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Recent Advances in Fruit Species Transformation

Written By

Hülya Akdemir, Jorge Gago, Pedro Pablo Gallego and Yelda Ozden Çiftçi

Submitted: 29 September 2011 Published: 07 March 2012

DOI: 10.5772/35139

From the Edited Volume

Transgenic Plants - Advances and Limitations

Edited by Yelda Özden Çiftçi

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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). Fruits and nuts have essential role to enhance quality of humankind life since a diet based on cereal grains, root and tuber crops, and legumes is generally lacked a wide range of products such as fiber, vitamin, provitamins or other micronutrients and compounds exist in fruit and nut species (Heslop-Harrison, 2005). According to last FAOSTAT statistics, totally about 594.5 million t fruit crops (except melons) were produced in the world in 2009 (http://faostat.fao.org). Because an increase demand exists in global food production, many economically important fruit crops production need to be improved, however, conventional breeding is still limited due to genetic restrictions (high heterozygosity and polyploidy), long juvenile periods, self-incompatibility, resources restricted to parental genome and exposed to sexual combination (Akhond & Machray, 2009; Malnoy et al., 2010; Petri et al., 2011). Thus, there is an urgent need for the biotechnology-assisted crop improvement, which ultimately aimed to obtain novel plant traits (Petri & Burgos, 2005).

Plant genetic engineering has opened new avenues to modify crops, and provided new solutions to solve 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 (Chilton et al., 1977; 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).

Currently, public concerns and reduced market acceptance of transgenic crops have promoted the development of alternative marker free system technology as a research priority, to avoid the use of genes without any purpose after the transformation protocol as selectable and reporter marker genes. Typically, it is employed for the selection strategy that confers resistance to antibiotics and to herbicides (Miki & McHugh, 2004; Manimaran et al., 2011). A large proportion of European consumers considered genetically modified crops as highly potential risks for human health and the environment. European laws are restrictive and do not allow the deliberate release of plant modified organism (Directive 2001/18/EEC of the European Parliament and the Council of the European Union). Under these premises, great efforts have also been realized to develop alternative marker free technologies in fruit species. Recently, it was demonstrated in apple and in plum, that transgenic plants without marker genes can be recovered and confirmed its stability by molecular analysis (Malnoy et al., 2010; Petri et al., 2011). In 2011, for first time it was described authentically “cisgenic” plants in apple cv. Gala (Schouten et al., 2006a,b; Vanblaere et al., 2011).

Efficient regeneration systems for the generation of transgenic tissues still appear as an important bottleneck for most of the species and cultivars. In the literature, different protocols were described to transform fruit cells using various DNA delivery techniques, however the attempts generally focused on transformation via Agrobacterium or microprojectile bombardment. In this chapter, a detailed application of these techniques in fruit transformation is summarized together with usage of proper marker and selection systems and in vitro culture techniques for regeneration of the transgenic plants.

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2. Techniques used to transform fruit species

Improvement of the plant characteristics by transfer of selected genes into fruit plant cells is possible mainly through two principal methods: Agrobacterium-mediated transformation and microprojectile bombardment (also called “biolistic” or “bioballistic”). 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 (Pawlowski & Somers, 1996; Klein & Jones, 1999; 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). Also, it was described that transgenic explants regenerated can be chimeric (Sanford et al., 1990). 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 via Agrobacterium-mediated and direct gene transfer techniques are presented.

2.1. Agrobacterium-mediated gene transfer

2.1.1. A complex relationship

In the decade of the 80´s, the first reports were published related to the introduction of foreign DNA in plant genome thanks to the Ti plasmid of Agrobacterium tumefaciens (De Block et al., 1984; Horsch et al., 1985). After more than 25 years, Agrobacterium-mediated gene transfer is still the most used method for fruit species transformation including apple, almond, banana, orange, grapevine, melon etc. (Table 1; Rao et al., 2009).

Plant transformation by using Agrobacterium has some advantages since the technique is relatively simple; transfer and integration of foreign DNA sequences with defined ends (left and right borders of T-DNA) is precise; stable transformation is high; transgene silencing is typically low and long T-DNA sequences (>150 kb) can be transferred (Veluthambi et al., 2003). However, it is still far from to be a routine transformation application in plants because of its host-range restrictions (Gelvin, 1998).

The initial drawback of Agrobacterium-mediated transformation method is the host-range restrictions. However, the bacterium and the target tissue can be manipulated to overcome this obstacle (Trick & Finer, 1997). These authors proposed a new approach to facilitate Agrobacterium penetration into plant tissues, the sonication assisted Agrobacterium-mediated transformation (SAAT) method. This method consists the use of ultrasounds to produce cavitations on and below the plant surfaces and into the membrane cells, wounding the tissues to enhance Agrobacterium infection (Trick & Finer, 1997, 1998).. Also, this method can be combined with vacuum infiltration to promote bacteria agglutination around the tissues to increase the Agrobacterium infection as it was demonstrated in kidney bean (Liu et al., 2005) and in woody plants as Eucalyptus (Villar et al., 1999; Gallego et al., 2002; Gallego et al., 2009). Today, application of these modifications solely or in combination with other approaches has made it possible to transfer foreign DNA via Agrobacterium even to monocots (Hiei et al., 1994; Ishida et al., 1996; Hensel et al., 2011) which were initially transformed with direct gene transfer methods since Agrobacterium is not a natural host. Following its first successful usage in soybean and Ohio buckeye (Trick & Finer, 1997), SAAT was also applied recently to fruit species including orange (Oliveira et al., 2009); banana (Subramanyam et al., 2011) and grapevine (Gago et al., 2011). In the last paper the developed efficient methodology that combined SAAT with vacuum infiltration allowed to obtain reporter gene expression in different newly formed organs such as stems, petioles and leaves. Expression was related to vascular tissues due to the EgCCR promoter of Eucalyptus gunnii and demonstrated that its activity is conserved and fully functional in grapevine as it was shown by uidA (GUS) and gfp reporter marker genes. Transgenic grapevine lines were verified by Southern blot analysis in five randomly chosen transgenic lines showing simple integration patterns in four lines with different band length indicating independent transformation events into the grapevine genome. We also applied the optimized protocol to pistachio nodes to reveal out if this method of transformation and vascular-specific promoter of eucalyptus, also works in this species. Histological observations of GFP activity presence in vascular bundles and leaves (Fig. 1) together with PCR amplification of 858 bp fragment of nptII and 326 bp of uidA (Fig. 2) genes confirmed not only gene integration but also showed that SAAT in combination with vacuum infiltration and vascular specific promoter could also be used for pistachio transformation. With PCR amplification, four out of five GFP+ putative transgenic shoots showed the amplified bands of nptII and uidA genes (Fig 2).

