The Journal of Genetics. Photon 115 (2014)146-163
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Original Research Article. ISJN: 4255-7158: Impact Index: 4.28
The Journal of Genetics
Ph ton
Genetic engineering in fruit crops
Disket Dolkar*, Parshant Bakshi, V. K Wali
Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Main Campus, Chatha, Jammu and Kashmir-180009, India
Disket Dolkar*, Parshant Bakshi, V. K Wali receive International Horticulture Research Award-2014 Article history:
Received: 7 August, 14 Accepted: 12 August, 14
Available online: 25 November, 2014 Keywords:
Diseases resistance, Abiotic stress, Genetic transformation
Corresponding Author: Dolkar D.*
Ph.D. Scholar
Email: [email protected] Bakshi P
Associate Professor, Fruit Science V. K Wali
Professor & Head, Fruit Science Abstract
Genetic engineering extends tremendous scope and opportunities in fruit production by providing new genotypes for breeding purpose, supply of healthy and disease free planting material, improvement in fruit quality, enhancing shelf-life,
availability of biopesticides, biofertilizers, etc. Integration of specially desired traits through genetic engineering has been possible in some horticultural crops. Recent advancements in molecular biology and genetics transformation have made it possible to identify, isolate and transfer desirable genes from any living organism to plants. The introduction or enhancement of desirable traits is traditionally done by breeding but it is time consuming and is not very precise. On the other hand, genetic engineering creates plants with specific changes in the background of a proven cultivar without disturbing their genetic constitution. Expression of undesirable genes can be blocked by the application of antisense gene technology and RNAi technology. Genetic transformation provides the means for modifying horticultural traits in various horticultural crops without altering their phenotype.
Citation:
Dolkar D., Bakshi P., V.K Wali., 2014. Genetic engineering in fruit crops. The Journal of Genetics. Photon 115,146-163
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1. Introduction
Rapid increase of human population together with global climate variability resulted in increased demand of plant based food and energy sources (Varshney et al., 2011). Fruit being a major source of micronutrients (e.g. vitamins and minerals), phytonutrients (e.g. antioxidants) and dietary fibers have essential role to enhance quality of humankind life since a diet on cereal grains and legumes is generally lacked a wide range of product that exist in fruit species (Heslop-Harrison, 2005). However, production has been often suppressed by climate change and various diseases caused by fungi, bacteria, and viruses etc. Therefore, developing fruit crops that are better adapted to biotic and abiotic stress is a key issue for the plant scientist to increase fruit yield in many parts of the world
today. During the past, some progress was made through conventional breeding with regard to these problems. However, has had little success in improving fruit plants and is constrained (a) due to long juvenile period, breeding programs for such plants can involve the professional lifetimes of several generations of scientists (b) erosion of naturally occurring genetic variability (c) transfer of undesirable genes along with desirable traits and (d) reproductive obstacles that limit the transfer of favorable alleles from diverse genetic resources (Varshney et al., 2011; Gambino and Gribaudo, 2012; Rai and Shekhawat, 2013). Tissue culture based technologies including somatic hybridization (Grosser et al. 2010), in vitro selection (Rai et al., 2011), haploid and double haploid
production (Germana, 2011), encapsulation technology (Rai et al., 2009) have also been applied to some fruit plants for crop improvement. Genetic engineering methods have opened new avenues to modify crops and provide new solution to slove specific needs (Rao et al., 2009). Contrary to conventional plant breeding, this technology can integrate foreign DNA into different plant cells to produce transgenic plants with new desirable traits (Newell, 2000). These biotechnological approaches are a great option to improve fruit genotypes with significant commercial properties such as increased biotic (resistance to disease of virus, fungi, pests and bacteria) (Ghorbel et al., 2001; Fagoaga et al., 2001; Fagoaga et al., 2006; Fagoaga et al., 2007) or abiotic (temperature, salinity, light, drought) stress tolerances (Fu et al., 2011); nutrition; yield and quality (delayed fruit ripening and longer shelf life) and to use as bioreactor to produce proteins, edible vaccines and biodegradable plastics (Khandelwal et al., 2011). In the last 20 years, genetic transformation of fruit crops has focused mainly on enhancing disease resistance (viruses, fungi, and bacteria), increasing tolerance of abiotic stresses (drought, frost, and salt), modified plant growth habit and fruit quality, although there are few cases of field evaluation and commercial application of these transgenic plants (Gomez-Lim and Litz, 2004; Petri and Burgos, 2005; Gambino and Gribaudo, 2012; Litz and Padilla, 2012). The present communication provides an overview on the progress achieved in recent years on the development of genetically engineered fruit plants tolerant to biotic and abiotic stresses, as well as the efforts made to improve fruit quality, extend post-harvest shelf life of fruits and reduction of generation time.
