IJSAR, 4(7), 2017; 103-112

International Journal of Sciences & Applied Research

www.ijsar.in

Transgenesis of tomato: Relevance in molecular research today

Somnath Mondal and Surekha Kundu*

Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta 35, Ballygunge Circular Road, Kolkata-700019, India. Corresponding author: *Surekha Kundu, Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta 35, Ballygunge Circular Road, Kolkata-700019, India. ______Abstract Tomato is not only one of the most important of vegetable crops but also is an important dicotyledonous model plant for scientific research. Genetic transformation of tomato is often the first step of various scientific researches to follow. Along with the view to generating tomato lines with diverse desirable traits transformed tomato serve as the background of doing functional studies. There are many transformation protocols for tomato and these have their advantages and dis-advantages. The selection of the desirable traits, the appropriate promoter/terminator sequences, suitable selectable markers and reporter genes are to be critically considered. A number of different transgenic lines of tomato are available today that have improved nutritional content, better shelf life, resistance to biotic/abiotic stresses and even for development of vaccines and as tools in immunotherapy. The importance of transgenesis of tomato in molecular researches today is discussed.

Keywords: Tomato, transformation, Agrobacterium, biotechnology, binary vector, tissue culture

Introduction be taken into consideration are the Tomato (Lycopersicon esculentum Mill), a receptiveness of tissue to foreign DNA member of the Solanaceae family, is not integration, availability of vectors and only one of the most important vegetable screenable markers and reproducible crops in the world but it is also a model regeneration of intact plantlets from plant for basic research on dicotyledonous transformed tissues. plants. The entire tomato genome consists of 12 chromosomes. Genetic transformation of Major methods of transformation of tomato is often the first step of various tomato scientific researches along with the view to Transformation methods followed in tomato generating tomato lines with diverse are mainly of two types namely direct desirable traits (Hansen et al, 1994). transformation and Agrobacterium-mediated However, for any genetic engineering transformation. strategy to become successful, several protocol factors need to be optimized for the Direct or non-Agrobacterium-based particular system. Other factors that need to methods

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IJSAR, 4(7), 2017; 103-112 These methods again can be discussed under Agrobacterium mediated transformation the following heads: This method includes infection of suitable explants using Agrobacteium tumefaciens Protoplast based transformation, in which carrying suitable recombinant binary the desirable gene is transferred into the vectors. protoplast in the presence of calf thymus carrier DNA and polyethylene glycol. The Agrobacterium strains and binary vectors prerequisite of this method is the preparation Agrobacterium mediated gene of receptive protoplasts from tomato tissues transformation is an effective and widely mainly leaf tissues. These have successfully used approach to introduce foreign DNA been used by many workers initially and into a dicotyledons plants like tomato. The transgenic plants have been produced (Jones ability of particular Agrobacterium strains to et al, 2005). transform plant cells is defined by their chromosomal and plasmid genomes which Non-protoplast based transformation, in between them must encode all the which, unlike the use of Agrobacterium, is machinery necessary for attachment and plant genotype-independent and relies upon DNA transfer (Vander et al, 2010). the bombardment of accelerated noble metal There are several significant advantages to particles coated with DNA. Most commonly transferring DNA via Agrobacterium, used instruments for accelerating DNA including a low transgene copy number, the coated particles are those powered by burst stable integration with fewer rearrangements of helium generated by a rupture membrane of long molecules of DNA with defined mechanism or by a shock wave generated by ends and the ability to generate lines which high voltage discharge through a watered are without selectable marker genes (Khan droplet (Jones et al, 2005). et al, 2012). The current protocols used for tomato transformation are based on shoot Agrobacterium infection process is divided regeneration from leaf disk/cotyledon tissue into two steps co-cultivated with disarmed Agrobacterium The first step includes, a short period, tumefaciens harboring binary vector. The normally a few minutes to a few hours, of efficiency of such procedures is generally inoculation by immersion of suitable low because most of the transformed leaf explants in an Agrobacterium suspension. cotyledon cells could not develop into Then, after the majority of Agrobacterium shoots. Agrobacterium mediated cells are removed by pouring or pipetting, transformation in tomato has been studied the explants are co-cultivated for a further on different media and conditions have been 1–3 days. One or both these steps are carried optimized. The current protocols used for out in darkness at approximately 25°C, tomato transformation are based on shoot although a two temperature co-cultivation regeneration from leaf disk/cotyledon tissue step has also been tried with one day at 27°C co-cultivated with disarmed Agrobacterium then two days to 25°C (Velcheva et al, tumefaciens harboring binary vector. The 2011). efficiency of such procedures is generally low because most of the transformed leaf Over view of Agrobacterium –mediated cotyledon cells do not develop into shoots tomato transformation over view (Jones et al, 2005). In general, the Agrobacterium method is considered preferable to the gene gun, because of the greater frequency of single- 104