Figure 1.

Expression of GFP in pistachio transformed with EgCCR-GFP-GUS construct. Fluorescent images of different tissues and organs were taken 6 months after Agrobacterium-mediated transformation. GFP fluorescence in shoot apex (A-B, bars represent 5µm) and transverse section of the transgenic microshoot (C-D, bars represents 100µm) were carried out using a 480/40 nm exciter filters, and two-barrier filter >510 nm (wide range) and 535/550 nm (specific filter for GFP fluorescence). (Abbreviations: vascular bundles (vb), pith, p; cortex, c).

Figure 2.

Analysis of putative transgenic pistachio plants by PCR amplification using primers designed for 858 bp fragment of the nptII gene (A) and for a fragment of 326 bp of uidA gene (B). (M: DNA 1000 bp ladder, C+: positive control, C-: untransformed plant, T1-T5 putative transgenic shoots lines).

Agrobacterium-mediated transformation is highly genotype dependent for many plants (Pérez-Piñeiro, 2012) but also for fruit species. Different reports described that some cultivars were completely found to be as highly recalcitrant for transformation process whereas others are completely successful (Galun & Breiman, 1998; Petri & Burgos, 2005). This problem is widely described in different fruit species as apricot, grapevine and others (López-Pérez et al., 2008; Wang, 2011). López-Pérez and collaborators (2009) described that grapevine cultivars “Crimson Seedless” and “Sugraone” obtained different transformation efficiencies depending on the optical densities tested. Transformation of hypocotyls obtained from germination of mature seeds and nodal explants of apricot cultivars Dorada, Moniquí, Helena, Canino, Rojo Pasión and Lorna resulted in different transformation efficiencies (Wang, 2011). Some authors pointed out that one of the main goals of plant genetic engineering must be the development of genotype-independent transformation procedures, however due to this highly complex plant-pathogen interaction it will be very difficult to achieve this with the currently available technologies (Petri & Burgos, 2005).

SpeciesAimPlasmidTransgenesReferences
Apple
Malus x domesticaMethod optimisationpBIN6nptII, nosJames et al., 1989
M. x domesticaInvestigation of early events in transformationpDM96.0501sgfp, gusA,nptIIMaximova et al., 1998
M. x domesticaInvestigation of influence of rolA gene on shoot growthpMRK10rolA, nptIIHolefors et al., 1998
M. x domesticaScab resistancep35S-ThEn42, pBIN19ESRech42, nptIIBolar et al., 2000
M. x domesticaResistance to fireblightpLDB15attE, nptII, gusAKo et al., 2000
M. x domesticaImprove rooting abilitypCMB-BrolB, nptII, gusAZhu et al., 2001
M. x domesticaScab resistancepBIN(Endo+Nag)ech42, nag70, nptIIFaize et al., 2003
M. x domesticaSelf-fertilitypGPTV-KANS3RNase, nptIIBroothaerts et al., 2004
M. x domesticaMethod optimisation
Enhance rooting
pCMB-BrolB, nptII, gusARadchuk & Korkhovoy, 2005
M. x domesticaMethod optimisationpNOV2819pmi, nptII, gusADegenhardt et al., 2006
M. x domesticaInvestigation of function of ARRO-1 in adventitious rootingpK7GWIWG2 (II)ARRO-1, nptIISmolka et al., 2009
M. x domesticaStability of scab resistancepMOG402.hth.gus.intronHth, nptII, gusAKrens et al., 2011
M. x domesticaDevelopment of selection systempCAMBIAVr-ERE-GUSVrERE, gusAChevreau et al., 2011
M. x domesticaTransformation without selectable marker genepPin2Att.35SGUSint+.n pPin2MpNPR1.GUS−.nptIIMalnoy et al., 2010
Almond
Prunus dulcisMethod optimisationpBI121mgfp-5-ER
pNOV2819
nptII
pmi
Ramesh et al., 2006
Avocado
Persea americana Mill.Method optimisationpMON9749, pTiT37-SEnptII, gusACruz-Hernandez et al., 1998
Banana
Musa spp.Method optimisation (Agro + SAAT+ Vacuum infiltration)pCAMBIA1301hptII, gusASubramanyam et al., 2011
Musa spp.Resistance to Fusarium wiltpBI121-PFLPpflp, nptIIYip et al., 2011
Blueberry
Vaccinium spp.Method optimisationp35SGUS-intgusACao et al., 1998
Blueberry
V. corymbosum L.Method optimisationpBISN1nptII, gusASong & Sink, 2004
Grapevine
Vitis viniferaMethod optimisationNrgusA, nptIINakano et al., 1994; Gago et al., 2011
V. rootstocksResistance to viruses and crown gallpBIN19
pGA482G
mutant virE2, nptII
GLRaV-3cp
Xue et al., 1999
V. viniferaResistance to fungal pathogenspBI121nptII
rice chitinase gene
Yamamoto et al., 2000
V. viniferaFungal resistancepGJ42chitinase, rip, nptIIBornhoff et al., 2005
V. viniferaMethod optimisationpGA643nptII, GFLVCPMaghuly et al., 2006
V. viniferaMethod optimisationNregfp, nptIIDutt et al., 2007
V. viniferaResistance to powdery mildewpGL2ricechitinase gene, hgtNirala et al., 2010
V. viniferaMethod optimisationpBin19-sgfpnptII, sgfpPérez-López et al., 2008
V. viniferaMethod optimisationpSGNnptII, egfpLi et al., 2006
V. viniferaMethod optimisationpCAMBIA2301nptII, gusAWang et al., 2005
Grapefruit
Citrus paradisiResistance to Citrus tristeza viruspGA482GGCP, RdRp, gusA, nptIIFebres et al., 2003
C. paradisiResistance to Citrus tristeza viruspGA482GGCP, gusA, nptIIFebres et al., 2008
Kiwifruit
Actinidia spp.Hairy root inductionA722,C58, ICMP8302,
ICMP8326, ID1576,
LBA 4404, A4T
gusA, nptIIAtkinson et al., 1990
Actinidia spp.Method optimisationpLAN411, pLAN421gusA, nptIIUematsu et al., 1991
A.deliciosaImproved rootingpBIN19nptII, rol A,B,CRugini et al., 1991
A. erianthaMethod optimisationpART27-10gusA, nptIIWang et al., 2006
A. deliciosaManipulation of plant architecturepBI121İptHonda et al., 2011
Mango
Magnifera indica L.Method optimisationpTiT37-SE::pMON9749nptII, gusAMathews et al., 1992
M.indica L.MethodoptimisationpGV3850::1103nptIIMathews et al., 1993
M.indica L.Mediate ethylene biosynthesispBI121nptII, gusA antisense ACC oxidase, antisense ACC synthaseCruz Hernandez et al., 1997
M. indica L.Rooting enhancementNrrol BChavarri et al., 2010
Melon
Cucumis meloResistance to ZYMV, TEV, PVYFLCP core ASnptII, ZYMV coatpr.Fang & Grumet, 1993
C. meloSalt resistancepRS655nptII, gusA, hal1Bordas et al., 1997
C. meloResistance to ZYMVpBI-ZCP3’UTRnptII, ZYMV coatpr.Wu et al., 2009
Nectarberry
Rubus arcticusMethod optimisationpFAJ3001gusAKokko & Kärenlampi, 1998
Orange
Citrus sinensisMethod optimisation
(Agro + SAAT+ vacuum infiltration)
pGA482GGgusA, nptIIOliveira et al., 2009
C. sinensisResearch on expression of Mt-GFPpBI. mgfp4.coxIVMt-gfpXu et al., 2011
C. sinensisInfluence of methylation on gene expressionpBIN.mgfp5-ERgfp, nptIIFan et al., 2011
C. sinensisModification of gibberellin levelspBinJIT-CcGA20ox1-sense pBinJITCcGA20ox1- antisensenptII, CcGA20ox1
nptII, CcGA20ox1
Fagoaga et al., 2007
C. sinensisResistance to fungipBI121.P23nptII, PR-5Fagoaga et al., 2001
C. aurantifoliaResistance to viruspBin19-sgfpnptII, sgfp, p23Fagoaga et al., 2006
Poncirus trifoliateEnhanced salt tolerancepBin438nptII, AhBADHFu et al., 2011
Papaya
Carica papayaResistance to PRSVpRPTWPRSV replicase gene, neoChen et al., 2001
Pear
Pyrus communisAlter growth habitpGA-GUSGFrolC, gusA, nptIIBell et al., 1999
P. communisMethod optimisationpPZP
pME504
gusA, nptIIYancheva et al., 2006
P. communisMethod optimisationPBISPGnptII, gusASun et al., 2011
Peanut
Arachis hypogaeaProduction of edible vaccines for Helicobacter pyloripBI121.Oleosin-UreBureB, nptIIYang et al., 2011
A. hypogaeaImprovement of salt and drought resistancepGNFA-(pAHC17)AtNHX1Asif et al., 2011
A. hypogaeaProduction of vaccines for Peste des petits ruminants (PPR)pBI121HnKhandelwal et al., 2011
Plum
Prunus armeniacaMethod optimisationpBIN19-sgfpnptII, gfpPetri et al., 2004
P. armeniacaMethod optimisationpBIN19-sgfp, p35SGUSintnptII, gfp/nptII, gusAPetri et al., 2008
P. domesticaTransformation of marker free plantspCAMBIAgfp94(35S)/ pGA482GGi ihpRNAE10´nptII, gfp, gusA, ppv-cpPetri et al., 2011
P. domesticaNew selection system with hygromycinpC1381, pC1301, pC2301gusA, hpt, nptIITian et al., 2009
P. domesticaControl of PPV infectionpGA482GGnptII, gusA, PRVcpScorza et al., 1995
P. salicinaMethod optimisationpCAMBIA2202nptII, gfpUrtubia et al., 2008
Pomegranate
Punica granatumMethod optimisationpBIN19-sgfpnptII, gfpTerakami et al., 2007
Strawberry
Fragaria spp.Method optimisationpBI121nptII, gusABarcelo et al., 1998
Fragaria x ananassa Duch.Modulation of fruit softeningpBI121antisense of endo-β-1,4-glucanaseLee & Kim, 2011
White mulberry
Morus albaMethod optimisationpBI121nptII, gusAAgarwal & Kanwar, 2007