2. The technique
Transgenic plants have been developed through a number of gene delivery methods. but Agrobacterium- and particle bombardment (also called “biolistic” or “bioballistic”) mediated gene transfer are the most popular methods for the development of transgenic plants. The technique chosen for transformation has its own characteristics that need to be evaluated. In nature the soil-borne Gram negative bacteria of the genus Agrobacterium infect a wound surface of the plants via a plasmid called Ti-plasmid containing three genetically important elements; Agrobacterium chromosomal virulence genes (chv), T-DNA (transfer DNA) and Ti plasmid virulence genes
(vir) that constitute the T-DNA transfer machinery. Since Ti plasmid encodes mechanisms of integration of T-DNA into the host genome, it is used as a vector to transform plants. Since direct gene transfer procedures involve intact cells and tissues as targets, in some species breaching of the cell wall is needed in order to enable entrance of DNA to cell (Petolino, 2002). This is accomplished by making some degree of cell injury or totally enzymatic degradation of the cell wall. Advantages of microprojectile bombardment can be summarized as i) transfer of multiple DNA fragments and plasmids with co-bombardment, ii) unnecessity pathogen (such as Agrobacterium) infection and usage of specialized vectors for DNA transfer (Veluthambi et al., 2003). Although microprojectile bombardment eliminates species-dependent and complex interaction between bacterium and host genome, stable integration is lower in this technique in comparison to Agrobacterium-mediated transformation (Christou, 1992). Moreover, the existence of truncated and rearranged transgene DNA can also lead transgene silencing in the transgenic plants (Paszkowski & Witham, 2001). On the other hand, other important requirement for this technique is that the explants or target cells have to be physically available for the bombardment (Hensel et al., 2011). Nevertheless, application of both of the techniques for the transfer of foreign DNA results in “transient” or “stable” expression of the DNA fragment. In the following sections, recent advances in genetic transformation of fruit species are presented. 3. Target traits for crop improvement 3.1 Disease resistance
In the last two decades, considerable efforts have been made on the development of transgenic fruit plants utilizing a broad range of genes to enhance disease resistance against fungal, bacterial and viral pathogens. Plants are generally susceptible to virus diseases and perennial crops, in particular, are more exposed to infection because of their long life span. One of the major limitations of transgenic approaches for disease resistance is that some defense responses are only effective against certain pathogens (Punja, 2001). Furthermore, there is a huge variation (taxonomic and physiological) in lifestyle among fungal, bacterial and viral pathogens, which made it impossible to develop effective broad spectrum disease resistance. There are also some challenges like risks to the environment and the consumer that need to be
addressed when introducing disease resistance trait into a plant species (Collinge et al., 2010).
Despite a number of social, environmental and biological concerns as well as experimental barriers, many fruit plants have been transformed by introducing a broad range of genes to develop disease resistant plants against fungal, bacterial and viral pathogens. Protection of plants against viral infection was one of the first and fundamental purposes of transgenic research on fruit trees and is also one of the first commercial applications of transgenic technology in plant. However, early attempts to achieve virus resistance by introduction of virus-derived sense or antisense sequences were not always successful. In retrospect, some of the unexplained effects observed in coat protein (CP)-mediated resistance techniques seem to have been caused by post-transcriptional gene silencing (PTGS) by accidental formation of double-strand RNA (dsRNA).
Among early and most important applications of transformation in fruit trees was the transgenic papaya resistant to Papaya ringspot virus (PRSV). PRSV is a Potyvirus non-persistently transmitted by aphids, which destroys the photosynthetic capacity of the canopy leading to reduction of fruit quality and yield, loss of vegetative vigour, and eventual mortality. Fitch et al. (1990) obtained transgenic plants expressing the sense CP gene of PRSV, and showed that line 55-1 was highly resistant to PRSV in greenhouse and field experiments. Two cultivars developed from line 55-1 (‘SunUp’, homozygous for the CP gene, and ‘UH Rainbow’, a F1 hybrid between ‘SunUp’ and the nontransgenic ‘Kapoho’ cultivar) were produced, authorized, successfully cultivated, and commercialized, and contributed to saving the papaya industry in Hawaii. Unfortunately, line 55-1 is susceptible to a number of PRSV isolates from other geographic regions, and several laboratories developed more transgenic lines effective against other PRSV isolates (Bau et al., 2003).
In papaya it is necessary to fix the transgenic resistance through backcross with parental lines to obtain hermaphrodite plants with desirable horticultural traits, the progenies then being used for generation of commercial hybrid cultivars. To significantly shorten this time-consuming breeding program, Kung et al. (2010) successfully transformed for PRSV and Papaya leaf-distortion mosaic virus (PLDMV)
resistance different commercial hermaphrodite papaya cultivars with desired horticultural features. Ming et al. (2008) reported a 39 draft genome sequence of ‘SunUp’ papaya, the first virus resistant transgenic fruit authorized for field cultivation and commercialization.
After this first success, many other transgenic fruit plants protected against viruses were produced. For instance, Citrus was transformed with the CP gene of Citrus tristeza virus (CTV), an aphid-transmitted Closterovirus, the causal agent of one of the most economically important diseases of Citrus. Dominguez et al. (2002) demonstrated PDR (pathogen derived resistance) in transgenic Mexican lime (C. aurantifolia (Christ.) Swing.) carrying the CTVCP gene. Mexican limes were transformed also with other regions of the CTV genome in sense, antisense, and hairpin forms (Lopez et al., 2010). All sense, antisense, and empty-vector transgenic lines were susceptible to CTV, whereas several hairpin lines had CTV resistance.
The authors showed that the resistance was always associated with the presence of transgene-derived siRNAs, but was not related to their levels. CTV resistance was also correlated with low accumulation of the transgene-derived transcripts rather than with high accumulation of siRNAs. The authors suggested that only a fraction of transgene derived siRNAs are indeed competent for RNA Lopez et al. 2010. silencing, the others being quickly degraded. Other approaches against CVT utilized in recent years are the production of cisgenic plants and transformation with single-chain variable fragment (scFv) antibodies. Cervera et al. (2010) reported the first example of successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies. scFv constructs against CTV p25 major CP were inserted in Mexican lime. The transgenic lines were challenged by CTV by graft-inoculation and most transgenic citrus lines had resistance and/or tolerance.
However, the resistance in most lines was only partial, probably because of the inoculation system used: graft-inoculation was very aggressive in terms of inoculum dose and permanent source of virions, whereas aphid transmission under natural conditions results in inoculation of a limited number of virions. The immune-modulation strategy seems to be effective and may be an alternative to the use of the viral genome (i.e. cross-protection or
PDR) for the control of CTV. However, the mechanisms that confer CTV-resistance by scFv are not completely understood: probably the recombinant antibodies interfere with virus assembly, spread, and pathogenesis, but further experiments are required to elucidate this aspect. In addition, the recombinant antibody approach could raise further ethical objections against open-field culture of the resulting transgenic plants, because of the non-plant origin of the transgenes.
