Genetic Engineering in Fruit Crops

K. Kumar¹', R. Kaur², and Shilpa²

• 1 Faculty of Agriculture, Shoolini University, Bajhol-Solan (H.P.) 173229, India
• 2 Department of Biotechnology, Dr Yashwant Singh Parmar University of Horticulture and Forestry,

Nauni - Solan (H.P.) 173230, India

1. Introduction

Fruit crops are an important agricultural produce, adding billions of dollars annually to the overall economy and are major sources of income for developing countries. Approximately 100 million acres of land has been devoted to their production worldwide and the livelihood of millions of fanning families depends on continued global trade. Fruits provide essential nutrients, vitamins, antioxidants and fibres in individual’s diet (Rai and Shekliawat, 2014). However, the present world fruit industry is based on a few genotypes. Intense selection and fixation of genotypes by clonal propagation leads to a narrow germplasm base (Janick, 1992). Thus, it was needed to change the cultivars. This can be achieved by conventional breeding, but it is hampered by the long generation time and juvenile periods, complex reproductive biology, high levels of heterozygosity, limited genetic sources and linkage drag of undesirable traits from wild relatives. Chemical controls and different managements have also been practically used in commercial production of different fruits in order to overcome disease infection, to improve shelf- life and to generate dwarf trees. However, these practices are either inefficient or unfriendly to human health and environment. In contrast, genetic engineering offers a better possibility to improve plant traits (Zhu et al, 2004). Dining the last about two decades, genetic engineering has been successfully used to improve tolerance to biotic and abiotic stresses, increased Suit yield, improved shelf-life of fruit, reduced generation time and production of fruit with superior nutritional value. Selectable marker genes are widely used for the efficient transformation of crop plants. Mostly antibiotic or herbicide resistance marker genes are preferred because they are more efficient. But due to consumer concerns, considerable effort is being put into developing strategies (homologous recombination, site-specific recombination, and co-transformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. The absence of selectable marker gene in the final product and the introduced gene(s) derived from the same plant will increase the consumer’s acceptance (Rrens et al., 2004). This can be fiilfilled by new genetic engineering approaches, like cisgenesis or intragenesis. Still, the development of transgenic fruit plants and their commercialisation are also hindered by many regulatory and social issues. Hence, the future use of transformed horticultural crops on a commercial basis depends upon thorough evaluation of the potential environmental and public health risks of the modified plants, stability of transgene over a long period of time and the effect of the gene on crop and fruit characteristics.

2. Methods of Genetic Transformation

Tissue culture plays a critical role in genetic transformation of a plant species because the initial requirement for most gene transfer systems is efficient regeneration of plantlets from cells carrying a foreign gene. Besides plant tissue culture, various methods of gene transfer are also necessaiy to obtain transgenic plants. Successful generation of transgenic plants requires a combination of the following:

• • A cloned gene with elements, such as promoters, enhancing and targeting sequences for its regulatory expression
• • A reliable method for delivery and stable integr ation of DNA into cells
• • A ‘tissue culture’ method to recover and regenerate intact plant from transformed organs or tissues
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Till date, a number of strategies have been developed for identification, isolation and cloning of desirable gene sequences from almost any organism. Next step is to deliver the desired gene sequence into the cell of target organism. This can be achieved by using one of the following methods:

• • Agrobacterium - mediated gene transfer
• • Particle bombardment or biolistic delivery
• • Electroporation
• • PEG-induced DNA uptake
• • DNA delivery via silicon carbide fibres
• • Laser-mediated DNA transfer
• • Microinjection
• • Liposome mediated gene transfer or lipofection
• • DEAE-dextran (diethylaminoethyloethyl-dextran) mediated transfection
• • Agroinfiltration
• • Vacuum infiltration

Besides these methods, there are many other transformation methods, such as ultrasound, seed imbibitions and electrophoresis, but these are not as successful as the methods mentioned earlier. Since the methods listed above have proved successful and applicable to several plant species, their principles will be explained in brief.

2.1. Agrobacterium-med/afed Gene Transfer

Most transgenic plants produced to date were created through the use of the Agrobacterium system. A. tumefaciens is the agent of crown gall disease and produces crown galls on infected species. The usefulness of this bacterium as a gene transfer system was first recognised when it was demonstrated that the crown galls were actually produced as a result of transfer and integration of genes from the bacterium into the genome of the plant cells (Hamilton and Fall, 1971). Virulent strains of Agrobacterium contain large Ti-(tumour inducing) plasmids, which are responsible for the DNA transfer and the following disease symptoms. Ti-plasmid contains two sets of sequences necessary for gene transfer to plants: one is T-DNA (transferred DNA) region that is transferred to the plant, and another is the Vir (virulence) genes which are triggered by phenolic compounds, especially acetosyringone and hydroxy acetosyringone secreted by wounded cells. The T-DNA regions is flanked by 25 base pairs on right and left borders and these are responsible for the T-DNA transfer to infected plant cells. Undesirable DNA sequences, e.g. genes for auxin, cytokinin or opine biosynthesis (sugar derivatives) present in the T-DNA of wild type strains have been deleted as phytohormones synthesis interferes with the plant regeneration and replaced with marker gene or desirable gene.

In addition to A. tumefaciens, A. rhizogenes is also capable of plant transformation by a similar process. Instead of tumours, A. rhizogenes causes ‘hairy-roots’ upon infection due to production of auxins in transformed cells. A. rhizogenes strains have also been engineered for transformation but are not widely used.

Agrobacterium-mediated transformation being simple and widely applicable, remains the method of choice to transform plants. Along with a suitable protocol for regeneration of complete plants from transformed cells, a large number of transformants can be generated. The whole procedure of DNA transfer and integration is very precise. Additionally, the resources required for transformation are not very difficult. The accurate site of integr ation of T-DNA into the host chromosome seems to be random. Independent transgenics for the same gene show wide variations in the levels of expression of the transgene. Hence, proper evaluation of transgenics for stable expression over generations is necessary.

2.2. Biolistic-mediated Transformation

Transformation of plant cells through microprojectile coated with DNA was developed by Sanford and Johnston (1985). In this method, the DNA to be used for transformation is coated with 1-2 pm tungsten or gold particles which are then mechanically ‘shot’ directly into the plant tissue by custom-made device, using either an electrical discharge, high gas pressure (such as compressed air, nitrogen or helium) or gun powder. The particles penetrate the cells and the adsorbed DNA is delivered into the cells. Use of biolistic gun is relatively simple and rapid. Its main advantage is its non-specificity resulting from the physical properties of the method. Moreover, any type of cells can be targetted, including embryos, pollen grains, microspores, leaf, stem, apical meristem, etc. Though particle bombardment has yielded success with a range of plant species, however, the high cost of equipments makes it expensive for individual laboratories with limited resources and also the recovery of transgenics is rather low.

2.3. Electroporation

Electroporation is perforation caused in the cell membranes by the use of electric current. Suitable ionic solution containing linearised recombinant plasmid DNA is used to suspend the protoplasts. This electroporation mixture is then exposed to the suitable voltage-pulse combination which depends upon the plant species and the source of protoplast. Protoplasts are then cultured in order to obtain cell colonies and plants. This method was initially developed for bacteria, yeast and animal cells and later adapted to plants. With the development of protoplast technology, electroporation was used to transform protoplasts (Fromm et ah, 1985). Later, the technique was found applicable to even intact tissues (D’Halluin et ah, 1992). This method has received less attention because of the wide success of particle bombardment technology. Yet, this method is widely valued for introduction of macromolecules into cells and to study transient expression of introduced DNA sequences.