IJSAR, 4(7), 2017; 103-112 site insertions of the foreign DNA, making it of Agrobacterium infection and easier to monitor (Chaudhury et al, 2009). transgenesis. However a new strain of Agrobacterium tumefaciens is a soil Agrobacterium expressing gamma-amino- bacteria that has the ability to infect plant butyric acid transaminase activity showed cells and transfer a fragment of its DNA into higher rate of tomato transgenesis in the host cells. When the bacterial DNA is presence of low-GABA (Nonaka et al, 2017) integrated into a plant chromosome, it effectively hijacks the plant's cellular Difficulty in identifying and Locating machinery and uses it to ensure the proteins Genes for Plant Traits to be used in required for the proliferation of the bacteria tomato transformation in the host cells. Identifying and locating genes for The infecting DNA in an A. tumefaciens cell agriculturally desirable traits is presently the is contained in the bacterial chromosome most limiting step in the transgenic process. and also in Ti (tumor-inducing) plasmid. We still know relatively little about the The Ti plasmid contains a stretch of DNA specific genes required to enhance plant termed T-DNA (~20 kb long) that is growth, improve stress tolerance, modify integrated in to the plant genome during the specific properties of the harvested product, infection process. A. tumefaciens can only or otherwise affect plant characters. Usually infect a plant through wounds. Under natural identifying a single gene involved with a conditions, when a plant root or stem is desirable trait is not adequate. Scientists wounded it gives off certain chemical must have an insight as to how the gene is signals. In response to those signals, the vir regulated, what other effects it might have genes of A. tumefaciens get activated and on the transformed plant, and how it direct a series of events which results in the interacts with other genes active in the same transfer of the T-DNA from the Ti plasmid biochemical pathway. Public and private to the plant genome. research programs are investing heavily into Different vir genes help in copying the T- new technologies to rapidly sequence and DNA to which a leader sequence is attached. determine functions of genes of the most For protection of the T-DNA it is coated important crop species. These efforts should with protein. A channel is opened in the result in identification of a large number of bacterial cell membrane, through which the genes potentially useful for producing T-DNA moves out. transgenic varieties for scientists to have a The T-DNA then enters the plant cell wider range of choices (Sederoff et al, through the wound. It is not clear how the 1999). bacterial DNA moves from the cytoplasm to the nucleus of the plant cell, nor how the T- Designing Genes for Insertion during DNA becomes integrated into the plant transgenesis chromosome. Once a gene has been isolated it must be To exploit the T-DNA as a transgene vector, cloned into a small cloning vector for scientists have removed the tumor-inducing maintenance. Typically E coli is the choice section of T-DNA, while retaining the left organism for this purpose of maintaining the and right border regions and the vir genes. vector with the cloned gene. Thereafter it The transgene is inserted between the T- must undergo several modifications before it DNA border regions, where it is transferred can be effectively inserted into a plant. to the plant cell and becomes integrated into the plant's chromosomes. Gamma-amino- butyric acid (GABA) is a negative regulator 105