Table 1.

Some important reports on genetic transformation of fruit species via A. tumefaciens or A. rhizogenes.

Some abbreviations: AtNHX1: a vacuolar type Na+/H+ antiporter gene; gfp: green fluorescent protein coding gene; hal1: yeast salt tolerance gene; hpt: hygromycin phosphotransferase coding gene; ipt: isopentyl transferase gene; neo: neomycin phosphorate transferase coding gene; nos, nopaline synthase coding gene; nptII, neomycin phosphtransferase II coding gene; pmi: phosphomannose isomerase coding gene; ppv: Plum pox virus; prsv: papaya ringspot virus; pvy: potato virus Y; tev: tobacco etch virus; gusA (uidA): β-glucuronidase coding gene; UreB: antigen gene; zymv: zucchini yellow mosaic virus.

2.2. Direct gene transfer

Direct gene transfer techniques include microprojectile bombardment, microinjection, electroporation, and usage of whiskers. Among them, microprojectile bombardment is an alternative technique of Agrobacterium-mediated transformation since its physical nature overcomes biological barriers and enables naked DNA delivery directly into host genome or alternatively into mitochondria and chloroplasts. In this technique, plasmid or linearized DNA-coated metal microparticles (gold or tungsten) at high velocity is bombarded to intact cells or tissues (Sanford et al. 1987; Klein et al. 1987; Sanford, 1988). Furthermore, biological projectiles such as bacteria (i.e., E. coli, Agrobacterium), yeast and phage associated with tungsten can also be used in microprojectile bombardment (Bidney, 1999; Kikkert et al. 1999).

Microprojectile bombardment was developed in the 1980s for transformation of plants which were recalcitrant to Agrobacterium–mediated transformation (Paszkowski et al., 1984) such as agronomically important cereals. Following the development of the first particle delivery system (Sanford et al. 1987; Sanford 1988), different effective devices such as PDS-1000/He, Biolistic® particle delivery system; particle inflow gun; electrical discharge particle acceleration; ACCELL™ technology and microtargeting bombardment device were also evolved to improve transformation capacity. Among them, PDS-1000/He, Biolistic® particle delivery system (BIO-RAD), which is a modified version of Sanford’s system, is the most used system for biolistic transformation due to its efficient and relatively simple application and acquisition of reproducible results between laboratories (Taylor & Fauquet, 2002). Particle inflow gun can be an alternative to other biolistic systems due to its very low cost and it was used successfully in banana transformation (Becker et al., 2000). Electrical discharge particle acceleration, ACCELL™ technology uses high voltage electrical discharge into a droplet water to generate shock waves and project microprojectiles to different cell layers of target tissues (McCabe & Christou, 1993). Microtargeting bombardment device was designed for shoot meristem transformations (Sautter, 1993) but it is not widely used for plant transformation. All of the microprojectile bombardment systems are not depend on any plant cell type but target cells which will be bombarded need to be physically accessible (Hensel et al., 2011).