2.4. Polyethylene Glycol (PEG)-mediated Uptake of DNA

PEG is widely used for fusion of animal cells and plant protoplasts (Kao and Michayluk, 1974). PEG precipitates the DNA on to the outer surface of plasma lemma and this precipitate is taken up by endocytosis. In this approach, protoplasts are suspended in a transformation medium followed by addition of linearised plasmid DNA containing the gene construct. After this, PEG is added to the mixture with pH adjustment of 8. The application of PEG to protoplast suspension leads to contraction of their volume. This results in endocytic vesiculation. DNA-cation complexes adsorbed to the membrane thus get introduced into the cells. PEG-mediated transformation is perhaps the least expensive of all the methods of transformation available at present.

2.5. Silicon Carbide Fibres

Silicon carbide fibre-mediated transformation involves vortexing of cells and fibres in a buffer that contains DNA. The fibres create holes in the cells and through the force of vortexing, DNA enters into the cell. This is a relatively recent method and hence has not been widely tested. The simplicity of the technique is its great desirability; however, the routine use of this method is hampered by hazardous natur e of silicon fibres.

2.6 Microinjection

Microinjection is the direct mechanical introduction of DNA into cells under microscopical control. In this method, a glass needle or micropipette of fine tip (0.5-1.0 micrometre diameter) is directly used to inject the DNA into plant protoplasts or cells (specifically into the nucleus or cytoplasm). This method is used to introduce DNA into large cells, such as oocytes, eggs and the cells of early embryo. By examination through a microscope, a cell is held in place with gentle suction while being manipulated with the use of a blunt capillary. A fine pipette is then used to insert the DNA into the cytoplasm or nucleus. This technique is more effective and easier to transform protoplasts as compared to cells because of interference caused by the cell wall. To obtain high rates of transformation, the DNA should be introduced into the nucleus or cytoplasm. This method is more successful in non-vacuolated embryonic cells with dense cytoplasm than large vacuolated cells because in the presence of large vacuoles, the DNA is delivered into the vacuole which is then degr aded. But this technique is not in routine use because it is more demanding and time consuming.

2.7. Liposome-mediated Gene Transfer or Lipofection

Liposomes are artificial circular lipid vesicles surrounded by a synthetic membrane of phospholipids. They have an aqueous interior which can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adhere to the protoplast membranes to fuse with them and transfer the DNA fragments. Thus, the DNA enters the cell and then the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells, but is not in much use because of its low transformation frequencies. This process generally involves three steps:

• • Adhesion of liposomes to the protoplast surface
• • Fusion of liposomes at the site of adhesion
• • Release of plasmid inside the protoplast or animal cell
• 2.8. DEAE-dextran (Diethylaminoethy Loethyl-dextran)-mediated Transfection

DEAE-dextran transfection is a method for introduction of DNA into the eukaryotic cells. DEAE- dextran facilitates DNA binding to cell membranes and entiy of the DNA into the cell via endocytosis. As DEAE-dextran is toxic to cells, the transfection conditions for individual cell lines may require careful optimisation for both DEAE-dextran concentration and exposure times. In general, DEAE-dextran- mediated transfection is successful in transient, but not stable transfection of cells. At higher DEAE- dextran concentrations, the exposure time to cells can be shortened in order to minimise cell death.

2.9. Agroinfiltration

Agroinfiltration is a method in plant biology to induce transient expression of genes in a plant or to produce a desired protein. In this method, a suspension of A. tumefaciens is injected into a plant leaf, where it transfers the desired gene to plant cells.

First step of the protocol is to introduce a gene of interest to a strain of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a suitable buffer solution. This solution is then placed in a syringe (without a needle). The tip of the syringe is pressed against the underside of a leaf while simultaneously applying gentle counter pressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf. The benefits of agroinfiltration, when compared to traditional plant transformation, are its speed and convenience.

2.10. Vacuum Infiltration

Vacuum infiltration is another way to penetrate Agrobacterium deep into plant tissue. In this procedure leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied, forcing air out of the stomata. Wien the vacuum is released, the difference in pressure forces the solution through the stomata into the mesophyll.

Once inside the leaf, the Agrobacterium remains in the intercellular space and transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed (no selection for stable integration is performed). The plant can be monitored for a possible effect in the phenotype, subjected to experimental conditions and then harvested and used for purification of the protein of interest.

2.11. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)

Recently, in 2012, a new technique of targetted genome editing through engineered nuclease emerged, known as clustered regularly interspaced short palindromic repeats (CRISPRs) (Sharrna et ah, 2017). In general, CRISPR is a family of DNA sequences in bacteria containing snippets of DNA from virus that have attacked the bacteria which are used by the bacteria to detect and destroy DNA from further attack by similar virus. This basic technique now has been used in many fruit crops, like apple and grapes, for successful alteration of characters by delivering cas9 nuclease complexed with synthetic guide RNA (gRNA) into the cell. The cell’s genome can be cut at a desired location, allowing the existing gene to be either silenced or removed and new ones added. Main steps followed in this technique are: 1) cas9 protein forms a complex with gRNA in the cell. 2) This complex matches to gDNA adjacent to a spacer. 3) cas9-RNA complex cuts the double strand of DNA. 4) Programmed DNA may be inserted at the cut. The fruit crop-wise progress made through genetic transformation is presented as under:

Apple

Apple has been successfully transformed using GUS (beta-glucuronidase), up til (Neomycin phosphotransferase II) and the nopaline synthase gene from A.tumefaciens. In apple, genetic transformation has been earned out for various traits, like disease resistance, improved phenotypic characters, enhanced nutrition level and salt tolerance.

(i) Disease Resistance

A lot of emphasis has been laid on transformation of apple for resistance to scab (Venturia inequalis). Attempts have been made to incorporate scab resistance by introducing various resistance genes, viz. a wheat puroindoline В (pinB) gene coding a protein with antifungal activity (Faize el ah, 2004), Vfal, Vfa2, or Vfa41 (Mahioy et ah, 2008), barley hordothiouin gene (hth) (Kreus et al., 2011).

Another disease which causes great damage to apple crop is fire blight caused by Erwinia amylovora. Several studies were carried out to overcome this disease through genetic transformation in apple cultivars with resistance genes, namely, viral EPS-depolymerase gene (dpo) (Flachowsky et al., 2008), fbjnrS originating from crab apple accession Malus robustaS (Mr5) (Giovanni et a!., 2014). To increase resistance to this disease, a very recent technique, i.e. CRISPR has been used by Malnoy et al. (2016) in which they targeted DIPM-1, DIPM- 2 and DIPM-4 genes in the Golden Delicious variety.

Successful genetic transformation has also been done to provide powdery mildew (caused by Podospliaera leucotricha) resistance by Chen et al. (2012), who introduced Malus hupehensis-derived nprl (Mhnprl) gene into the ‘Fuji’ apple. Four transgenic apple lines were verified by PCR and RT-PCR. Furthermore, the transgenic apple plants resisted infection by Podospliaera leucotricha better than the wild-type plants. Rihani et al. (2017) modified flavonoid pathway in apple evs i.e. ‘Holsteiner Cox’ and ‘Gala’ via Agrobacteriwn-vasdiuXtd transformation by over-expressing the MdMyblO transcription factor to increased effect on plant disease resistance.