In another case, Plum pox virus (PPV), the causal agent of the Sharka disease, is a major constraint to Prunus production, and genetic transformation of Prunus has focussed almost exclusively on the induction of resistance to PPV (Petri and Burgos, 2005). Scorza et al. (1994) transformed plum (Prunus domestica L.) and apricot (Prunus armeniaca L.) with the CP of PPV. The transgenic plum clone C5 had high resistance to PPV whether inoculated by aphids or by chip budding. The transgenic plum clone C5 were characterized by the production of a long-size class of 24-nt siRNAs where as non-transformed plum trees react to viral infections by initiating PTGS-like mechanisms involving the production of 21-nt siRNAs (Hily et al., 2005).
Field tests on C5 and other transgenic lines performed in Poland, Romania, and Spain demonstrated that C5 trees exposed for several years to natural infection did not become infected, whereas susceptible transgenic and wt trees developed severe symptoms within the first year (Hily et al., 2004). Although highly resistant in field tests, C5 trees could be artificially infected by chip budding or via susceptible rootstock. Even 8 years after virus inoculation, infected C5 trees had only a few mild symptoms on single isolated shoots, indicating the long-term nature and high level of resistance to PPV (Malinowski et al., 2006).
Scorza et al. (2013) after extensive testing and risk assessment in laboratory, greenhouse and in the field for over 20 years, ‘HoneySweet’ plum, a plum pox virus (PPV) resistant transgenic plum, has now been validated for cultivation in the USA. Despite several positive results, induction of resistance in transgenic plants does not always result in effective protection against the target virus. In grapevines (Vitis spp.) at least sixty different viruses have been documented (Martelli, 2009), infecting these species and causing estimated global losses of over 1 billion US dollars. Notwithstanding the substantial efforts
made in recent years, and with the partial exception of some rootstocks resistant to Grapevine fan leaf virus (GFLV) (Vigne et al., 2004), transgenic virus-resistant grapevines have not yet been obtained, confirming the complexity of this plant–pathogen relationship. Transgenic grapevines containing the GFLV-CP gene and with transgene silencing were unable to withstand spread of the virus after simple graft inoculation (Gambino et al., 2010). In these transgenic grapevines lines the correlation between accumulation of siRNAs, transgene methylation, and RNA silencing could not be confirmed.
Highly methylated cytosine residues were detected in the GFLV-CP transgene, in the terminator, and in the 35S promoter of grapevines without transgene expression, but no detectable level of siRNAs was recorded. The susceptibility to GFLV could be because of both the high viral inoculum and the constant viral pressure from the rootstock applied to relatively young and small plants. Under these conditions, transgenic grapevines might be unable to suppress GFLV replication, as reported above after the graft-inoculation of transgenic Mexican lime (Cervera et al., 2010) and plum (Hily et al., 2004).
In addition to viruses, another important purpose of research on fruit trees is resistance to fungal and bacterial infections. For fungal and bacterial diseases, the following four strategies have been applied to make disease resistant transgenic plant: transgenic disease-resistant plants used (1) genes encoding pathogenesis-related proteins (PR proteins), antimicrobial peptides or antimicrobial metabolites (2) genes that encode detoxification mechanisms (3) genes that have a role in pathogen recognition and (4) genes that regulate defense mechanisms (Collinge et al. 2010).
The most widely used transgenic approach to enhance resistance against fungal diseases has been based on the over-expression of PR proteins (Kovacs et al., 2013). The production of hydrolytic enzymes like chitinase and glucanase, best characterized class of PR proteins, capable of degrading the cell wall of invading pathogenic fungi is an important component of the defense response in plants against fungal pathogens (Neeraja et al., 2010; Ceasar and Ignacimuthu, 2012). Transgenic plants expressing chitinase and glucanase gene showed enhanced resistance to fungal disease in many fruit plants (Nookaraju and Agrawal, 2012; Gambino and
Gribaudo, 2012; Litz and Padilla, 2012). Gentile et al. (2007) introduced the chit42 gene from Trichoderma harzianum into ‘Femminello siracusano’, one of the best Italian lemon (Citrus limon (L.) Burm.f.) cultivars. Significantly less lesion development was observed for leaves of transgenic lemon plants inoculated with Botritis cinerea than for wt leaves.
Overexpression of the transgenic fungal gene enhanced transcript levels of genes associated with production of reactive oxygen species (ROS) and induced establishment of systemic resistance whereas expression of native chitinase and glucanase genes involved in systemic acquired resistance (SAR) was down-regulated. In recent years, the use of antimicrobial peptides constitutively expressed in plant tissues has been recommended for the genetic engineering of plants for disease resistance against fungal and bacterial pathogens (Collinge et al. 2010).
Defensins, one of the classical examples of small antimicrobial peptides, play an important role in the plant defense response against fungal targets. Defensins interact with fungal-specific membrane components and subsequently permeabilize them to inhibit fungus growth (Stotz et al., 2009; Coninck et al., 2013). Recently, Ghag et al. (2012) reported the over-expression of two Petunia floral defensin genes (PhDef1 and PhDef2) in transgenic banana plants. In vitro and ex vivo bioassays performed in this study clearly indicate that the transgenic banana plants were resistant against fungal pathogen
Fusarium oxysporum f. sp. cubense. There are
some other examples also where the antimicrobial protein originates from insects or other animal system. Many non-plant antimicrobial proteins like Attacin, Cecropin, Magainin with antimicrobial activity and their expression in transgenic fruit plants have been reported (Cardoso et al., 2010; Collinge et al., 2010; Mondal et al., 2012).