Particle bombardment were carried out not only to optimize plant transformation but also to transfer gene constructs encoding for various antimicrobial peptides or proteins for fungal resistance against to Fusarium oxysporum f. sp. cubense and Mycospaerella fijiensis or preharvest and postharvest diseases Verticillium theobromae or Trachysphaera fructigen (i.e., Remy et al., 2000; Sagi et al., 1998; Tripathi, 2003), virus (i.e., Fitch et al., 1992; Tennant et al., 1994; Gonsalves et al., 1994; Scorza et al., 1996), pest (i.e., Serres et al., 1992) and herbicide tolerance (i.e., Zeldin et al., 2002). This technique has been applied to transformation of various fruit species including banana, cranberries, citrus, grapevine, melon, papaya and peanut (Table 2).

SpeciesAimTransfer systemPlasmidTransgenesReferences
Apple
Malus x domesticaMethod optimisationPEG-mediatedpKR10GfpMaddumage et al., 2002
Banana
Musa spp.Method optimisationParticle bombardmentpUbi-BtintORF1
pBT6.3-Ubi-NPT
pUbi-BTutORF5
pBT6.3-Ubi-NPT
pUGR73
pDHkan
nptII, gusA, BBTVBecker et al., 2000
Musa spp.Tolerance to Sigatoka leaf spotParticle bombardmentpYC39ThEn-42, StSy, Cu, Zn-SODVishnevetsky et al., 2011
Musa spp.Resistance to virusParticle bombardmentpAB6, pAHC17,pH1gusA, bar,ubi, BBTV-G-cpIsmail et al., 2011
Cranberry
Vaccinim macrocarponMethod optimisation &
Pest control
Particle bombardmentpTvBTGUSnptII, gusA, BtSerres et al., 1992
V. macrocarponHerbicide resistanceParticle bombardmentpUC19bar, aphIIZeldin et al., 2002
Grapevine
Vitis viniferaMethod optimisationBiolisticpBI426nptII, gusAHebert et al., 1993
V. viniferaMethod optimisationParticle bombardment & AgropGA482GGnptII, gusA TomRSV-CPScorza et al., 1996
V. viniferaMethod optimisationBiolisticpSAN237nptII, magainin,PGLVidal et al., 2003
V. viniferaComparison of minimal cassette with standard circular plasmidsBiolisticpSAN168, pSAN237Magainin, nptIIVidal et al., 2006
Kiwifruit
Actinidia spp.Method optimisationPEG 4000pDW2CatOliveira et al., 1991
Actinidia spp.Method optimisationElectroporationpB1121, pTi35SGUSgusA, nptIIOliveira et. al., 1994
A. deliciosaMethod optimisationPEG 4000p35SGUSgusARaquel & Oliveira, 1996
Melon
Cucumis meloProtection againstParticle bombardment
İnfection & Agro
pGA4822GG/CPnptII, gusA, CMV-WLCPGonsalves et al., 1994
Papaya
Carica papayaPRV resistanceParticle bombardmentpGA482GGPRV, nptIIFitch et al., 1992
C. papayaPRV resistanceParticle bombardmentpGA482GGnptII, gusA, cpPRVHATennant et al., 1994
C. papayaControl of PRSVParticle bombardmentpGA482GGcpPRSV-pHA5, nptII, gusACai et al., 1999
C. papayaMethod optimisationParticle bombardmentpCAMBIA1303
pML202
hpt, nptII, mgfp5’Zhu et al., 2004
C. papayaUse of PMI/ManParticle bombardmentpNOV3610PmiZhu et al., 2005

Table 2.

Some important reports on genetic transformation of fruit species via direct gene transfer.

A successful protocol was studied very recently in banana cv. Williams apical meristems with microprojectile bombardment of a new construct pRHA2 plasmid containing bar and coat protein of banana bunchy top nanovirus (BBTV-cp) genes that encoded the viral coat protein by using BiolisticTM PDS-1000/He system, 650 psi helium pressures and 5 µg DNA/shot for acquisition of virus resistance (Ismail et al., 2011). After bombardment, 62% of apical meristems were survived on the selective medium and 80% of explants produced shoots in the following first subculture and all shoots were rooted (Ismail et al., 2011). In addition to those disease-based studies, others were also carried out in order to develop efficient transformation protocols via biolistic transformation (Sagi et al., 1995; Becker et al., 2000). Among them, Sagi and co-workers (1995) reported the transformation of embryogenic cell suspensions of cooking banana ‘Bluggoe’ (ABB genome) and plantain ‘Three Hand Planty’ (AAB genome) via particle bombardment. Then, Cavendish banana cv. Grand Nain embryogenic suspension cells were co-bombarded with the plasmid containing nptII selectable marker gene under the control of BBTV promoter or the cauliflower mozaic virus (CaMV) 35S promoter, the β-glucuronidase (gusA) reporter gene and BBTV genes under the control of the maize polyubiquitin promoter by using particle inflow gun and stably integration was obtained in all of the tested transformed plants (Becker et al., 2000). Very recently, microprojectile bombardment was also applied to induce tolerance to Sigatoka leaf spot caused by Mycosphaerella fijiensis in banana by transferring endochitinase gene of ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase gene (StSy) under the control of 35 S promoter and the inducible PR-10 promoter, respectively (Vishenevetsky et al., 2011). Moreover, in order to improve scavenging of free radicals generated during fungal attack, the superoxide dismutase gene (Cu, Zn-SOD) of tomato was also included to this gene cassette under the control of ubiquitin promoter. Both PCR and Southern blot analysis confirmed the stable integration of the transgenes and 4-year field trial showed that several transgenic banana lines had improved tolerance not only to Sigatoka but also other fungus such as Botrytis cinerea. Gene transfer via microprojectile bombardment was also carried out in American cranberry (Vaccinium macrocarpon) firstly to increase productivity by transferring Bacillus thuringiensis subsp. Kurstaki crystal protein gene (Bt) for pest resistance (Serres et al., 1992), and latter on, by bar gene to confer tolerance to the phosphinothricin-based herbicide glufosinade (Zeldin et al., 2002). Although preliminary bioassays for efficiency of the Bt gene against an important lepidopteran demonstrated no consistently effective control in former, stable transmission and expression of herbicide tolerance was observed in both inbred and outcrossed progeny of cranberry trans clone in latter.