(ii) Improved Phenotypic Characters

In apple, a breakthrough was made in the reduction of juvenile phase using transgenic approach. Kotoda et al. (2000) cloned the ‘MdtfT (Malus x domesticatf) gene homologous to Arabidopsisterminal flower 1 (tfll) gene which suppresses floral meristem identity genes, leafy (Ify) and apetalal (apl). Transgenic apples, expressing antisense ‘Mdtfl’ gene, flowered at about eight to 15 months after grafting on rootstocks; on the other hand, non-transformed control plants did not flower in five years. Expression of genes, i.e. ‘Bpmads4’ gene of silver birch (Flachowsky et ah, 2007), ‘florigen-like’ gene flowering locus T (ft) fr om poplar (Flachowsky and Hanke, 2012) also showed acceleration in the onset of flowering in apple plants.

The apple rootstock M26 (Malus pumila) is a very popular rootstock with semi-dwarf habits and gives high quality fruit at a young agern but it is prone to poor prop ability in soil. So young trees require staking in windy locations. To combat this problem, ‘rolC’ gene was introduced into M26 by A. tumefaciens LBA4404 for dwarfism and enhanced rooting ability. The ‘rolC’ transgenic lines showed reduced stem length and increased root number in vitro. Rooting ability was also examined in an isolated greenhouse after mound layering (Kim et ah, 2009). When compared with non-transgenic M26, ‘rolC’ transgenic line showed higher rooting ability.

A recent study showed knocking out of S-gene expression in the pistil and overcoming of selfincompatibility in apple cultivar (Broothaerts et ah, 2004). The apple cultivar ‘Elstar’ was transformed with T-DNA constructs containing the S3-RNase cDNA in anti-sense orientation. The resultant transgenic lines were screened for their ability to set fruits following self-pollination under controlled greenhouse conditions. In 12 lines, complete ‘S3 S-RNase’ gene modified the self-incompatibility behaviour of the plant, leading to either intermediate or complete self-fertility (Dreesen et ah, 2012).

Apple fruits turn brown quickly after slicing. Though a natural phenomenon, browning has been considered an undesirable trait that causes consumer inconvenience and unnecessary waste. To solve this issue of fruit browning in apple, Okanagan Specialty Fruits (OSF), a Canadian firm located in British Columbia, developed a series of new apple varieties from widely gr own existing apple varieties, such as Golden Delicious, Granny Smith, Gala and Fuji, which do not turn brown for over two weeks under appropriate conditions. To achieve the non-browning trait in these apples, gene silencing was used to turn down the expression of polyphenol oxidase (PPO), an enzyme that causes browning, by transformation with ‘pgas’(a hybrid sequence carrying four gene groups, i.e. PP02, GP03, AP05 and pSR7) transgene, which was directly derived from the apple genome. These genetically-engineered nonbrowning apples have been named as Arctic Apples. Compared with their non-transgenic controls, Arctic Apples showed 76-82 per cent reduction in PPO activities in leaves and 90-91 per cent of reduction in the mature fruit. Because of high percentage reductions in PPO activities in mature fruit, Ar ctic Apples show little browning (Carter, 2012; Xu, 2013) (Fig. 1). OSF is looking for market access in Canada and the US for Ar ctic Golden Delicious and Arctic Granny Smith since the last few years (Lehnert, 2011). hi 2012, a field test application was approved to conduct study of the Arctic Apple in the state of Washington followed by USDA approval in February, 2015 (Pollack, 2015) and by FDA in March, 2015 (FDA, 2015). It became the first genetically-modified apple approved for US sale (Tracy, 2015). In 2016, three varieties were approved by the USDA (Arctic Golden, Ar ctic Granny and Arctic Apple Fuji) and are expected for retail sale in 2017 with each apple bearing a ‘snowflake’ logo.

Figure 1. Production of Arctic Apple (Source: www.arcticapple.com)

(Hi) Enhanced Nutrition Level and Salt Tolerance

Nutrition is another important factor among the consumers. To fulfil this aspect, Cation Exchangers 1 (caxl) gene from Arabidopsis thaliana was introduced into Korean apple cv. ‘Hongro’byA. tumefaciens to obtain transgenic apple with enhanced ‘calcium’ levels. Southern blot analysis of ‘Hongro’ transformants showed that two putative transgenic lines were integrated with ‘caxl ’ gene in genomic DNA. The caxl comparative expression levels of two transgenic lines were higher than that of non-transformants, when evaluated using a real-time PCR. These two lines were multiplied in vitro and micro-grafted on apple rootstocks M9 in the greenhouse. After two years of micro-grafting, the fruits came into bearing (Kim et al., 2010).

Soil salinity is a major factor limiting apple production in some areas. Tonoplast Na+/H+ antiporters play a critical role in salt tolerance. Li et al. (2010) isolated Mdnhxl, a vacuolar Na+/H+ antiporter from Luo-2, a salt-tolerant rootstock of apple and introduced it into apple rootstock M26 by Agrobacterium- mediated transformation. RT-PCR analysis indicated that the gene was highly expressed in transgenic plants, which conferred high tolerance to salt stress.

Apricot

Genetic transformation studies in apricot (Prunus armeniaca) are relatively few and whatever attempts had been made were aimed at enhancing transformation protocols and incorporating resistance to Sharka (plum pox virus) disease. In an attempt, Petri et al. (2006) developed an efficient regeneration and transformation protocol in callus of mature tissues of apricot cultivars by integration of transgenes (Green

Fluorecsent Protein gene igfp) and nptll marker genes). It was observed that a low selection pressure applied during the first days of co-culture, followed by a higher selection pressure afterwards, greatly improved transformation and selection efficiencies. Putative transformed shoots were in vitro multiplied and then rooted to confirm 'gfp'' expression in the roots.

It was observed that transformation protocols are genotype dependent. Wang et al. (2009, 2013) developed genotype independent protocols using meristematic cells and by direct shoot regeneration from the proximal zone of mature cotyledons.

(i) Disease Resistance

Sharka (plum pox virus) is a dreaded viral disease of genus Prunus. The integration of ^lPPV-cp' gene via Agrobacterium-mediated transformation into apricot genome has been confirmed using both GUS staining and PCR analysis (Machado et al, 1992) in immature embryos. The resultant transformed plants were observed to be resistant to virus infection. These results raised the possibility of transforming commercial apricot cultivars for different traits (Sansavini, 2004).

Avocado

Genetic transformation has played an important role in avocado to pror ide disease resistance, herbicide resistance and improved shelf life. Various strategies have been followed to improve the efficiency of genetic transformation in avocado (Cruz et al, 1998; Ahmed et al., 2012).

(i) Disease Resistance

In an experiment, antifungal protein (afp) gene was used by Litz et al. (2007) to transform cultivars ‘Suardia’ and ‘Hass’ of avocado, using embryogenic cultures to enhance resistance to root diseases. From transformed cultures, enlarged and opaque somatic embryos were obtained by plating on to semisolid MS medium with 20 per cent coconut water. But the rate of normal germination was low and most somatic embryos produced only small shoots. Therefore, micrografting on to in vitro seedlings was done to rescue shoots from somatic embryos. To obtain plants ex vitro, in vftr o-derived shoots were also grafted on to three-week old seedlings. Transformation was confirmed by PCR and transgenic plants grew luxuriantly in the greenhouse.