IJSAR, 4(7), 2017; 103-112 Modification of cloned genes more so in red ripe fruits of tomato (Sharma Sometimes, the cloned gene is modified to et al, 2016) achieve greater expression in a plant. For example, the Bt gene for insect resistance is Importance of terminator sequence of bacterial origin and has a higher The termination sequence signals to the percentage of A-T nucleotide pairs cellular machinery that the end of the gene compared to plants. Plant translational sequence has been reached. Current machinery prefers G-C nucleotide pairs. terminator sequences used are nopaline During modification A-T nucleotides were synthase terminator or rubisco terminator substituted with G-C nucleotides in the Bt (Basu et al, 2015). gene without significantly changing the amino acid sequence. This ensured in Current use of selectable marker genes enhanced production of the Bt gene protein Selectable marker genes are added to the in plant cells (Deist et al, 2014). gene construct in order to ―mark‖ plant cells or tissues that have successfully integrated Importance of promoter sequence the transgene for easy identification and A promoter sequence must be added up selection. This is necessary because stream of the cloned gene to be correctly achieving incorporation and expression of expressed or in other words translated into a transgenes in plant cells is a rare event, protein product (Agarwal et al, 2016). The occurring in just a few percent of the promoter is the switch that controls when targeted tissues or cells. Selectable marker and where in the plant the gene will be genes encode proteins that provide expressed. To date, most promoters in resistance to chemicals that are normally transgenic crop varieties have been inhibitory to plant cells, such as antibiotics constitutive, causing genes to be expressed or herbicides. Only those plant cells that in all parts of the plant and throughout the expresses the selectable marker gene will life cycle of the plant across different survive when grown on a medium developmental stages. The most commonly containing the antibiotic or herbicide within used constitutive promoter is CaMV35S, limited ranges. Just like other genes, the from the cauliflower mosaic virus, which marker genes also require promoter and generally results in a high degree of termination sequences for proper expression. expression in plants. Other promoters are Currently the most commonly used more specific and respond to cues in the selectable marker gene for tomato is plant's internal or external environment. An neomycin phosphor transferease (nptII) gene example of a light-inducible promoter is the that imparts Kanamycin resistance. promoter from the cab gene, encoding the major chlorophyll a/b binding protein or Selection and Regeneration of transgenic wound inducible promoters (Feng et al, tomato 2014). Tissue specific promoters are The selection of transgenic tissues from important tools for site-specific expression expalnts is followed by regeneration of of genes and improvement of crops. Fruit whole plants. ripening is an important part of crop yield and is genetically regulated by irreversible Selection of successfully transformed changes in the fruit. Such a promoter of tomato tissues Ripening induced protein 1 (RIP 1) in Following the gene insertion process, plant tomato is specifically expressed in fruits, tissues are transferred to a selective medium containing an antibiotic or herbicide, 106