In tangelo (Citrus reticulata Blanco × C. paradisi Macf.) cv. Page embryogenic suspension cells were bombarded with tungsten coated plasmid containing gusA and nptII genes (Yao et al., 1996). Following to bombardment, 600 transient and 15 stable transformants were obtained and integration of the interest genes confirmed by PCR and Southern blot analyses. A large of kanamycin-resistant embryogenic calli showed also GUS activity. In another study, Kayim and associates (1996) bombarded tungsten-coated plasmid (pBI221.2) containing the gusA gene into lemon cv. Kütdiken nucellar cells by biolistic device and expression of the gusA gene was histochemically confirmed.

Feronia limonia L. is important fruit tree because of its edible fruits. It is suitable for cultivation in semi-arid tropics and also can be used for reforestation and wasteland reclamation projects (Sing et al., 1992; Purohit et al., 2007). Feronia limonia L. hypocotyl segments were also bombarded with tungsten-coated plasmid pBI121 having gusA reporter gene driven by CaMV35S promoter and nptII as a selective marker under control of nos promoter using BiolisticTM PDS-1000/He particle delivery system at different rupture disc pressures (1100 and 1350 psi) and target distances (6 and 9 cm) (Purohit et al., 2007). This study revealed that 1100 psi/6 cm and 1350 psi/9 cm were the optimal bombardment condition with supplying a maximum 90% of GUS transient expression.

In grapevine, the initial transformation studies via microprojectile bombardment were performed for method optimization with transferring nptII and gus genes as selective and reporter marker genes, respectively (Hebert et al., 1993; Kikkert et al., 1996; Scorza et al., 1996). Later, Vidal and co-workers (2003) studied the efficiency of biolistic cotransformation in grapevine for multiple gene transfer of nptII and antimicrobial genes (magainin and peptidyl-glycine-leucine). The stable transformation was confirmed by gus gene expression, followed by PCR and Southern blot analyses of nptII and antimicrobial genes showed. Three years later, same research group (Vidal et al., 2006) reported the efficient biolistic transformation of grapevine by using minimal gene cassettes, which are linear DNA fragments lacking the vector backbone sequence.

Papaya is economically important and preferred another fruit species because of its nutritional and medicinal properties grown in tropical and subtropical regions (Tripathi et al., 2011). Papaya ringspot virus (PRSV) is major limiting factor in papaya production in Hawaii (Gonsalves, 1998; Fuchs & Gonsalves, 2007). First PRSV resistant papaya plants (cv. SunUp) were obtained by PDS/1000-He particle bombardment device of cv. Sunset with the transformation vector pGA482GG/cpPRV4 containing the prsv coat protein (CP) gene (Fitch et al., 1992). The PRSV resistant papaya has been commercialized, reached to end user and improved papaya is now under production in Hawaii (Tripathi et al., 2008). This study was followed by other reports mainly on improvement of PRSV tolerance in papaya via microprojectile bombardment-based transformation (Tennant et al., 1994; Cai et al., 1999; Guzman-Gonzalez et al., 2006). The deployment of transgenic papayas has showed that virus CP protein supplies durable and stable resistance to homologous strains of PRSV (Fermin et al., 2010). Moreover, no ecological influence of transgenic papayas on adjacent non-transgenic papaya trees, microbial flora and beneficial insects was evident (Sakuanrungsirikul et al., 2005). However, political and social factors have negatively affected the technology in Thailand (Davidson, 2008).

Although there are various wild peanut species having disease resistance traits, hybridization between wild and cultivars is difficult due to self-incompatibility, low frequency of hybrid seed production and linkage drag (Stalker & Simpson, 1995) and because of that genetic transformation is a practical tool to improve disease resistant cultivars. Singsit and associates (1997) transformed peanut somatic embryos with gold-coated plasmid constructs containing both Bacillus thuringiensis cryIA(c) and hph genes driven by CaMV35S promoters by PDS 1000 He biolistic device for resistance lepidopteran insect larvae of lesser cornstalk borer. The embryogenic cell lines showed hygromycin resistance and integration of hph and Bt genes were confirmed by PCR and/or Southern blot analyses in regenerated plants and a progeny. 18% CryIA(c) protein of total soluble protein was detected by ELISA immunoassay in the hygromycin resistant plants. Production of peanut stripe virus (PStV) resistant peanut is another attempt for biotechnologists since the virus negatively affects seed quality and yield in Asia and China (Higgins et al., 1999). Somatic embryos of peanut cv. Gajah and cv. NC-7 were transformed by co-bombardment of hph gene and one of two forms of the PStV coat protein genes and both of the transgenic plants showed high level resistance to the homologous virus isolate (Higgins et al., 2004). Transfer of anti-apoptotic genes originated from mammals, nematods or virus into plants is another approach for enhancement of plant resistance against to biotic and abiotic stresses (Chu et al., 2008a). With this aim, peanut cv. Georgia Green embryogenic callus was bombarded with anti-apoptotic Bcl-xL gene by microprojectile bombardment. Although Bcl-xL protein was detected in four transgenic lines, just one transgenic line (25-4-2a-19) had stable protein expression and showed tolerance to 5µM paraquat (commercial herbicide) (Chu et al., 2008a). Around 0.6% of total population in USA is affected of IgE-mediated allergic reaction following to peanut consumption (Sicherer et al., 2003). To produce hypoallergenic peanut, peanut cv. Georgia Green embryogenic cultures were also transformed via microprojectile bombardment and silenced peanut allergens (Ara h 2 and Ara h 6) by RNA interference. Expression of these allergens was not decreased effectively but, binding of IgE to the two allergens, significantly declined (Chu et al., 2008b).

Apart from microprojectile bombardment, electroporation (Oliviera et al., 1994) and PEG-mediated transformation were also carried out in apple (Maddumage et al., 2002) and kiwifruit (Raquel & Oliveira, 1996) in order to optimize transformation protocol by transferring gusA, gfp and/or nptII.