(ii) Herbicide Resistance

Attempts were made to introduce resistance to herbicide basta in somatic hybrid of cultivar ‘Fuerte’ using genes glucanase, chitinase, bar, uidA (Rahatjo et al, 2008). In another study, the same team of workers transformed cultivar ‘Hass’ using afp, npt II and uidA for herbicide resistance.

(Hi) Improved Shelf-life

Another problem with avocado fruits is that they ripen on the tree and have a poor shelf-life. To overcome this problem, avocado was transformed to extend the ‘on-the-tree’ stay of mature fruits and to increase the shelf-life of fruits. For this embryogenic cultures were genetically transformed using A. tumefaciens strain EHA101 harboring SAM (S-adenosyl-L-methionine) hydr olase, a gene that blocks ethylene biosynthesis (Efendi, 2003; Litz et al, 2007). Transformation was confirmed using PCR. Transgenic plants were recovered successfully and were transferred to the greenhouse, where they showed good growth.

Banana

Genetic transformation of banana focused on disease resistance, abiotic stress tolerance and control of fruit ripening. To achieve these goals, genetic transformation was achieved in several ways. It was observed that the most successful procedure is Agrobacterium-mediated transformation of embryogenic suspension cultures (Hernandez et al, 1999). Embryogenic cultures are usually induced from immature male (Escalant et al, 1994) and female flowers (Grapin et al, 2000).

(i) Disease Resistance

Bananas have also been suggested as an appropriate vehicle for edible vaccines (Mor et al, 1998) and bananas containing malaria epitopes have been generated (Hassler, 1995). The transgenic bananas will thus be first edible vaccines that hopefully protect millions of children in the developing world against bacterial and viral infections (Gleba et ah, 2004).

To induce resistance to BBTV, banana cv. ‘Dwarf Brazilian’ was transformed using A. tumefaciens containing replicase-associated protein (rep) gene of the Hawaiian isolate of BBTV. Twenty-one transformants were found resistant to BBTV challenge and showed no bunchy top symptoms, whereas all of the control plants were infected with BBTV after a period of six months monitored in greenhouse (Bortli et al., 2009; Krishna et al., 2013).

Another most devastating disease of banana is wilt. To overcome this disease a number of genes viz., soybean endo- beta -1,3-glucanase gene (pROKla-Eg) (Maziah et al., 2007), sweet pepper hypersensitivity response-assisting protein (hrap) gene (Tripathi et al., 2010), rice thaumatin-like protein (tip) gene (Hu et ah, 2013), were transferred to banana through Agrobacterium-mediated transformation to produce wilt resistant plants. Bioassay of transgenic banana plants with Fusarium wilt pathogen showed that expression of tip enhanced resistance to Fusarium as compared to control plants.

Tripathi et al. (2017) demonstrated transgenic banana expressing sweet pepper genes, i.e. Hrap and Pflp providing resistance to Xanthomonas wilt. They also enhanced resistance to mixed population of nematodes by expressing cysteine proteinase inhibitor and synthetic peptide.

(ii) Abiotic Stress Tolerance

Several reports show rise in transgenic bananas being developed with salt tolerance. Musa Stress Associated Proteins 1 (sapl) gene (Sreedharan et al., 2012) and plasma membrane intrinsic protein gene (Musapipl;2) (Sreedharan et al., 2013) were used to transform banana plants to provide abiotic stress tolerance. It was noted that greenhouse hardened transgenic plants were tolerant to drought, salt, cold, heat and oxidative stress and also showed faster recovery towards normal growth and development.

(Hi) Shortening of Life Cycle

A long juvenile cycle of banana affects the crop productivity. To overcome this problem, Talengera et al. (2009) transformed embryogenic cells of banana cv. ‘Sukalindiizi’ with ‘Arabidopsis thalianacyclin D2;l ’ gene. Presence, integr ation and transcription of the transgene were confirmed by PCR, Southern blot and reverse transcriptase PCR analyses. Regenerants showed improved leaf elongation in two lines.

Cherry

Genetic transformation is mainly aimed at development of enhanced phenotypic characters. Several transformants have been regenerated on the dwarfing cherry rootstock Tumil’ (P. incisa x serrula) with Agrobacterium rhizogenes. Improvement in grafting and rooting rate was reported on further grafting on two wild cherry scions (Druart and Gruselle, 2007).

Investigations were also carried out to transform pear rootstock (Cydonia oblonga) and sweet cherry rootstock (Primus cerasus x P. canescens) plants with rooting stimulating ‘rolB ’ gene. After PCR analysis it was confirmed that the gene integr ated in one transformant of Cydonia and eight transformants of sweet cherry'. All transformants rooted in vitro. It was established during the investigations that rot В transgene did not influence proliferation rate of genetically modified sweet cherry rootstocks in vitro. An increase in cold hardiness of sweet cherry rootstock was noted under in vitro conditions, though no significant difference was observed in transformed and control plants of Cydonia for cold hardiness (Rugienius et al., 2009).

Pmnus necrotic ringspot virus (PNRSV) is a major pollen-disseminated virus that adversely affects many Primus species. In this study, Song et al. (2013) used RNA interference (RNAi)-mediated silencing with vector containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid cherry rootstocks, ‘Gisela 6’ (GI 148-1) and ‘Gisela 7’(GI 148-8)’. One year after inoculation with PNRSV and Prune Dwarf Virus, non-transgenic ‘Gisela 6’ exhibited no symptoms but a significant PNRSV titre, while the transgenic ‘Gisela 6’ had no symptoms and minimal PNRSV titre (Fig. 2).

Figure 2. Transformation of cherry rootstocks (a) shoot regeneration from leaf explants; (b) proliferation of a putative transgenic; (c) rooting of transgenic plants; (d) growing of transgenic plants. The white arrows are showing three procumbent plants derived from one transgenic event of ‘Gisela 6’ (Source: Song et at., 2013)

Citrus

A lot of transformation work has been done on citrus and this helped to overcome several serious problems like disease resistance, abiotic stress tolerance, enhanced flower and fruit characters.

(i) Disease resistance

Development of commercial cultivars with greater resistance to citrus canker is the best strategy for effective disease management. Several attempts were made to impart resistance against citrus canker by transforming citrus with different genes, viz. insect-derived attacin A gene (attA) (Boscariol et al., 2006), Arabidopsis nprl gene (At nprl), (Zhang et ah, 2010), dennaseptin coding sequence (Furman et al., 2013) and Fls2 from Nicotiana benthamiana (Hao et al., 2016).

Citrus mosaic virus (CiMY) is one of the most harmful diseases of citrus. Rootstocks with coat protein gene were transformed which were tolerant to citrus mosaic virus (CiMY) (Iwanami, 2010). Azevedo et al. (2006) produced transgenic Rangpur lime plants with the ‘bO’ gene, for fungus resistance via A. tumefaciens-mediated transformation and evaluated these plants for Phytophthora nicotianae resistance. One of the two transgenic lines showed gr eater tolerance to the fungal pathogen as compared to the control.

RNA-silencing is another genetic engineering technique to knock down the expression of gene. This technique has been used to knock down the ‘acrtsl' transcripts encoding a hydroxylase involved in the biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata. A genomic ВАС clone, containing a portion of the ACRT cluster, was sequenced which led to identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. The functional role of one of these open reading frames, ‘acrtsl ’ encoding a putative hydroxylase, in ACR-toxin production was studied by homologous recombination-mediated gene disruption. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic (Izumi et al., 2012).

