for a plant to grow from

regenerative tissue and produce fruit (Yang et al., 2022; Bao et al., 2022, Naing et al., 2019).

Conversely, a benefit of transforming tissue that can regenerate new plants is to observe the

phenotypic changes that come with editing the plant throughout development. Ultimately, the

choice to regenerate transgenic plantlets or transgenic fruit lies in the goals of the study itself.

For tomato in particular, a plant that is better known for its commercial use than scientific use

(Gerszberg et al., 2015), the regeneration of whole transgenic plants is a more attractive option

for many researchers. This method not only allows scientists to confirm that their editing

protocol works but also that the edited tissue is still viable and can regenerate full plants (Frary

and Van Eck, 2004; Horsch et al., 1985; McCormick et al.,1986; Gerszberg et al., 2015). On the

other hand, transforming cotyledon or leaf tissue with the goal of regeneration relies on the

tissue being able to regrow and generate transgenic events after undergoing the stress of

bacterial infection. This means that some explants that contain positive events may not be able

to regenerate plantlets due to stress even though they contain the desired edit (Frary and Van

Eck, 2004).

For projects interested in the molecular mechanism of transformation and its direct outcome on

the explant, injecting the fruit with the virulent Agrobacterium and observing the changes is a

common way to quickly get results (Yang et al., 2022; Bao et al., 2022, Naing et al., 2019).

These experiments typically use a visual marker which makes analyzing the rate of genome

editing simple. The PDS gene is commonly knocked out to achieve an albino phenotype, an
obvious visual marker (Naing et al., 2019). Since tomato is a diploid plant, the expression of a

visual marker requires editing on both alleles. Using the fruit for this rather than regenerative

tissue is useful because dying regenerative tissue often lacks pigment, so unsuccessfully

transformed tissue can potentially be mistaken for a biallelic event. This is common in PDS

knockout transformations in all plants. Poddar et al. discuss how they initially mistook dying

tissue for positive results and vice versa. When using fruit as the explant, however, the explant

already has strong pigment, and change in the pigment of the explant is what determines

whether or not the genome editing was successful (Bao et al., 2022, Naing et al., 2019).

Few studies have used mature leaf tissue as an explant since the first two papers regarding

tomato transformation (Horsch et al., 1985; McCormick et al., 1986). Since then, cotyledon and

mature fruit have become the primary explants of choice. McCormick et al. achieved similar

rates of genome editing and regeneration as more modern studies using cotyledon tissues

which poses the question of why researchers seem to prefer cotyledon tissue over mature plant

tissue. Additionally, depending on whether or not editing mature fruit results in all tissues of that

fruit being edited, it may be possible to regenerate whole plantlets using this method by

germinating the seeds in the transformed tomato. Being able to successfully edit explants

outside of those routinely, namely experiments that produce transgenic reproductive tissue, may

open new avenues for genome editing efficiencies and transgenic tissue regeneration.

Target Genes

Due to the agricultural and economic importance of the tomato, many projects target genes that

improve the flavor, size, or nutrients available in the fruit (Gerszberg et al., 2015). This cannot

be achieved if the protocol for gene editing is not streamlined. It is easier to optimize and perfect

a protocol using genes of interest that are easy to identify and select for. These usually include

an antibiotic resistance gene and a visual marker (Gupta and Van Eck, 2016; Naing et al., 2019;
Yang et al., 2022). Both are typically used in the case that specific genes are being targeted

rather than simply inducing a specific mutation. Due to the diploid nature of the tomato genome,

edits in the genome can occur either on one or both alleles. Inducing an edit in the genome that

has a distinct visual outcome is useful in this scenario because you can easily distinguish

between biallelic mutations and the rest of the transformed explants (Naing et al., 2019).

PDS knockout, knockdown, or overexpression is used as a visual marker in studies in tomato,

as well as wheat, and rice. This visual marker is used in tandem with antibiotic resistance to

ensure that the transformation was successful and that the Cas9 endonuclease edited the

genome at the correct loci (Tanaka et al., 2022; Poddar et al., 2023; Naing et al., 2019;

Illa-Berenguer, LaFayette, and Parrott, 2023). The PDS gene is involved in the biosynthesis of

carotenoids, and a knockdown in this gene causes plants to express an albino phenotype. The

advent of the CRISPR-Cas9 complex changed the field of genome editing by allowing scientists

to make edits to specific genes, rather than just insert genes randomly throughout the genome.

The use of the antibiotic resistance gene and the PDS edit is to ensure that the constructed

gene was inserted correctly into the genome as well as confirm that the CRISPR-Cas9 complex

then edited the genome in the correct place to induce a phenotypic change. All explants in

which the transformation was successful will survive on the selection medium that contains the

antibiotic, and those that are genetically edited will survive on the medium and exhibit a color

change: albino in the case of knockout/knockdown and enhanced pigmentation in the case of

overexpression (Naing et al., 2019).