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3. Markers and selection of transformants

3.1. Reporter genes

Reporter genes or non-selectable marker genes are commonly used components of the plasmid constructs allowing the verification of transformation and the detection of the putative transformed cells. In many fruit transformation studies, histochemical analyses of transformed cells are visualized by using β-glucuronidase (GUS) expression as a reporter gene (Jefferson, 1987; Table 1). This enzyme is encoded by E. coli uidA (gusA) gene and histochemical localization of the gene expression is detected in subcellular levels (Daniell et al., 1991). High levels of GUS is not toxic for plant and the enzyme is very stable in cells, however, the assay is destructive to plants (Miki & McHugh, 2004). gusA generally co-transformed with other selective marker genes to enable the selection of transformants. The gene gfp encodes for the protein green fluorescent protein (GFP) (Chalfie et al., 1994). This is one of the mostly used reporter marker gene in fruit transformation protocols for monitoring transformed cells in vivo and in real time just by application of UV-light for the excitation of the fluorescent protein. GFP has not any cytotoxic effect on transformed plant cells (Stewart, 2001; Manimaran et al., 2011). In vivo detection may permit the manual selection of transformed tissues with focusing in the areas where the signal is more brightly. Fusion of GFP with other proteins of interest provides precise visualizing of intracellular localization and transport in transformed plant (Miki & McHugh, 2004; Manimaran et al., 2011). In some fruit species, it is reported that chlorophyll red autofluorescence can mask GFP expression making the detection really difficult or even impossible in species as apricot, peach and plum (Billinton & Knight, 2001; Padilla et al., 2006; Petri et al., 2008; Petri et al., 2011). However, it was described as an efficient reporter gene in other woody fruit plants, such as citrus (Ghorbel et al., 1999) and peach (Pérez-Clemente et al., 2004). These contrary results confirm the highly variability of the reporter gfp gene which is described by Hraška and co-workers (2008). Other reporter gene, luciferase (luc) (Gould & Subramani, 1988) also let the monitorization of the transgene putative cells in living tissues, however, it is not so widely employed as the gfp (van Leeuwen et al., 2000; Miki & McHugh, 2004).

3.2. Selection systems, a critical step

Selection of transformed regenerants is a critical step in any transformation procedure (Burgos & Petri, 2005). Selection systems can be classified as positive or negative, and conditional or non-conditional. Positive selection systems are those that promote the growth of transformed cells and tissues, by the contrary, negative selection systems are those that promote the death of the transgenic cells. Both systems can be conditioned by an external substrate to perform their activity. Currently, negative selection systems are used in combination with positive selection systems to eliminate transformed cells with incorrect molecular programmed excision of the T-DNA (Schaart et al., 2004; Vamblaere et al., 2011). Typically, in positive conditional selection systems the selectable marker gene encodes for an enzyme conferring resistance to some specific toxic substrate that enable the growth of the transformed cell, tissues and inhibiting or killing non transformed tissues (more information in the comprehensive review of Miki & McHugh, 2004). In the literature approximately 50 selection marker genes are described for genetic plant transformation, however, just only three genes of positive conditional selection system (nptII and hpt, resistance to the antibiotics kanamycin and hygromycin, respectively, and bar gene encoding resistance to herbicide phosphinothricin) are commonly employed in more than 90% of the papers (Miki & McHugh, 2004). These three selectable genes are also the most used ones to transform fruit species as it can be seen in Table 1 and Table 2. Escherichia coli nptII gene (also known neo) encoded protein (neomycin phosphotransferase, NPTII) inactives aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (G418), and paramomycin that inhibit protein translation in the transformed cells (Padilla & Burgos, 2010). Hygromycin B is another aminoglycoside antibiotic that inhibits protein synthesis with a broad spectrum activity against prokaryotes and eukaryotes and especially it is very toxic in plants. Escherichia coli hpt (aphIV, hph) gene codes for the hygromycin phosphotransferase to detoxify hygromycin B by phosphorylation via an ATP-dependent phosphorylation of a 7’’-hydroxyl group and it is generally used as another selection marker gene when nptII was not effective in plant transformation studies (Twyman et al., 2002; Miki & McHugh, 2004).

Similar to antibiotics, herbicides have different specific target sites in plants. The resistance can be achieved by various mechanisms such as usage of natural isozyme or generation of enzyme mutagenesis or detoxification of the herbicides by metabolic processes. Phosphinothricin (PPT; ammonium glufosinate) is an active component of commercial herbicides formulations and analogous to glutamate, the substrate of glutamate synthase. In plants, this enzyme catalyzes the conversion of glutamate to glutamine by removing ammonia assimilation from the cell. Inhibition of the enzyme results in ammonia accumulation and disruption of chloroplast and finally cell death due to photosynthesis inhibition (Lindsey, 1992; OECD, 1999). In plant transformation studies, as herbicide resistance selection marker gene, pat from S. viridochromogenes (Wohlleben et al., 1988) and bar gene from S. hygroscopicus (bialophos resistance; Thompson et al., 1987) encoding the enzyme phosphinothricin N-acetyltransferase (PAT) are extensively used for resistance to PPT. PAT converts PPT to a non-herbicidal acetylated form by transferring the acetyl group from acetyl CoA to the free amino group of PPT (Miki & McHugh, 2004).

Currently, an alternative to these highly employed “toxic” approaches conditional positive selection markers based on the promotion of a metabolic advantage to transformed cells are used. Some authors mentioned that this kind of selection can improve considerably the selection of the transformants, since others such as antibiotics generally cause considerable necrosis (produced by the death of non-transformed cells) that often inhibits regeneration from adjacent tissues (Petri & Burgos, 2005). Previously, results obtained with this approach demonstrated higher yields than when the toxic selective agents were employed, and seems to be broadly applicable to crop plants (Miki & McHugh, 2004). Some of the most widely used are the AtTPS1/glucose (Leyman et al., 2006); galT/galactose (Joersbo et al., 2003); xylose isomerase (Haldrup et al., 1998); D-aminoacid/dao1 (Alonso et al., 1998) and the pmi/mannose (Joersbo et al., 1998). Probably, one of the most used one in fruit species is the gene pmi that encodes the enzyme phosphomannose isomerase (EC 5.3.1.8) that catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. This enzyme is present in bacteria as E. coli and also, in humans, however it is not present in plants, as exception of soybean and other legumes. Using a media with mannose as the unique carbon source, only transformed cells can grow and develop. Glycolysis is inhibited due to the accumulation of mannose-6-phosphate converted from mannose by hexokinase with preventing cell growth and development in non-transformed cells (Miki & McHugh, 2004). Sensitivity to the toxic effect of mannose-6-phosphate is different between species, and can be avoided by combining with other sugars such as sucrose, maltose and fructose (Joersbo et al., 1999). Diverse fruit trees were selected with this system, alone or in combination with sucrose, i.e., 12 g/L mannose and 5 g/L sucrose in orange (Ballester et al., 2008); 30 g/L mannose without any sugar more in papaya (Zhu et al., 2005); 2,5 g/L mannose and 5 g/L sucrose in almond (Ramesh et al., 2006) or 1-10 g/L mannose and 5-30 g/L in apple (Degenhardt et al., 2006). In Citrus sinensis, the best results were obtained when 13 g/L mannose as unique source of carbon was added into the selection media. Mannose combined with other sugars promoted reduction in transformation efficiencies and escapes (Boscariol et al., 2003). Apricot cv. Helena and Canino required the lower combination of mannose with sucrose (1,25 g/L mannose and 20 g/L sucrose) in comparison with other woody fruit trees to obtain the most effective selection procedure. Moreover, safety assessments were revealed that there is no any adverse effect of the enzyme on mammalian allergenicity and toxicity (Reed et al., 2001).