Phytoalexins, an excellent example of antimicrobial metabolite, are an important component of plant defense and many studies demonstrated that phytoalexins contribute significantly to resistance against pathogens (Ahuja et al., 2012). However, the production of antimicrobial metabolites usually requires the coordinated action of a number of biosynthetic enzymes and many genes are necessary to encode the subunits of these enzymes. Furthermore, biosynthetic pathways for many of antimicrobial proteins have not
been well characterized; therefore the genes encoding these enzymes are not often available (Collinge et al., 2010).
Resveratrol is a phytoalexin produced naturally in a number of plants against the growth of fungal or bacterial pathogens. In grapevine, Fan et al., (2008) found that transgenic plants expressing stilbene synthase (STS) gene showed fivefold more resveratrol content than non-transgenic plant. stilbene synthase gene (Vst1) from V. vinifera is responsible for the synthesis of the phytoalexin resveratrol in grapevines and the introduction of a single gene was sufficient to synthesize the compound in heterologous plant species. In addition to the antifungal activity, resveratrol is regarded as having beneficial effects on human health, because of its antiinflammatory, antiplatelet, and anticarcinogenic activity (Delaunois et al., 2009).
Apple scab, caused by the fungus Venturia inaequalis (Cooke) G. Wint., is the most widespread disease in apple orchards worldwide. Although scab-resistant apple cultivars (e.g. ‘Liberty’ and ‘Florina’) have been bred by introgressing the Vf gene for scab resistance from the small fruited crabapple Malus floribunda clone 821; these cultivars were not commercially successful, partly because of insufficient fruit quality. Szankowski et al. (2009) obtained cisgenic apples resistant to scab by inserting the HcrVf2 gene from Malus floribunda 821 and its native promoter into susceptible apple cultivars.
Highly scab resistant ‘Elstar’ and ‘Gala’ plants were obtained, proving that the HcrVf2 gene controlled by its native promoter is effective in conferring resistance to V. inaequalis similarly to Vf introgressed in apple through classical breeding. Joshi et al. (2011) demonstrated that the resistance provided by the Vf cluster is only from HcrVf2 (not from HcrVf1) and that high levels of gene expression did not help in conferring resistance against Vf virulent isolates of V. inaequalis, but is helpful for resistance against Vf avirulent isolates. NPR (non-expressor of pathogenesis-related genes) was identified as a key factor in transducing the signal leading to SAR and has been demonstrated to induce enhanced fungal and bacterial resistance through elevated expression of PR genes. Malnoy et al. (2007) cloned and inserted the MpNPR1-1 gene in apple to increase resistance to V. inaequalis and also to Erwinia amylovora, responsible for
bacterial fire blight. Among the bacterial diseases affecting perennial fruit species, fire blight caused by Erwinia amylovora is particularly destructive for apple and pear (Pyrus communis L.).
Against fire blight, in addition to classical lytic enzymes, for example cecropins and magainins (Petri and Burgos, 2005; Bhatti and Jha, 2010), Flachowsky et al. (2008) transformed apple with a gene coding for an extracellular polysaccharide (EPS)-depolymerase from the E. amylovora phage phi-Ea1 h. Bacteriophages such as phi-Ea1 h carry an EPS-depolymerase which binds to the capsular EPS and degrades the bacterial polysaccharide that is a major virulence factor of E. amylovora. Insertion of the Leaf Colour (Lc) gene from maize (Zea mays) induced strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins) in transgenic apple (Flachowsky et al., 2010).
These transgenic plants had altered phenotypes (reduced size, trichome development, and shoot diameter, and abnormal leaf development with fused leaves), and, interestingly, higher resistance against fire blight and apple scab.
Asiatic citrus canker, induced by the bacterial pathogen Xanthomonas axonopodis pv. citri, causes high economic losses in Citrus-producing countries. In recent years, transformation of citrus species has focussed particularly on resistance to this bacterium. In addition to classical antimicrobial genes, for example attA, rice Xa21, and genes Shiva A and Cecropin B, new genes have been tested. Yang et al. (2011) transformed sweet orange with the carboxyl terminal portion of the pthA gene (essential for the pathogen to cause hyperplastic canker symptoms) which encodes three nuclear localization signals (NLS) that are critical for gene function and localization to the host cell nucleus. Plants transformed with the gene in sense orientation had less disease incidence in comparison with the wt and the antisense NLS transgenic clones. Mendes et al. (2009) transformed C. sinensis cv Hamlin with the hrpN gene from E. amylovora that encodes an harpin protein which elicits HR and SAS in plants. The hrpN gene was driven by gst1, a pathogen-inducible promoter from potato that is functional in some heterologous species and also responds to different types of pathogen. Although reduced susceptibility to citrus canker was observed for several
transgenic lines, severe leaf curling and abnormal growth was observed for most lines, and only one had high tolerance of the bacterium and was without leaf abnormalities. Apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange and transgenic lines were less susceptible to citrus canker. This low susceptibility after X. axonopodis inoculation was related to increased production of polyamines, triggering HR and activation of defencerelated genes (Fu et al. 2011). This is the first example of improving disease resistance in a perennial fruit crop via transformation with a gene involved in polyamine biosynthesis. The polyamines are, therefore, not only involved in improving abiotic stress, but also seem to have interesting prospects for engineering biotic stress tolerance. As reported for virus diseases, genetic engineering against bacterial or fungal diseases does not always result in complete resistance, but often gives only partial protection. Some authors have also reported the effect of cultivar genetic background where these genes were inserted. Cardoso et al. (2010) and Mendes et al. (2009) observed that the effectiveness in citrus canker resistance of the constitutive expression of attA or Xa21 in different sweet orange cultivars varied according to the original susceptibility of the cultivar. Recently it has been demonstrated that terpene down-regulation might be a strategy to generate broad spectrum resistance of fruit against pests and pathogens. Silencing of the limonene synthase gene in orange plants led to reduced accumulation of limonene in the fruit peel (Rodrı´guez et al. 2011). Transgenic fruits had marked resistance against
Penicillium digitatum and Xanthomonas citri
subsp. citri, and males of the citrus pest medfly (Ceratitis capitata) were less attracted by transgenic fruits. Limonene in the peel of citrus fruit seems to be involved in the trophic interaction between fruits, microorganisms, and insects.