(ii) Abiotic Stress Tolerance

Genetic transformation for different abiotic stresses, viz. salt and drought tolerance was earned out. Cervera et al. (2000b) transformed cv. ‘Carrizo’ citrange with the yeast gene ^lhal2 which helps to tolerate salt stress. To enhance drought tolerance and antioxidant enzymatic activity, transgenic ‘Swingle’ citrumelo rootstocks were transformed with the P5CSF129A gene coding for key-enzyme for proline synthesis, under water deficit. It was observed that transgenic plants were able to survive with water deficit better than non-transformed controls. Also proline diminished the deleterious effects caused by oxidative stress due to its ability to increase the activity of antioxidant enzymes (Carvalho et al., 2013).

(Hi) Enhanced Flower and Fruit Characters

Enhanced flowering is an important aspect of fruit production. To achieve this aspect, flower initiation genes, i.e. Arabidopsis leafy (Ify) or the apetalal (apl) (Pena et al., 2001) and Citrus flowering locus T (Cift) (Endo et al., 2009) were genetically engineered in citrus. It was noted that transgenic plants flowered extremely early and started to produce normal fruit within two years of genetic transformation by A. tumefaciens infection.

Another factor contributing to fiuit character is parthenocarpic or seedless fruits. To fulfil this goal, parthenocarpy gene (DefH9-iaaM) and chimeric ribonuclease gene (eg/-400) were introduced into citrus to obtain possible seedless transformants. After A. tumefaciens-mediated transformation, many transgenic citrus lines with seedless trait were obtained (Tan et al., 2012).

Grapes

In the 90s, development of transgenic grapevine rootstocks and scion cultivars (Nakano et al., 1994; Franks et al., 1998) took place. It was noted that efficiency of transformation in grapes was highly influenced by Agrobacterium strain, the genotype of grape and culture conditions (Torregrosa et al., 2002).

(i) Biotic Stress Tolerance

Powdery mildew is a fungus that scars the mature fruit, infects buds and leaves a white powdery coat over grape leaves, leading to leaves falling off that the plant and can no longer produce enough sugar to create wine-quality grapes. To overcome this problem, Yamamoto et al. (2000) introduced rice chitinase gene (RCC2) into the somatic embryos of grapevine by Agrobacterium-vae&iaXed infection. Two transformants showed enhanced resistance against powdery mildew and slight resistance against Elisinoe ampelina inducing anthracnose, leading to reduction in disease lesions. Bomhoff et al. (2000) also attempted to improve fungal resistance by transforming grape cv. Seyval blanc with genes for chitinase and RIP (ribosome-inactivating protein). Transgenic plants were also generated by transferring rice chitinase gene to raise disease-resistant plants. The transgenic plants showed delayed onset of the disease and only small lesions formed after in vitro inoculation of powdeiy mildew. The transgenic plants grew well in the greenhouse without any phenotypic alterations (Nirala et al., 2010). In another study, a susceptible gene 'MLO-T was targeted by Malnoy et al. (2016) in grape cultivar Chardoimay to increase powdeiy mildew resistance. Efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimised alongwith analysis of targeted mutagenesis insertion and deletion rate using targeted deep sequencing. This led to the conclusion that direct delivery of CRISPR/Cas9 ENPs to protoplast system enables targeted gene editing together with generation of DNA-free genome edited grapevine plants.

Another approach of transformation is cisgenesis, which involves isolation and modification of genetic elements from the host genome, which are reinserted to develop plant varieties with unproved characteristics. As a first step towards production of fungal-disease-resistant cisgenic grapevines, the Fitis vinifera thaumatin-like protein (vv?/-l) gene was isolated from cv. ‘Chardonnay’ and re-engineered for constitutive expression in ‘Thompson Seedless’. Among the engineered plant lines of ‘Thompson Seedless’, two exhibited seven to 10 days delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests (Dhekney et al., 2011).

Silencing of conserved root-knot nematodes (RKN) effector gene was achieved through RNA interference (RNAi) for inducing nematode resistance in grape. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, a conserved RKN effector gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic haiiy root lines, carrying either of the two RNAi constructs, showed less susceptibility to nematode infection as compared with control (Yang et al., 2013).

(ii) Abiotic Stress Tolerance

Ferritin helps to protect plant cells from oxidative damage which is induced by abiotic stresses. Keeping in view this point, Medicago sativa (alfalfa) ferritin gene (MsFer) was used to transform Vitis berlandieri x Vitis rupestris cv. ‘Richter 110’ grapevine rootstock lines. The transformants exhibited increased production of ferritin, which led to improved abiotic stress tolerance (Zok et al., 2010).

(Hi) Phenotypic Characters

Key genes responsible for flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), for flavonoid hydroxylation (and also for their stability, colour and antioxidant capacity) have been cloned in red grapevine, cv. Shiraz. Also ectopic expression of their functionality was proven in Petunia hybrida (Bogs et al., 2006). In grape, two kinds of anthocyanin active transporters localiszed to the tonoplast were discovered: two belonging to the Multidrug And Toxic Extrusion (MATE) family called anthoMATEl-3 (AMI andAM3), which can bind acylated anthocyanins and translocate them to the vacuole in the presence of MgATP (Gomez et al., 2009) and an ABC-type transporter, ABCC1, shown to perform the transport of glucosylated anthocyanidins (Francisco et al., 2013). More recently, three GSTs (VviGSTl, VviGST3, VviGST4) have been tested for their ability to bind glutathione and monomers of different phenylpropanoids (anthocyanin, PAs, and flavonols). All the three genes displayed the binding activity with distinct specificity according the phenylpropanoid class (Perez-Diaz et al., 2016).

CRISPR/Cas9 system has been used for efficient knockout of the L-idonate dehydrogenase gene (IdnDH), involved in the tartaric acid pathway (Ren et ah, 2016). In grape, a computational survey of all the CRISPR/Cas9 sites available in the genome revealed the presence of 35,767,960 potential CRISPR, Cas9 target sites, distributed across all chromosomes with a preferential localisation at the coding region level (Wang et ah, 2016). A Grape-CRISPR website of all possible protospacers and target sites was created and made available to the public (http://biodb.sdau.edu.cn/gc/index.html), so that future research can be facilitated (Gascuel et ah, 2017). Nakajima et ah, 2017 also reported successful targeted mutagenesis in grape cv. Neo Muscat using the CRISPR/Cas9 system. They targeted Vitis vinifera phytoene desaturase (VvPDS) gene. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants (Fig. 3).

Kiwifruit

Genetic transformation in kiwifruit has provided many improved varieties with disease resistance, enhanced salt tolerance, altered flower and fruit characters.

(i) Disease Resistance

Kiwifruit was transformed with a soybean [3-1,3-endoglucanase cDNA (Nakamura et ah, 1999) and with a stilbene synthase (sts) gene, responsible for synthesis of resveratrol - an antifungal phytoalexin from Vitis (Kobayashi et ah, 2000). Although only an increased resistance to grey mould disease (Botiytis cinerea) was reported for plants transformed with the endoglucanase enzyme, the plants transformed with ‘sts’ gene from Vitis produced piceid resveratrol - a glucoside, which may confer some beneficial effects on human health to the transformed kiwifruit plants.