Other selective strategies were developed as positive non-conditional systems, or in other words, using selectable marker genes that “promote” plant regeneration. Currently, there is more information about the genetic and biochemical control of organogenesis than embryogenesis for plant regeneration. Because of this, commonly genes related with cytokinins synthesis are employed for shoot organogenesis. More efforts are required to discover molecular mechanisms of embryogenesis to use these strategies in species highly dependent on embryogenesis regeneration to develop transgenic plants. Genes as cki1 or the most employed isopentenyl transferase ipt gene encoding the enzyme IPT, catalyze the synthesis of isopentyl-adenosine-5-monophosphate, which is the first step in cytokinin biosynthesis (Miki & McHugh, 2004). This gene modify the endogenous balance between cytokinins and auxins, stimulating cell division and differentiation of the cells that promote an altered morphology, development and physiology of transgenic plants (Sundar & Sakthivel, 2008). Some authors observed that the ipt gene improved transformation efficiency in apricot leaf explants in comparison with the selection through nptII (López-Noguera et al., 2009).

3.3. A differential transgene expression: Constitutive versus specific promoters

Currently, an important debate is carrying out about the risks of the “unpredictable” behavior and recombinogenic potential of constitutive promoters (Gittins et al., 2003) and to avoid the public concerns about the risks of ubiquitous transgene expression in crops.

Commonly, most of the fruit species have been transformed with plasmidic constructions harbouring constitutive or ubiquitous promoters, as the Cauliflower Mosaic virus 35S (CaMV35S). In this sense, different authors described that constitutive expression may be harmful for the host plant, causing sterility, retarded development, abnormal morphology, yield penalty, altered grain composition or transgene silencing (Cai et al., 2007 and references therein) and its expression level is dependent on the cell type, the developmental stage and on the perception of environmental triggers (Hensel et al., 2011). Moreover, under constitutive promoters reporter and selectable marker, and genes of interest are expressed continuously in all tissues without any temporal control. In this sense, specific-promoters appear as an alternative approach to avoid the undesirable side effects of constitutive promoters and to target transgene expression in a spatial or temporal specific way (Gago et al., 2011; Hensel et al., 2011).

Recently, vascular specific promoter EgCCR from Eucaliptus gunnii was checked in pistachio in this study as mentioned above as well as other fruit species such as kiwifruit and grapevine (Paradela et al., 2006; Gago et al., 2011) and results demonstrated that this promoter is conserved and fully functional in these species. Vascular promoters can drive resistance to biotic or abiotic stresses related with vascular tissues. Specific promoters could be useful to synchronize transgene activity spatially and/or temporally to control with more accuracy the pathogenic process (Gago et al., 2011).

3.4. Alternative transformation systems: Transgenics without marker genes

A highly desirable approach to promote public acceptation for future commercialization of transgenic plants and products is focused on the elimination of marker genes from transformed plants or the direct production of marker-free transgenics (Kraus, 2010). These newly and promising approaches are highly dependent on previously established highly efficient regeneration protocols that may be based on organogenesis or embryogenesis (Petri et al., 2011). There are various technologies such as homologous recombination, co-transformation, site-specific recombination (Cre/loxP site specific recombination system, R/RS system, FLP/FRT system etc) or marker elimination by transposons to remove selective marker genes (Hao et al., 2011; Manimaran et al., 2011). However, there are still few marker-free fruit species transformation protocols.

Strawberry leaf explants were transformed with site-specific recombinase for the precise elimination of undesired DNA sequences and a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants (Schaart et al., 2004).

MAT (multi-auto-transformation) (Ebinuma et al., 1997) combined with the Agrobacterium oncogene ipt gene, for positive selection with the recombinase system R/RS for removal of marker genes acting as “molecular scissors” after transformation were used as alternative approach in citrus plants (Ballester et al., 2007; 2008). Also, in apricot (López-Noguera et al., 2009) a similar strategy was used. Regeneration of apricot transgenic shoots was significantly improved to non-transformed plants (regenerated in non-selective media). Moreover, it was significantly higher in comparison with previous published data using resistance to kanamycin mediated by nptII gene. The lack of ipt differential phenotype promoted difficulties to assess the excision of the marker genes, that require periodic assays. Complete excision of marker genes ranged from 5 to 12 months, however, only 41% of the regenerated transgenic shoots R-mediated recombination occurs correctly. In Citrus sp., it was also reported that anomalous excision of marker genes promoting failures in the expression of the reporter genes (Ballester et al., 2007, 2008).

Apple (Malnoy et al., 2010) and pineapple sweet orange (Ballester et al., 2010) transformation using ‘‘clean’’ binary vector including only the transgene of interest were carried out to create marker-free transformants. Very recently, melon (C. melo L. cv Hetao) was transformed with a marker-free and vector-free antisense 1-aminocyclopropane-1-carboxylic acid oxidase construct via the pollen-tube pathway and transgenic lines are choosen by PCR without using any selectable marker agent (Hao et al., 2011).

In plum (Prunus domestica), transformation was carried out without reporter or selectable marker genes using a high-throughput transformation system (Petri et al., 2011). Previously, authors checked the efficiency of the regeneration of transformed shoots using conventional constructs harbouring reporter marker such as gusA and gfp, and nptII gene. Transformation efficiency varied from 5.7-17.7%. Using a marker free construct, the intron-hairpin-RNA (ihpRNA) harbouring the Plum Pox Virus coat protein (ppv-cp) gene, these authors regenerated five transgenic lines confirmed by Southern blot. It is important to take into account that this kind of free marker strategy is widely dependent on highly yields in regeneration systems.

3.5. Cisgenesis, the P-DNA technology and multigene transformation

Other relevant advance in fruit species transformation was the proposal made by Schouten and coworkers (2006), the “cisgenesis”. This term means the use of recombinant DNA technology to introduce genes from crossable donors plants, isolated from within the existing genome or sexually compatible relative species for centuries therefore, unlikely to alter the gene pool of the recipient species. Cisgenesis includes all the genetic events of the T-DNA as introns, flanking regions, promoters, and terminators (Vanblaere et al., 2011).