IJSAR, 4(7), 2017; 103-112 depending on which selectable marker was Delayed ripening imparted by used. Only transgenesis plants expressing the selectable marker gene Tomatoes have been used as a model will survive, as shown in the figure, and it is organism to study the fruit ripening of assumed that these plants will also possess climacteric fruit. To understand the the transgene of interest. Thus, subsequent mechanisms involved in the process of steps in the process will only use these ripening, scientists have genetically surviving. engineered tomatoes. In 1994, the Flavr Savr became the first Regeneration of whole tomato plants commercially grown genetically engineered To obtain whole plants from transgenic food to be granted a license for human tissues such as immature embryos, they are consumption (Francis et al, 2017). A second grown under controlled environmental copy of the tomato gene polygalacturonase conditions in a series of media containing was inserted into the tomato genome in the nutrients and hormones. For tomato the antisense direction. During ripening the regeneration media usually contain a polygalacturonase enzyme degrades the cytokinin and auxin. The regenerating plants pectin of the tomato cell wall, causing the are transferred onto fresh rooting media with fruit to soften. When the antisense gene is antibiotic. Once whole plants are generated expressed it interferes with the production of and produce seed, the progeny is evaluated. the polygalacturonase enzyme, thereby This regeneration step has been a stumbling delaying the ripening process. The Flavr block in producing transgenic plants in Savr failed to achieve commercial success many species, but for tomato this step is and was withdrawn from the market in nowadays fairly standardized (Khan et al, 1997. Similar technology, but using a 2012). truncated version of the polygalacturonase gene, was used to make a tomato paste. Genetically modified tomato available DNA Plant Technology (DNAP), Agritope today and Monsanto developed tomatoes that The tomato originated from South America delayed ripening by preventing the and was brought to Europe by the Spanish in production of ethylene, a hormone that the 16th century. Wild tomatoes are small, triggers ripening of fruit. All three tomatoes green and largely unappetizing but after inhibited ethylene production by reducing centuries of breeding there are now the amount of 1-aminocyclopropane-1- thousands of varieties grown worldwide. carboxylic acid (ACC), the precursor to Agrobacterium-mediated genetic ethylene. DNAP's tomato, called Endless engineering techniques were developed in Summer, inserted a truncated version of the the late 1980s that could successfully ACC synthase gene into the tomato that transfer genetic material into the nuclear interfered with the endogenous ACC genome of tomatoes. Genetic material can synthase. Monsanto's tomato was engineered also be inserted into a tomato cell's with the ACC deaminase gene from the soil chloroplast and chloroplast plastosomes bacterium Pseudomonas chlororaphis that using biolistics. This is called organelle lowered ethylene levels by breaking down transformation. Tomatoes were the first food ACC. Agritope introduced an S- crop with an edible fruit where this was adenosylmethionine hydrolase (SAMase) possible. encoding gene derived from the E. coli bacteriophage T3, which reduced the levels of S-adenosylmethionine, a precursor to 107

IJSAR, 4(7), 2017; 103-112 ACC. Endless Summer was briefly tested in Na+/H+ antiport (AtNHX1) from A. thaliana the marketplace, but patent arguments lead to salt accumulating in the leaves of the forced its withdrawal (Bawa and plants, but not in the fruit and allowed them Anilakumar, 2013). to grow more in salt solutions than wildtype Scientists in India have delayed the ripening plants. They where the first salt-tolerant, of tomatoes by silencing two genes edible plants ever created. Tobacco osmotic encoding N-glycoprotein modifying genes overexpressed in tomatoes produced enzymes, α-mannosidase and β-D-N- plants that held a higher water content than acetylhexosaminidase (Meng et al, 2016). wildtype plants increasing tolerance to The fruits produced were not visibly drought and salt stress. damaged after being stored at room In another report, transgenic tomato plants temperature for 45 days, whereas with choline oxidase gene (codA) conferred unmodified tomatoes had gone rotten. In more resistance under salt stressed India, where 30% of fruit is wasted before it conditions. They accumulated more glycine reaches the consumers due to a lack of betaine than wild type plants. The refrigeration and delayed conveyance, photosynthetic rates were also higher in genetic engineering of the tomato may transgenic plants. Under salt-stress the decrease wastage. Na+/K+ ratio is lower in transgenic plants than wild plants as the Na+ efflux is greater Environmental stress tolerance than K+ influx in the roots of transgenic Abiotic stresses like frost, drought and lines. Accumulation of glycine betaine increased salinity are a limiting factor to the induced the expression of K+-transporter, growth of tomatoes. While no genetically Na+/H+ antiporter and H+-ATPase genes. modified stress tolerant plants are currently Hence in transgenic tomato plants codA commercialised, transgenic approaches have plays critical role in regulation of ion been researched. An early tomato was channels and transporters (Wei et al, 2017). developed that contained an antifreeze gene Sucrose is the most common form of sugar (afa3) from the winter flounder with the aim transported within plants. Sucrose of increasing the tomato's tolerance to frost. transporters (SUTs) play a key role in The antifreeze protein was found to inhibit sucrose partitioning. More over sucrose ice recrystallization in the flounders blood, molecules can act as a signal to different but had no effect when expressed in pathways to manipulate gene expression and transgenic tobacco. The resulting tomato physiological adaptation under abiotic stress was never commercialized, but raised conditions. Over-expression of apple-SUT, ethical questions over adding genes from MdSUT1 in tomato conferred stress one kingdom to another. tolerance under low temperature. More Other genes from various species have been anthocyanin pigmentation was observed inserted into the tomato with the hope of under low temperature in MdSUT1- increasing their resistance to various transgenic tomato plants than wild type. In environmental factors. A gene from rice addition to abiotic stress tolerance the (Osmyb4), which codes for a transcription transgenic tomato lines also showed better factor, that was shown to increase cold and sucrose uptake, early flowering and fruit- drought tolerance in transgenic Arabidopsis ripening (Ma et al, 2017). thaliana plants was inserted into the tomato. This resulted in increased drought tolerance, Transgenesis of tomato for resistant to but did not appear to have any effect on cold biotic stress tolerance. Overexpressing a vacuolar 108