Kiwifruit was also transformed with osmotin gene to attain resistant to Botiytis cinerea causing grey mould and Cadophora luteo-olivacea, causing post-harvest problem. Experiment was earned out to evaluate the resistance of stored fruits after separate artificial inoculation with B. cinerea and C. luteo- olivacea. A divergent degree of resistance to fungi and post-harvest damage was detected among the transgenic clones (Rugini et ah, 2011).

(ii) Enhanced Salt Tolerance, Flower and Fruit Characters

For maintaining high Na+/H+ ratio, ‘At nhxP from Arabidopsis, was transferred into kiwifruit by

Figure 3. Schematic representation of transformation and regeneration process for gene editing using CRISPR cas9. Red arrowheads indicate the point of mutation detection (Source: Nakajima el ah, 2017)

Agvobacterium-mediated protocol. Under salt stress, these transgenic lines accumulated more Na+ than control, due to an increased Na+/H+ antiporter activity (Tian et al., 2011). Flowering and fruiting of transgenic plants was obtained within two years of transformation in greenhouse. GUS activity indicating stable expression of the uidA gene was observed in leaf, stem, root, petal and fruit tissues. Transgenic phenotypes were inherited in seedling progeny (Wang et ah, 2012).

Litchi

In litchi, genetic transformation was aimed at imparting disease resistance and to produce parthenogenic fruits.

(i) Disease Resistance

Rice chitinase gene was introduced for antifungal response. The transgenic plants showed delayed onset of the disease and smaller lesions following in vitro inoculation of die-back, leaf spots and blight pathogen (Phoinopsis sp.). The transgenic plants were adapted to the greenhouse and no phenotypic variations were recorded (Das and Rahman, 2010, 2012).

(ii) Parthenocarpic Fruits

One of the objectives of litchi transformation is recovery of parthenocarpic fruits (Yao et ah, 2001). For production of parthenocarpic fruits, embryogenic cultures of ‘Brewster’ (‘Chen Tze’) litchi derived from leaves of a mature tree were transformed with the pistillata (pi) cDNA in antisense orientation through Agrobacterium-mediated transformation. Transgene integration was confirmed by conventional and quantitative PCR (Padilla et ah, 2013) in four transformed lines.

(Hi) Improved Shelf-life

Das et al. (2016) isolated SAMDC cDNA from Datura stramonium and introduced into litchi genome by Agrobacterium tumefaciens through zygote disc transformation. Transgenic plants expressing Datura SAMDC showed 1.7- to 2.4-fold higher levels of spermidine and spermine than wild-type plants demonstrating that increasing polyamine biosynthesis in plants may be a means of creating improved fruit shelf-life.

Mango

Genetic transformation of mango has been based on embryogenic cultures derived from the nucellus of young fruits (Litz, 1984).

The feasibility of gene transfer using the beta-glucuronidase (GUS) reporter gene showed that osmotic treatment and particle acceleration pressure had a major effect on GUS transitory expression (Cruz-Hemaudez et al., 2000: Samanta et al., 2007).

(i) Disease Resistance

Rivera et al. (2011) performed the genetic transformation of somatic embryos of mango cv. ‘Ataulfo’ with the Bell pepper ‘J1 defensin’ gene. In vitro tests showed that protein extracts from transformed embryos inhibited the growth of CoUetotrichum gloeosporioides, Aspergillus niger and Fusarium sp.

Papaya

Papaya is perhaps the only successful example among fruit crops where transgenic plants are being produced commercially and various transformation studies were conducted for biotic stress tolerance and to enhance fruit quality.

(i) Biotic Stress Tolerance

Papaya ring-spot virus is a devastating virus that causes severe damage to the papaya industry. Transgenic papaya cvs. ‘Rainbow’ and ‘SunUp’ resistant to papaya ringspot virus (PRSV) were released in Hawaii in 1998 (Manshardt, 1998). These transgenics were the joint venture of Cornell University, University of Hawaii, the USDA and Upjohn Company in USA to save papaya industry from destruction of PRSV. Some other attempts have also been made till date to provide resistance in papaya by various genes, viz. PRSV (Papaya leaf-distortion mosaic virus) coat protein (cp) (Wei et al., 2008; Rola et al., 2010). No changes to endogenous gene function and no allergenic reactions were predicted from analysis of the insertion site and flanking genomic DNA in transgenics, thus supporting a positive review of the appeal for importing and consumption of transgenic cv. ‘Rainbow’ and its derivatives (Fig. 4).

Several fungal diseases cause great damage to the papaya industiy. So transgenic papaya plants were generated with stilbene synthase gene cloned from grapevine to impart fungal disease resistance. Greenhouse studies showed that the disease levels in transgenic plants were reduced to 35 per cent of the disease levels in non-transformed control plants (Zhu et al., 2010).

Commercial papaya cv. ‘Kapoho’, which is highly susceptible to mites, was transformed with the snowdrop lectin (Galanthus nivalis agglutin [gyta]) gene having insecticidal activity towards sap-sucking insects. A laboratory bioassay using carmine spider mites recorded improved pest resistance in the transgenic papaya plants (McCaffeity et al., 2008).

(ii) Enhanced Fruit Quality

Through anti-sense RNA technology, down regulation of the ‘ACC (Aminocyclopropane-1-carboxylic acid) oxidase’ gene (responsible for the last step in ethylene formation) resulted in the suppression of ethylene production, thereby delaying fruit ripening thereby allowing more accumulation of amino acids and sugars, resulting in better quality papaya (Che et al., 2011).

Passionfruit

Transformation of passionfruit mediated by A. tumefaciens as well as direct methods has been reported for disease resistance and to enhance phenotypic characters.

(i) Disease Resistance

Trevisan et al. (2006) transformed passionfruit with a sequence derived from the replicase and coat protein (cp) genes from passionfruit woodiness virus (PWV) and the preliminary results suggested that this strategy could be used to control this virus disease. Particle bombardment method of gene transfer was used to transfer the bactericide ‘ attacin A ’ gene driven by 3 5 S promoter to yellow passionfruit (Vieira et al., 2002), conferring resistance to Xanth от on as campestris pv. passiflorae.

(ii) Enhanced Phenotypic Characters

A. rhizogenes-mediated transformation of passionfruit species was checked using suspension culture. Hairy roots, differentiated at the inoculation sites to establish individual root clones were used to initiate long-term cultures on semi-solid medium. The clones retained their high growth rates and antibiotic

Figure 4. The process of genetic engineering in papaya (Source: Wieczorek and Wright, 2012)

resistance phenotypes. The regenerated roots displayed typical features of hairy roots, such as hairiness, branching and growth habit (Reis et al., 2007).

Peach

Though several reports of Agrobacterium-mediated genetic transformation of mature and immature peach tissues have been reported in the past, however, no transgenic plants were recovered and limited data were presented as evidence of transformation and stable integration of foreign DNA into the peach genome. Nevertheless, work is going on for development of efficient transformation protocols in peach.

Based upon green fluorescent protein (gfp) technology, Perez-Clemente and workers (2004) have developed A. tumefaciens based efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. Survived shoots exhibited high-level of 'gfp' expression mainly visible in the young leaves of the apex. In vivo monitoring of ‘gfp’ expression reported an early, rapid and easy discrimination of both transgenic and escape buds. High levels of 'gfp' expression were also maintained in the second generation of transgenic peach plants. Hence it is now possible to produce transgenic peach plants throughout the year without the need to isolate immature seeds from which the regeneration of transgenic plants and recovery of non-chimeric plants is difficult.