3.2 Abiotic stress tolerance
In recent years, several approaches, using different genes, have been reported to improve resistance to different abiotic stress. However, in all cases in which the molecular bases of abiotic resistance were examined, it was seen that this resistance or tolerance was linked to increased antioxidant capacity of the tissues through control of the genes involved in ROS metabolism. Abiotic stress including salinity, drought, heat, flood, frost and other abiotic stress, are often interconnected with
oxidative stress, trigger a series of biochemical, physiological and molecular changes in plants, and may induce similar cellular damage (Ahmad et al., 2012; Rai et al., 2011) that cause reduced growth and productivity in plants.
Drought, saline soils, and cold or high temperatures are the most common stresses that plants encounter. Plant responds to drought and/or salinity in mainly two phases, primarily as. To protect from the negative consequences of abiotic stresses, such as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell and oxidative stress which may cause denaturation of functional and structural proteins (Munns and Tester 2008; Jewell et al. 2010), the plants have evolved many biochemical and molecular mechanisms.
These diverse environmental stresses trigger the cell signaling process, transcription controls and cellular responses such as the production of a number of stress proteins, expression and activities of antioxidants and accumulation of compatible solutes which activate stress-responsive mechanisms to reestablish homeostasis and thus protect or repair damaged proteins and membranes (Wang et al. 2003). Usually, abiotic stress tolerance through detoxification mechanism or accumulation of metabolites is likely to involve many genes at a time. Therefore, the attempts to develop transgenics for abiotic stress tolerance involved ‘‘single action genes’’ i.e. genes involved in biosynthesis of a single metabolite that would confer increased tolerance, are unlikely to be sustainable (Bhatnagar-Mathur et al., 2008). In recent years, several studies, using different genes, with diverse function and mechanism were employed for the development of transgenic fruit plants to improve resistance/tolerance to different abiotic stresses (Table 2).However, in most cases this resistance/tolerance to abiotic stresses was linked to increased antioxidant capacity of the tissues or accumulation of compatible solutes through control of the genes involved in ROS metabolism.
For instance, enhancing of drought stress in apple was achieved by inserting the Osmyb4 gene from rice (encoding a transcription factor belonging to the Myb family) that improves adaptive responses to drought and cold stress, most probably because of the constitutive activation of several stress-inducible pathways and the accumulation of several compatible solutes (Pasquali et al., 2008). Shekhawat et
al. (2011b) demonstrated the multiple abiotic stress tolerance in banana by overexpression of MusaWRKY71, a novel stress-responsive WRKY transcription factor gene from Musa spp. cv. Karibale Monthan (ABB group). Proline is an important osmoregulator and the overexpression of genes involved in its synthesis often results in drought tolerance. Transgenic Carrizo citrange (C. sinensis x
Poncirus trifoliate L. Raf.) plants constitutively
expressing a D1-pyrroline-5-carboxylate synthetase mutant gene (P5CSF129A), which encodes the rate-limiting enzyme of the proline biosynthetic pathway, grew normally in vivo and accumulated high levels of proline in the leaves.
The use of Citrus rootstocks with increased proline accumulation seems to be a promising approach to maintaining the productivity of plants under drought stress condition (Molinari et al., 2004). The same gene was inserted in ‘Swingle’ citrumelo rootstocks (C. paradisi Macf. x Poncirus trifoliata): transgenic plants had resistance to water deficit, and proline, in addition to action as an osmotic adjustment mediator, protected against ROS by modulating the antioxidant enzymes activity (de Campos et al. 2011). In another study, overexpression of (DREB)1b, a cold inducible transcription factor from Arabidopsis thaliana, was able to improve tolerance to cold stress in grapevine (Jin et al., 2009).
Stress salinity is an important factor limiting crop production in saline soil and in soils irrigated with non-potable water that contains higher levels of salt than normal. Tonoplast Na+/H+ antiporters are of critical importance in salt tolerance, and one of these transporters (MdNHX1) has been isolated from a salttolerant rootstock of apple and introduced it into the widely used rootstock M.26 (Li et al., 2010). The M.26 transgenic rootstock had improved salinity stress tolerance, compartmentalizing more Na in the roots and also maintaining a relatively high K+/Na+ ratio in the leaves compared with wt plants. Similar results were obtained by Tian et al. (2011), who transformed kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang and A. R. Ferguson with an Arabidopsis Na+/H+ antiporter (AtNHX1). Under salt stress, better osmotic adjustment, higher antioxidant capacity, lower membrane damage, and more vigorous growth were observed for these transgenic lines than for wt plants. Another approach to enhancing salinity tolerance has been reported for transgenic grapevine rootstock 110 Richter (V. berlandieri
x V. rupestris) containing the ferritin gene (MsFer) from Medicago sativa (alfalfa).