IJSAR, 4(7), 2017; 103-112 The Brassica juncea 2S seed storage protein precursor gene has been used to produce Improved nutritional content of tomato insect resistant tomato lines in India through transgenesis (Mandal et al, 2002). In this work the Tomatoes have been altered in attempts to novelty was that while the precursor of the improve their flavour or nutritional content. protein had trypsin inhibitor properties, the In 2000, the concentration of pro-vitamin A processed protein did not have this property, was increased by adding a bacterial gene thereby rendering the protein harmless encoding phytoene desaturase, although the during consumption. The insecticidal toxin total amount of carotenoids remained equal from the bacterium Bacillus thuringiensis The researchers admitted at the time that it has been inserted into a tomato plant (Koul had no prospect of being grown et al, 2014). When field tested they showed commercially due to the anti-GM climate. resistance to the tobacco hornworm More recently, scientists have increased the (Manduca sexta), tomato fruitworm production of anthocyanin, an antioxidant in (Heliothis zea), the tomato pinworm tomatoes in several ways. One group added (Keiferia lycopersicella) and the tomato fruit a transcription factor for the production of borer (Helicoverpa armigera). A 91 day anthocyanin from Arabidopsis thaliana feeding trail in rats showed no adverse (Zuluaga et al, 2008). Whereas another used effects, but the Bt tomato has never been transcription factors from snapdragon commercialised. Tomatoes resistant to a root (Antirrhinum) (Tohge et al, 2015). When the knot nematode have been created by snapdragon genes where used, the fruits had inserting a cysteine proteinase inhibitor gene similar anthocyanin concentrations to from taro A chemically synthesised ceropin blackberries and blueberries, and when fed B gene, usually found in the giant silk moth to cancer susceptible mice, extended their (Hyalophora cecropia), has been introduced life span. Another group has tried to into tomato plants and in vivo studies show increase the levels of isoflavone, known for significant resistance to bacterial wilt and its potential cancer preventative properties, bacterial spot. When the cell wall proteins, by introducing the soybean isoflavone polygalacturonase and expansin are synthase into tomato. prevented from being produced in fruits, they are less susceptible to the fungus Transformation of tomato for improved Botrytis cinerea than normal tomatoes. taste Cucumber mosaic virus (CMV-Fny) in When geraniol synthase from lemon basil combination with its satellite RNA (77- (Ocimum basilicum) was expressed in satRNA FN) causes severe necrotic disease tomato fruits under a fruit-specific promoter, in tomato. Systemin signal peptide hormone 60% of untrained taste testers preferred the is related to stress response in Solanaceae taste and smell of the transgenic tomatoes family and it induces proteinase inhibitors in (Bartoszewski, 2003). The fruits contained plants just like jasmonate (JA) responsive around half the amount of lycopene, pathways. In transgenic tomato lines over reducing the health benefits of eating. expressing prosystemin necrotic lesions were reduced in 50% of plants. In over Transgenic tomatoes for production of expression lines JA-biosynthetic genes like vaccines and vehicle of vaccine delivery LoxD, AOS and JA-induced genes like Pin I Tomatoes (along with potatoes, bananas and and Pin II were upregulated and salicylic other plants) are being investigated as acid (SA) responsive genes ere down vehicles for delivering edible vaccines. regulated considerably (Bubici et al, 2017). Clinical trials have been conducted on mice 109