Padilla et al. (2006) performed a strategic study using Agrobacterium mediated transformation and gfp markers to assess transformation efficiency of various bacterial strains and explants. Highest rates of transformation were achieved with a combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter in peach epicotyl intemodes (56.8 per cent), cotyledons (52.7 per cent), leaves (20 per cent), and embryonic axes (46.7 per cent). The study showed that intemodes, cotyledons, and embryonic axes were superior to embryonic hypocotyls. Still, further optimisation will be required to make peach transformation and regeneration a routine.

Pear

In pear, Agrobacterium-mediated genetic transformation was attempted to impart disease resistance, abiotic stress tolerance and to enhance phenotypic characters.

(i) Disease Resistance

Transformation for disease resistance in pear against fire blight (caused by Envinia amylovora) was done by Malnoy et al (2005) and Djennane et al. (2009), who used an exopolysaccharide (eps) depolymerase and ferritin gene from pea.

(ii) Abiotic Stress Tolerance

Over-expression of the apple spermidine synthase (Mdspdsl) gene in pear conferred salt and multiple abiotic stress tolerance by altering polyamine titers in the plants. Selected lines were exposed to salt, osmosis and heavy metal stresses for evaluating their stress tolerances. One of the transgenic lines which was revealed to have the highest spermidine synthase accumulation and expression level of ‘Mdspdsl ’, showed the strongest tolerance to these stresses (Wen et al, 2008, 2009).

(Hi) Enhanced phenotypic characters

Rooting ability of dwarfing sweet cherry hybrid rootstock (Prunus cerasus x P. canescens) and pear rootstock (Cydonia oblonga R Mill) was improved by transforming them with the ‘rolB ’ gene (cloned from A. rhizogenes plasmid pRiA4) using A. tumefaciens mediated gene transfer. As compared with the control, after co-cultivation with A. tumefaciens, the rooting rate of Cydonia regenerants in vitro increased by 6-44 per cent and of Prunus hybrid by 8-30 per cent. All transformants had well-formed roots (Staniene et al, 2007).

Matsuda et al. (2009) transformed European pear ‘La France’ and ‘Ballade’ with the citius flowering locus T (Cift) gene, which induces early flowering. Of the seven seedlings that expressed the ‘Cift’ gene, five flowered within 10 months after their transfer to greenhouse, indicating that the ‘Cift’ gene induced early flowering in the transformed pear plants. Usually pear plants flower after four to five years.

A transgenic line of ‘Spadona’, named Early Flowering-Spadona (EF-Spa), was produced using an ‘Mdtfll' RNAi cassette targeting the native pear genes ‘Pctfll-Г and ‘Pctfll-2’ to cut short the juvenile period. Pollination of ‘EF-Spa’ trees generated normal-shaped fruits with viable F₍ seeds. The greenhouse-grown transgenic Fj seedlings formed shoots and produced flowers one to 33 months after germination (Freiman et al, 2012).

To inhibit the browning process in fruits of Yali pear, Antisense gene technique was used to reduce the expression of polyphenol oxidase (ppo) gene. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased compared with the non-transformed shoots. This has paved the way for breeding new varieties of pears with browning resistance in future (Li et al, 2011).

Persimmon

There are very few studies on genetic transformation in persimmon. Some studies with respect to abiotic stress tolerance and enhanced phenotypic characters are as follows:

(i) Abiotic Stress Tolerance

Gao etal. (2000) were the first to successfully introduce the cod Agene for choline oxidase оiArthrobacter globiformis into persimmon cv. Jiro by Agrobacterium-mediated transformation using leaf discs as explants. Regenerants were recorded with the ability to synthesize glycinebetaine, hence, found salt stress tolerant.

(ii) Enhanced Phenotypic Characters

Studies conducted by Koshita etal. (2002) led to successful introduction of rolC gene from Л. rhizogenes into cv. Saijo, indicating the possibility of producing dwarf plants. Recently, persimmon has been transformed with the gene encoding the pear fruit polygalacturonase inhibiting protein (PGIP) using A. tumefaciens EHA101 to enhance shelf-life of the fruit (Tamura et ah, 2004).

Gao et al. (2013) transformed the Japanese persimmon with Arabidopsis flowering locus T gene (Atft), and ‘PmtflP gene- a Prunus mume ortholog of Arabidopsis terminal flower 1 (tfll) gene. Ten lines of transgenic ‘PmtflP shoot and two lines of transgenic Atft' shoot were obtained. The ‘Pmtfll’ transgenic in vitro shoots did not show a different appearance compared with non transformed ‘Jiro’ shoots, however, the Atft 'transgenic shoots showed a bushy phenotype with the short intemodes.

Pineapple

Pineapple has been transformed by microprojectile bombardment and by co-cultivation with A. tumefaciens to impart disease resistance, herbicide tolerance and enhanced quality traits.

(i) Disease Resistance

Stewart et al. (2001) cloned a polyphenol oxidase (ppo) gene from pineapple fruits under conditions that produce blackheart, a fruit defect. The ‘ppo’ gene has been silenced in transformed plants. A comparative analysis of transgenic pineapple lines [silenced for polyphenol oxidase (ppo)} gene and the untransformed control lines revealed that all of the control lines expressed blackheart and exhibited the greatest incidence and severity, while the transgenic lines were regarded as blackheart resistant, having no blackheart symptoms (Ко et al., 2013).

(ii) Herbicide Tolerance

Pineapple plants transformed with the ‘bar' gene for bialaphos herbicide resistance were evaluated for transgene stability, gene expression and tolerance to gluphosinate ammonium, which is an active ingr edient of herbicide BastaReg. X, under field conditions. Genetically-modified plants remained green and healthy following spraying with the herbicide. In contrast, non-transformed pineapple plants of the same cultivar became necrotic and died within 21 days of spraying of the herbicide (Sripaoraya et al., 2006).

(Hi) Quality- Traits

Leaf bases of Pineapple cv. Queen were transformed with soybean ferritin cDNA. Few of the transgenic plants were hardened in the greenhouse and were grown to maturity to determine the enhanced iron and zinc accumulation in the fruits (Mhatre et al., 2011).

In the USA, pineapples were genetically modified for viral and nematode resistance, delayed maturation, modified sugar composition and flowering time (Hanke and Flachowsky 2010). Recently, the Del Monte company obtained red-fleshed pineapple named ‘Rose’ by combining over-expression of a gene derived from tangerine and suppression of other genes in order to increase the accumulation of lycopene (Ogata et al., 2016).

Plum

Plum producers world-wide are facing multiple challenges including climate change, the need for reduced chemical inputs, the spread of native and exotic pests and pathogens, and consumer demands for improved fruit quality and health benefits. In an effort to develop new approaches genetic engineering was proved a successful technology.

(i) Disease Resistance

The USDA-ARS Appalachian Fruit Research Station fruit-breeding programme in collaboration with partners in the USA and Europe have developed a genetic-engineering approach to target resistance to Plum pox virus (PPV) by transferring Plum pox virus-coat protein gene (PPV-cp). This program has resulted in the development of a transformed plum cultivar ‘HoneySweet’ which has been tested for 15 years in the European Union and USA and is highly resistant to PPV. ‘HoneySweet’ has received foil regulatory approval in the USA and represents a new source of PPV resistance (Hily et ah, 2007; Mikhailov et al., 2012; Scorza et ah, 2013).