The transgenic lines had tolerance of NaCl and, in addition, increased tolerance of chemically generated oxidative stress induced by tert-butyl hydroperoxide (t-BHP) and paraquat (Zok et al., 2010). An apple spermidine synthase gene (MdSPDS1) has been inserted in pear, resulting in attenuated susceptibility to stress-inducing treatment (Wen et al., 2008). Results suggested that the line #32 over-expressing the SPDS gene substantially increased tolerance to NaCl, mannitol, and Cu stress by altering polyamine titres and activating the antioxidant pathways of the cells. In subsequent years other work has shown the increased resistance of this line to multiple abiotic stress: aluminium (a major cause of poor crop yields, particularly in those countries where acid soil predominates) and the heavy metals Cd, Pb, and Zn, the major industrial pollutants leading to phytotoxicity (Wen et al., 2010). In contrast, severe growth inhibition was observed for antisense pears for
MdSPDS1 after exposure to either NaCl or Cd
(Wen et al., 2011). In these lines the antioxidant system was not induced under stress and exogenous application of spermidine alleviated the stress. The results provided evidence of the central role of polyamines in alleviation of both Cd and salt stress in pear.
Cold tolerance is essential if temperate woody plants are to survive freezing winter temperatures. C-repeat binding factor (CBF/DREB) transcriptional activator genes have the ability to induce expression of a suite of genes associated with increased cold tolerance. The CBF gene from Arabidopsis increased the freezing tolerance of several species by at least of 2–30C. However, Dhekney et al. (2007) inserted this gene into papaya (papaya production is affected by low temperatures that occur periodically in the subtropics) but did not obtain the expected results, because the cold acclimation mechanism seems to be absent from papaya. The peach CBF gene (PpCBF1) was isolated and inserted in apple rootstock M.26 (Wisniewski et al. 2011).
Overexpression of this gene had an effect on photoperiod sensitivity of apple: growth cessation and leaf senescence were observed for transgenic apple lines in response to short daylength treatment. This work indicates that genes regulating dormancy and those that regulate cold hardiness may be more
interactive than expected, especially in woody plants. In grapevines, Jin et al. (2009) achieved cold resistance by overexpressing the dehydration response element binding (DREB)1b, a cold-inducible transcription factor from A. thaliana. Osmotin, a pathogenesis-related protein with cryoprotective functions, was inserted into the olive tree (Olea europaea L.), a frost-sensitive species lacking dormancy (D’Angeli and Altamura, 2007). The osmotin induced cold protection in transgenic olive trees by activation of plant cell death (PCD), regulation of cytoskeleton dynamics, and arrest of cold-induced calcium signalling. PCD seems to be a strong activator of cold acclimation (acquisition of freezing tolerance by exposure to non-freezing low temperatures) in this species.
Transgenic pineapple plants transformed with the bar gene for bialaphos resistance were developed (Sripaoraya et al., 2006) and evaluated for tolerance to herbicide Basta. Seven months after transfer to the field, plants were found tolerant to 1600 ml/rai of the herbicide Basta® X (stock concentration 15% w/v glufosinate ammonium), this being twice the dose recommended for field application of the herbicide. Transgenic plants tolerant to glufosinate ammonium should facilitate more effective weed control in pineapple plantations without damage to the crop. Plants, when exposed to abiotic stress conditions produce several pathogenesis-related proteins to compensate the effect of stress conditions. Among those proteins, osmotin is one of the important one released during abiotic stress conditions. Husaini and Abdin (2008) over-expressed tobacco osmotin gene in strawberry (Fragaria x ananasa Duch.) and found that the transgenic strawberry plants exhibited tolerance to salt stress.Researchers at the Horticultural Research International, the United Kingdom, have identified the genes which control the taste, smell and color of strawberries. As a result, it would now be possible to create super strawberries that will taste sweeter using transgenic approaches. Stewart et al. (2001) cloned a PPO gene from pineapple fruits under conditions that produce blackheart. The PPO gene has been silenced in transformed plants and transgenic plants are under field evaluation. Also, Park et al. (2005) demonstrated that fruit from tomato plants expressing Arabidopsis thaliana H+/ cation exchanger (CAX) gene have more calcium (Ca2+) and prolonged shelf life when compared to controls.
Table 1: Transgenic plants expressing genes for disease resistance in fruit plants: some recent reports
Plant Gene Resistance/disease Remarks References
Apple HcrVf2 Venturia inaequalis / apple
scab disease
Confers scab resistance in transgenic cultivated apple cv. Gala
Belfanti et al. (2004)
MpNPR1 Venturia inaequalis and
Gymnosporangium juniperi-virginianae
Increased disease
resistance by expression of pathogenesis - related (PR) genes
Malnoy et al. (2007)
dpo Erwinia amylovora/fire
blight
Encoding for an extracellular
polysaccharide (EPS) depolymerase
Flachowsky et al. (2008)
Lc Fire blight and apple scab - Flachowsky et al. 2010
BBTv-cp Virus resistant Encoding viral coat protein Ismail et al. (2011)
Avocado pdf1.2 – Encodes an antifungal
defensin
Raharjo et al. (2008)
Banana rcc2 or rcg3 class-I rice
chitinase gene
Mycosphaerella fijiensis / black leaf streak disease
Expression of the chitinase (RCG3) Protein
Kovacs et al. (2013) ThEn-42 along with StSy and
SOD
Botrytis cinerea / Sigatoka disease
Overexpression of fungal endochitinase
Vishnevetsky et al. (2011) PhDef1 and PhDef2 Petunia
floral defensins gene
Fusarium oxysporum /wilt disease
Encodes antimicrobial peptides ‘Defensins’
Ghag et al. (2012) ihpRNA-Rep and
ihpRNAProRep
Banana bunchy top virus (BBTV)/banana bunchy top disease
Transgenic plants
expressing small interfering RNAs targeted against viral replication initiation gene
Shekhawat et al. (2012)
Citrus hrpN Xanthomonas axonopodis
pv. Citri / citrus canker
encodes a harpin protein Mendes et al.
(2009)
attacin A Xanthomonas citri subsp.