IJSAR, 4(7), 2017; 103-112 using tomatoes expressing antibodies or vegetable crop in one of the two aspects for proteins that stimulate antibody production consideration. The other being, with the targeted to norovirus, hepatitis B, rabies, genome sequence now being available, HIV, anthrax, respiratory syncytial virus and tomato has become the model IgA antibody against rotavirus (Merlin et al, dicotyledonous plant of choice next to 2017). Korean scientists are looking at using Arabidopsis in transgenic research. the tomato to expressing a vaccine against On the whole it can be said that transgenesis Alzheimer's disease. Hilary Koprowski, of tomato is a part of the research work for who was involved in the development of the many laboratories all over the world. polio vaccine, is leading a group of Transgenic tomato with desirable traits is researchers in developing a tomato not only the end product of research, it is expressing a recombinant vaccine to SARS. often the background which is the starting The Hepatitis B surface antigen (HBsAG) point of other research to follow. has been reported to accumulate to 0.01% of soluble protein level in transgenic tomato Acknowledgement (MacKenzie, 1994). The antigens, delivered Partially funded by DBT, Government of in a macromolecular form, are known to India, UGC, India and UGC-CAS tolerate the digestive tract environment. Department of Botany, University of Interestingly, the recombinant HBsAG was Calcutta. able to form virus-like particles of 22 nm diameter (comparable to yeast-derived Conflict of interest HBsAG-based vaccine). Plant extract was There is no conflict of interest. used for parenteral immunization in mice. The immune response included all IgG References subclasses as well as IgM against hepatitis Agarwal, P., Kumar, R., Pareek, A., Sharma, B. A. K., 2016. Fruit preferential activity of the tomato RIP1 gene promoter in Tomato the popular model plants for transgenic tomato and Arabidopsis. Mol transgenic research Genet Genomics. DOI 10.1007/s00438- Tomato is an important vegetable crop for 016-1262-4. India. According to FAOSTAT (2008) total Bartoszewski, G., Niedziela, A., Szwacka, world production was 129,649,883 tones out M., Niemirowicz-Szczytt K., 2003. of which 10,260,600 tones were from India Modification of tomato taste in transgenic which ranked fourth. Yet heavy losses upto plants carrying a thaumatin gene from 50% is seen every year due to different Thaumatococcus daniellii Benth. Plant abiotic and biotic stress conditions. Adverse Breeding 122, 347—351 abiotic conditions include salinity, drought, Bubici, G., Carluccio, AV., Stavolone, L., heavy metal toxicity. The biotic stresses Cillo, F., 2017. Prosystemin including diseases like tomato leaf curl overexpression induces transcriptional (caused by tomato leaf curl virus), early modifications of defense-related and blight (caused by Alternaria solani f.sp. receptor-like kinase genes and reduces lycopersici) and insects are among the most the destructive (McGarvey et al 1995). On the Chaudhury, Z., Rashid, H., 2009. An same note soil borne diseases are least improved Agrobacterium mediated understood and cause great damage to transformation in tomato using tomato production (Chowdhury et al 2013). hygromycin as a selective agent. Thus the improvement of this important 110

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