Ravelonandro et ah (2011) studied the heritability of the virus transgenes engineered in ‘HoneySweet’ plum through different crosses with two commercial cultivars of Prunus domestica (‘Pranier d’Ente 303’ and ‘Quetsche 2906’) and one wild species, P spinosa 2862 rootstock using ‘HoneySweet’ plum as the pollen donor. As much as 46 per cent of the F_t progeny was transgenic. These results confirmed the high potential of ‘HoneySweet’ plum for PPV resistance breeding programmes.

Two transgenic lines of plum cv. ‘Stanly’ expressed Gastrodia antifungal gene (gafp-1) and recorded to exhibit resistance to the pathogen Phytophthora cinnamomi and the root-knot nematode Meloidogyne incognita. ‘gafp-Г lectin was identified within the roots, but not in the soft shoot or leaf tissues of the grafted, wild type scions. These results suggest that gafp-1 mRNA and protein are not moving into the wild type scion tissues of chimeric-grafted plum trees (Nagel et ah, 2010). Rootstocks created from such transgenic lines will be more readily accepted by consumers, proving that foreign gene products are not migrating into a grafted, nontransgenic scion on which fruit is produced and they express only in the rootstock portion where they are required.

(ii) Enhanced fruit quality

Plum hypocotyls were transformed with an antisense peach ‘ACC oxidase’ gene (responsible for the last step in ethylene formation). Fruit quality data consisting of fruit firmness, colour, date of ripening, brix, and size as well as ethylene production rates were measured for two years on the fruiting lines which suggested that in some transgenic lines, ethylene production as well as softening were delayed relative to the ‘Bluebyrd’ parental line (Callahan and Scorza, 2007).

The flowering locus T1 (ftl) gene from Populus trichocaipa under the control of the 35S promoter was introduced into the European plum. Transgenic plants expressing higher levels of ‘ft’ flowered and produced fruits in the greenhouse within one to ten months. Plums with ‘ft' gene did not enter dormancy after cold or short day treatments, yet field planted ft' plums remained winter hardy down to at least -10°C. The flowering and fruiting phenotype was found to be continuous in the greenhouse but limited to spring season and they tend to fall in the field (Srinivasan et ah, 2012).

Strawberry

Efficient genetic transformation protocols have been used to impart biotic and abiotic stress tolerance and enhanced fruit quality in strawberry.

(i) Biotic Stress Tolerance

The CP4EPSP synthase gene, which confers resistance to glyphosate, an active ingredient of herbicide Roundup, was introduced into cv. Camarosa through Agrobacterium-mediated transformation. The transformants showed a range of tolerance to the herbicides ranging from complete tolerance to death (Morgan et ah, 2002). 19 independent transgenic lines of cv. Firework and 15 lines of cv. Selekta were obtained via Agrobacterium-mediated transformation for taste improvement and enhanced disease resistance by introduction of thaumatin II (thaull) gene (Schestibratov and Dolgov, 2006).

Modification of strawberry plants by introducing the stilbene synthase (sts) gene resulted in production of the phytoalexin resveratrol and provided enhanced resistance against several pathogenic fungi (Hanlnneva et ah, 2009). An effective system for chitinase (chit42) gene transformation mediated by A. tumefaciens was determined to obtain a disease-resistant strawberry plant (Xie et ah, 2008).

Table 1. Genetic Transformation Work done in Fruit Crops

(ii) Abiotic Stress Tolerance

A foreign target-gene of cold-inducible transcription factor CBF1 (C-repeat binding factor) from Arabidopsis thaliana was introduced into strawberry by using the leaf-disc method via A. tumefaciens. All transgenic and control plants were subjected to a temperature of -2°C for seven days. It was noted that 60 per cent of transgenic plants and 85 per cent of control plants wilted at low temperature (Jin et al., 2007). Over-expression of tobacco ‘osmotin’ gene led to increased salt stress tolerance, total soluble protein and chlorophyll content as compared to the wild plants in strawberry (Husaini et al., 2012).

(Hi) Enhanced Fruit Quality

Improving the storing quality of strawberry fruit is a difficult problem. This is because of ethylene production which decreases shelf-life of fruit. Applying antisense gene strategy to control ethylene receptor is a new feasible method. Two antisense ethylene receptor genes Fragaria ananassa ethylene receptor ‘FaEtrl’ and ‘FaEr2’ were introduced into leaves of strawberry with A. tumefaciens EHA105. Northern blot analysis indicated that ‘FaEtrV and ‘FaEr2’ mRNA abundance was decreased in antisense strawberry plants (Zhu et al., 2009).

‘rolC’ lines of strawberry cv. Calypso were produced by genetic transformation using A. tumefaciens. Yield and fruit quality of the control and transgenic lines were measured under open-field conditions, which showed 30 per cent greater yields in transformants than controls, due to 20 per cent more fruits per plant and an increased fruit weight along with better fruit quality. No significant increase towards tolerance to Phytophthora cactorum and symbiosis with root arbuscular mycorrhizal fungi (AMF) was observed (Landi et al., 2009).

RNAi mediated transformation was used to down-regulate the genes ‘FaplC’ and ‘FaE3’ encoding a pectate lyase and a polygalacturonase enzyme, respectively. Notable reduction towards the loss of firmness at the red ripe stage was observed (Garcia et al., 2012; Youssef et al., 2013). Bulley et al. (2012) increased ascorbate (vitamin C) in strawberry, using a transgene, GDP-l-galactose phosphorylase (ggp).

3. Overview and Future

In fruit crops, widely used methods of transformation are Agrobacferinm-mediated gene transfer and microprojectile bombardment. In nature, Agrobacterium infects host plants where there is an injury and liberation of phenolic compounds. The addition to the culture medium of phenolic compounds like acetosyringone stimulates transcription of virulence genes in Agrobacterium. Together with the gene of interest, other genes required for transformation are transferred including marker genes that allow selection of transformed cells. However, due to public concern with the introduction of antibiotic resistance genes into food, methods to eliminate them from the transformed plants and strategies that avoid selection of transformed cells with antibiotics are being developed. These new alternative methodologies have only begun to be applied to the production of transformed fruit trees.

One of such methodology is production of marker-free transgenics. Marker-free transgenic crops confer several advantages over transgenic crops containing selection genes coding, for example for antibiotic resistance. Efforts are on to avoid or minimise the inclusion of transgenes or sequences by promoting the use of clean vector systems. A positive selectable marker gene such as mannose A (manA) that encodes for phosphomannose isomerase and provides transformed cells with a metabolic advantage over non-transformed cells was tested. The second strategy is the use of reporter genes, such as green fluorescent protein gene (gfp), b-glucuronidase (GUS), b-galactosidase (LacZ) and luciferase (LUC) for screening of transgenic plants.

In conclusion, transformation and regeneration of fruit crops especially tree crops is not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for breeding of fruit crops requires the development of genotype-independent procedures based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration- promoting genes. Yet another obstacle is that international laws will neither allow deliberate release of plants carrying antibiotic resistant genes nor their commercialiszation. Therefore, development of procedures to avoid the use of antibiotic-based selection or to allow elimination of marker genes from the transformed plant will be a research priority in the coming years.

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