Treatments for Editing Improvement

One common treatment used in other crops is subjecting the explants to heat for varying

amounts of time during co-cultivation (Illa-Berenguer, LaFayette, and Parrott, 2023; Poddar et

al., 2023; Clement et al., 2016; Gurel et al., 2009; Tanaka et al., 2022). This typically is in an
attempt to optimize the co-cultivation environment for endonuclease activity and tissue growth.

This has been done successfully in wheat, rice, and sorghum. While some of these studies used

gold particle bombardment instead of Agrobacterium-mediated transformation (Poddar et al.,

2023; Tanaka et al., 2022), the method of transformation is not as important in this instance

because the treatment is applied afterward. In experiments using particle bombardment,

however, the treatment is applied during the resting stage.

Heat treatment is only applicable in cases that utilize the CRISPR-Cas9 complex, or a similar

endonuclease (Illa-Berenguer, LaFayette, and Parrott, 2023). This is because the goal of

temperature treatment is to find a temperature where the endonuclease can function effectively

and tissue can proliferate. Tissue culture has an optimal temperature of 24°C while the Cas9

endonuclease has an optimum temperature of 37°C (Tanaka et al., 2022). Having the

endonuclease function optimally is irrelevant to transformation if the temperature is too high for

the transformed tissue to grow. Similarly, generating large amounts of tissue is not beneficial

unless the endonuclease can edit the plant’s genome first. Many of these papers have found

intermediate temperatures between 30°C and 34°C to be optimal (Poddar et al., 2023; Clement

et al., 2016; Gurel et al., 2009; Tanaka et al., 2022; Illa-Berenguer, LaFayette, and Parrott,

2023), but these were all done on other crops which may have different optimal temperatures

than tomato.

Compared to other crops, continuous improvement in tomato genome editing has been less

frequent due to its ease of transformation. Alternative protocols have incorporated hydrolytic

enzymes to enhance editing efficiency by damaging the target tissue, enabling the naturally

virulent Agrobacterium to attack the tissue (Clement et al., 2016; Weber et al., 2003). This

approach has the potential to expand the range of explants usable in tomato transformations.

Presently, most transformations focus on cotyledon or whole fruit tissues, but enzymatic

treatments could make it possible to edit more resistant tissues. Both heat and enzymatic
treatments have worked to improve editing efficiencies in other crops and should be used to

improve the current genome editing protocol for tomato.

Conclusion

Between the late twentieth century when plant transformation via Agrobacterium mediation was

first introduced to agricultural science and the discovery of CRISPR-Cas9 as a tool for genome

editing, the protocol for editing tomato was not improved upon heavily. As described by Horsch

et al. in 1985 and McCormick et al. in the following year, Agrobacterium-mediated

transformation using co-cultivation is still the primary method for tomato transformation. It has

been modified slightly, namely, the culturing scheme (Gupta and Van Eck, 2016) and delivery of

the bacteria (Bao et al., 2022), but for the most part, the transformation has remained the same

for the past three decades. In comparison, other crops such as rice, wheat, and sorghum whose

transformation protocols have been continuously improved upon. The biggest change in tomato

has come in the explants used as target tissues. While past scientists used mature leaf tissue, it

is now more common for cotyledon tissue to be in regenerative experiments (Frary and Van

Eck, 2004; Gerszberg et al., 2015), and mature fruits are even now used as an explant to

demonstrate genome editing (Yang et al., 2022; Bao et al., 2022, Naing et al., 2019). With the

expansion of explants and editing technology, it seems logical for scientists to turn to other parts

of the plant, and potentially back to the more recalcitrant mature leaf tissue.

Other crops such as rice, sorghum, sunflower, and wheat proved to be much more resistant to

transformation. This prompted researchers to find ways to improve the rate of genome editing

and transformation, utilizing hydrolytic enzymes (Clement et al., 2016; Weber et al., 2003) and

temperature treatments (Poddar et al., 2023; Clement et al., 2016; Gurel et al., 2009; Tanaka et

al., 2022; Illa-Berenguer, LaFayette, and Parrott, 2023). Both of these methods have improved

the output of transformations in the aforementioned crops. Applying these methods to

transformation and genome editing in tomato could allow for expansion in the field. Tomato was

the first crop to have a genetically modified line be approved for commercialization. This led to

the large variety of tomatoes available to the public today. Furthering the field of genome editing

in tomato may prove to be essential in the face of climate change, and being able to effectively

and efficiently edit the genome in order to improve pesticide resistance or drought tolerance is a

pressing issue in the field today. Using these techniques and treatments that have greatly aided

other crops in increasing editing efficiency may be the next step for tomato genome editing.

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