Citri/Asian citrus canker
Encodes Attacin A, an antimicrobial
Peptides
Cardoso et al. (2010)
AtNPR1 Xanthomonas citri subsp.
Citri/citrus canker
Key positive regulator of SAR
Zhang et al. (2010)
scFv Citrus tristeza virus (CTV) Overexpression of scFv
antibody fragments directed against epitopes of its major coat protein p25
Cervera et al. (2010)
pthA-nls Xanthomonas xonopodis pv.
citri/Citrus canker
Encodes three nuclear
localizing signals (NLS)
Yang et al. (2011)
pv. citri/citrus canker antibacterial peptide
MdSPDS1 Citrus canker Production of polyamines Fu et al. (2011)
Attacin E Elsinoe fawcettii/citrus scab Encodes Attacin, an
antimicrobial peptides acts against fungal pathogen
Mondal et al. (2012)
Grape vine
Stilbene synthase gene (STS) – Synthesis of the antifungal
phytoalexin resveratrol
Fan et al. (2008) GFLV ovement protein (MPc)
gene
Grapevine fanleaf virus (GFLV)
Post-transcriptional gene silencing (PTGS)
Jardak-Jamoussi et al. (2009)
Vst1 Systemic acquired
resistance (SAR)
Synthesis phytoalexin Delaunois et al. (2009)
VpSTS Uncinula necator / powdery
mildew
Involvement of stilbene
synthase promoter in
pathogen - and stress inducible expression
Xu et al. (2010)
Chitinase and b-1,3-glucanase Plasmopara iticola / downy
mildew
Increased activities of chitinase and b-1, 3-glucanase in transgenic lines
Nookaraju and Agrawal (2012)
Papaya PRSV coat protein (CP) Papaya ringspot virus Characterization of
Insertion Sites in Rainbow Papaya
Suzuki et al. (2008)
PRSV and PLDM coat protein (CP)
Papaya ringspot virus and Papaya leaf-distortion mosaic virus
Virus resistance mediated by RNA mediated post-transcriptional gene silencing (PTGS)
Kung et al. (2010)
DmAMP1 Phytophthora palmivora Encodes antimicrobial
peptides ‘Defensins’ acts against fungal Pathogen
Zhu et al. (2007)
Plum PPV (cp) C5 clone PPV Production of 24-nt siRNAs Hily et al. (2005)
Strawberry Chit-42, or β-1,3-glucanase Colletotrichum acutatum/
anthracnose crown rot
Transgenic lines showed significantly fewer anthracnose crown rot lesions compared to the controls
Mercado et al. (2007)
Apd – Encodes am antimicrobial
peptide-D
Table 2: Genes, mechanisms, and genetically modified fruit plant species implicated in plant responses to many abiotic stresses: some recent reports
Plant Gene Remarks Perform of
transgenic plants to abiotic stress
References
Apple Osmyb4 Encoding a transcription factor
belonging to the Myb family, accumulation of several compatible solutes
Drought and cold Pasquali et al. (2008)
MdNHX1 Tonoplast Na+/H+ antiporters Salt Li et al. (2010)
PpCBF1 C-repeat binding factor
(CBF/DREB), transcriptional
activator genes
Cold Wisniewski et al. (2011)
MdCIPK6L Encode a CBL-interacting protein
kinase (CIPK)
Salt, drought and Chilling
Wang et al. (2012)
Banana MusaDHN-1 Overexpression of dehydrin gene,
belonging to a broader class of LEA
proteins
Drought and salt Shekhawat et al. (2011a)
MusaWRKY71 Encodes a WRKY transcription
factor protein
Multiple abiotic stress
Shekhawat et al. (2011b)
MusaSAP1 Encodes a zinc finger protein i.e.
stress associated proteins (SAP)
Multiple abiotic stress
Sreedharan et al. (2012)
Citrus P5CSF129A Osmotic adjustment, protected
against ROS by modulating the antioxidant enzymes activity
Water deficit de Campos et al. (2011)
AhBADH Overexpressing AhBADH gene
regulates accumulate higher level of glycinebetaine
Salt Fu et al. (2011)
D1-pyrroline-5-carboxylate synthetase (P5CS)
Endogenous accumulation of
proline
Drought de Carvalho et al. (2013)
Grap-vine DREB1b Dehydration response element
binding gene, a cold inducible transcription factor
Cold Jin et al. (2009)
VvCBF4 C-repeat binding factor gene,
reduced freezing-induced
electrolyte leakage
Cold Tillet et al. (2012)
Kiwi-fruit AtNHX1 Maintaining a relatively high K+/Na+
ratio
Salt Tian et al. (2011)
Mul-berry hva1 Encodes a group 3 LEA protein Salinity and
drought
Lal et al. (2008)
proteins belonging to the plant PR-5 group of proteins
variety of fungal (biotic) pathogen
Papaya C-repeat binding factor
(CBF)
Transcriptional activator genes Cold Dhekney et al. (2007)
Pear SAMDC2 Encodes sadenosylmethionine
decaboxylase, transgenic plants expressing polyamines
Salt He et al. (2008)
SPDS1, SPDS Encodes spermidine synthase,
transgenic plants expressing
polyamines
Salt, multiple abiotic stress
Wen et al. (2008, 2009)
Straw-berry Osmotin Enhanced levels of proline, total
soluble protein
Salt Husaini and Abdin (2008)
Table 3: Genetic transformation of fruit plants with agronomic and horticultural traits: some recent reports
Plant Gene Remarks References
Apple rol B Effects of rol B transgenic rootstocks on growth,
flowering and fruit quality of non-transgenic scion cultivars grafted onto these rootstocks
Smolka et al. (2010)
BpMADS4 Transgenic apple was characterized and selected for its
use in a fast breeding program
Flachowsky et al. (2011)
PG1 Down regulation of PG1 expression caused fruit
softening in transgenic line
Atkinson et al. (2012)
MdTFL1-1 Reduced vegetative growth and generation time Flachowsky et al. (2012)
MYB10 GM apple have high concentrations of foliar, flower and
fruit anthocyanins, analysis of GM apples reveals effects on consumer attributes
Espley et al. (2013)
FLOWERING LOCUS T genes (PtFT1 and PtFT2)
Improved fast track breeding approach by heat-induced expression of PtFT1 and PtFT2 genes
Wenzel et al. (2013)
Citrus CcGA20ox1 (a key
enzyme
of GA biosynthesis)
Antisense expression reduced height of plant whereas sense expression promoted shoot length
Fagoaga et al. (2007)
Arabidopsis thaliana
MAC12.2 gene
Significantly less seeds in transgenic lines than the control
Tan et al. (2009) Grape-
vine
rol B Improved the rooting of grape rootstocks Geier et al. (2008)
VvMYBPA1/10023289 9
Specific regulation of proanthocyanidin biosynthesis thus increase nutrient content
Bogs et al. (2007) Kiwi-
fruit
GGP Increased ascorbic acid content in transgenic lines Bulley et al. (2012)
first report of the use of co suppression technology in a tropical fruit
Pear Citrus FLOWERING
LOCUS T gene (CiFT)
Early flowering in transgenic lines Matsuda et al. (2009)
Plum Phytoene desaturase
(pds)
Inhibition of carotenoid biosynthesis and chlorophyll photo-oxidation
Petri et al. (2008) FLOWERING LOCUS
T genes (PtFT1)
Transgenic lines produced fruits in the greenhouse within 1–10 months
Srinivasan et al. (2012) Straw-
berry
FaMYB10 Enhanced anthocyanin content in root, foliar, and fruit Lin-Wang et al. (2010)
FaEG3 Increased fruit firmness in transgenic line Mercado et al. (2010)
PFP Over-expression of pyrophosphate : fructose
6-phosphate 1-phosphotransferase, regulates glycolytic and gluconeogenic metabolism
Basson et al. (2011)
Endo-b-1,4-glucanase antisense gene (FraCel1)
Regulate starch content in fruit during ripening Lee and Kim (2011)
FaplC and FaEG3 antisense genes
Encoding a pectate lyase and a endo-b-1,4-glucanase, reduced rate of fruit softening
Youssef et al. (2013)
Research Highlights
Among early and most important applications of transformation in fruit trees was the transgenic papaya resistant to Papaya ringspot virus (PRSV). Kung et al. (2010) successfully transformed for PRSV and Papaya leaf-distortion mosaic virus (PLDMV) resistance different commercial hermaphrodite papaya cultivars with desired horticultural features. Ming et al. (2008) reported a 39 draft genome sequence of ‘SunUp’ papaya, the first virus resistant transgenic fruit authorized for field cultivation and commercialization.
In another case, Plum pox virus (PPV), the causal agent of the Sharka disease, is a major constraint to Prunus production, and genetic transformation of Prunus has focussed almost exclusively on the induction of resistance to PPV. Scorza et al. (2013) after extensive testing and risk assessment in laboratory, greenhouse and in the field for over 20 years, ‘HoneySweet’ plum, a plum pox virus (PPV) resistant transgenic plum, has now been validated for cultivation in the USA.
Apple scab, caused by the fungus Venturia inaequalis (Cooke) G. Wint., is
the most widespread disease in apple orchards worldwide. Highly scab resistant ‘Elstar’ and ‘Gala’ plants were obtained, proving that the HcrVf2 gene (from Malus floribunda 821) controlled by its native promoter is effective in conferring resistance to V. inaequalis.
Apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange and transgenic lines were less susceptible to citrus canker. This low susceptibility after X. axonopodis inoculation was related to increased production of polyamines, triggering HR and activation of defencerelated genes. This is the first example of improving disease resistance in a perennial fruit crop via transformation with a gene involved in polyamine biosynthesis. The polyamines are, therefore, not only involved in improving abiotic stress, but also seem to have interesting prospects for engineering biotic stress tolerance.
Enhancing of drought stress in apple was achieved by inserting the Osmyb4 gene from rice (encoding a transcription factor belonging to the Myb family) that improves adaptive responses to drought and cold stress, most probably because of the constitutive activation of several stress-
inducible pathways and the accumulation of several compatible solutes (Pasquali et al., 2008).
Shekhawat et al. (2011) demonstrated the multiple abiotic stress tolerance in banana by overexpression of MusaWRKY71, a novel stress-responsive WRKY transcription factor gene from Musa spp. cv. Karibale Monthan (ABB group).
Proline is an important osmoregulator and the overexpression of genes involved in its synthesis often results in drought tolerance. The use of Citrus rootstocks with increased proline accumulation seems to be a promising approach to maintaining the productivity of plants under drought stress condition (Molinari et al., 2004). The same gene was inserted in ‘Swingle’ citrumelo rootstocks (C. paradisi Macf. x Poncirus trifoliata): transgenic plants had resistance to water deficit, and proline, in addition to action as an osmotic adjustment mediator, protected against ROS by modulating the antioxidant enzymes activity (de Campos et al. 2011).
An apple spermidine synthase gene (MdSPDS1) has been inserted in pear,resulting in attenuated susceptibility to stress-inducing treatment (Wen et al., 2008). Plants, when exposed to abiotic stress conditions produce several pathogenesis-related proteins to compensate the effect of stress conditions. Among those proteins, osmotin is one of the important one released during abiotic stress conditions. Husaini and Abdin (2008) over-expressed tobacco osmotin gene in strawberry (Fragaria x ananasa Duch.) and found that the transgenic strawberry plants exhibited tolerance to salt stress. Reference
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