This methodology proposes to transfer the own plant DNAs, the P-DNAs. The use of this technology requires the construction of whole plant derived vector from the target species. Within the target species genome, it must be a DNA fragment with two T-DNA border-like sequences oriented as direct repeats ideally about 1-2 kb apart with suitable restriction sites for cloning of a desirable gene.

In the last years, different works were considered to step towards introducing regulatory elements and genes of interest from crossable donor plants, however with some foreign elements as marker genes in species as melon and apples (Benjamin et al., 2009; Joshi, 2010; Szankowski et al., 2009). Up to 2011 there is no any report of real “cisgenesis” plantlets, in agreement with Schouten et al. (2006) definition of the topic. In 2011, Vanblaere and coworkers developed apple cv. Gala cisgenic plants by expressing the apple scab resistance gene HcrVf2 encoding resistance to apple scab. Marker-free system was employed for the development of three cisgenic lines containing one insert of the P-DNA after removing by recombination with using chemical induction. These lines were not observed different from non-transformed cv. Gala plants.

Cisgenic plants are essentially the same as the traditionally bred varieties, and they might be easier to commercialise than the “problematic” transgenic plants (Schouten et al., 2006; Rommens et al., 2007). Critical opinions to these proposals also were clearly exposed, the uncontrolled P-DNA integration into the plant target genome can cause mutations or affect to the expression of other native genes, altering the behaviour of that cisgenic plants in an unpredictable manner (Schubert & Willims, 2006; Akhond & Machray, 2009). Recently, interesting approaches are being proposed for genome editing using ZFNs (Zinc finger nucleases) that can promote induction of double-strand breaks at specific genomic sites and promote the replacement of native DNA with foreign T-DNA (Weinthal et al., 2010).

The multigen transfer (MGT) methodology consist in introducing more than one gene at once. Commonly, most of the transgenic plants are generated by introducing just one single gene of interest, but now MGT are being developed to obtain more ambitious phenotypes as the complete import of metabolic pathways, whole protein complex and the development of transgenic fruit species with various new traits simultaneously transferred (Naqvi et al., 2009). In this sense, this technology would be highly desirable for commercial fruit species cultivars to obtain new traits related with large fruit size, high coloration of the fruit epidermis, flesh firmness and virus resistance (Petri et al., 2011) at the same time without the need of several rounds of introgressive backcrossing.

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4. In vitro culture techniques for the recovery of transgenic plants

Plants are complex, diverse organisms and have adapted evolutionarily to almost every ecological niche on the planet. Development of successful transformation protocol depends on a reliable and highly efficient regeneration system. Explant types are highly variable since it depends on the selected organogenetic process optimized for each species. Commonly, the genetic transformation protocols of fruit species employed explants such as ovules, anthers, seedlings, zygotic and somatic embryos, cotyledons, epicotyles, hypocotyles, leaf pieces, roots, meristems (Fagoaga et al., 2007; Lopez- Perez et al., 2008; Petri et al., 2008; Husaini, 2010; Malnoy et al., 2010; Bosselut et al., 2011; Petri et al., 2011; Gago et al., 2011). Typically, it is recommended that those tissues have high and active cell division to enhance the regeneration of the transgenic lines (Mante et al., 1991; Schuerman & Dandekar, 1993; Wang, 2011). Ideally, fruit species transformation must be done with somatic tissues such as leaves and roots to transform varieties already well known and accepted in the market by the consumers. Recently, some authors also proposed the possibility of the use of transgenic seedlings to develop new fruit varieties through subsequent cross-breeding. These transgenic seedlings can add new traits impossible to obtain in the species genome-pool (Petri et al., 2011).

Organogenesis was the strategy selected in different species to develop most of the known and efficient regeneration protocols for fruit species, concretely for fruit trees (Petri et al., 2011). Almond (Costa et al., 2006); apple (Smolka et al. 2009; Lau & Korban, 2010; Vanblaere et al., 2011); banana (Subramanyam et al., 2011); fig (Yancheva et al., 2005); kiwifruit (Tian et al., 2011); peach (Padilla et al., 2006); strawberry (Mercado et al., 2010); peanut (Asif et al., 2011); watermelon (Huang et al., 2011) and pear (Sun et al., 2011) are some examples of transformed cultivars for some fruit species that the transformed tissues were regenerated via organogenesis. Since organogenesis protocols are developed for many different fruit species, it is easier to adapt the regeneration system into genetic transformation methods (Frary & Eck, 2005). However, some risks also are assumed in using this regeneration system. Generally, it is considered that the origin of the new adventitious shoots is based on the involvement of few cells (George et al., 2008), enhancing the risks of chimera development.

Somatic embryogenesis that leads the formation of an embryo from somatic cells is another procedure to regenerate fruit transformants such as banana (Vishnevetsky et al., 2011); papaya (Zhu et al., 2001); grapevine (Nirala et al., 2010) and mango (Chavarri et al., 2010). Regeneration from transformed embryos can be achieved via direct germination or shoot organogenesis and the method is useful for large-scale and rapid propagation of transformants. In grapevine most of the approaches are being performed by using embryogenic cultures from different tissues such as zygotic embryos, leaves, ovaries and anther filaments to provide cells amenable to gene insertion and regeneration (Mezzetti et al., 2002; Dutt et al., 2007; López-Noguera et al., 2009). However, these techniques are highly genotype dependent and for many cultivars they have been difficult to obtain successful results (Dutt et al., 2007). Moreover, it is considered that anther filaments, as commonly employed in grapevine for embryogenic calli, are laborious, cultivar-dependent, depend on availability of immature flowers and may affect strongly the phenotype of the regenerated plantlets (Mezzetti et al., 2002). However, it is really interesting to take into account that regeneration from somatic embryos and secondary somatic embryos are currently assumed that they are derivatives of single cell origin.

In the decade of the 90´s some unsuccessful efforts were reported to transform meristems from micropropagated shoot tips due to high explant mortality and uncontrolled Agrobacterium overgrowth after coculture stages (Ye et al., 1994; Druart et al., 1998; Scorza et al., 1995). Mezzetti and co-workers (2002) described in grapevine the development of meristematic bulk tissues, a highly aggregate of meristematic cells produced after three months in increased concentrations of BA (N6-benzyladenine) and the removal of the apical meristem. After 90 days, under the previous conditions, these highly regenerative tissues produced easily adventitious shoots and can be transformed by Agrobacterium, being able to regenerate several transgenic lines. Other interesting approach was the genetic transformation of shoot apical meristems, previously subjected to a dark growth stage after wounding for transformation. Authors reported that 1% of shoot tips produced stable transgenic lines after weeks (Dutt et al., 2007). Ismail and co-workers (2011) transformed successfully banana apical meristems via microprojectile bombardment and regenerated 80.3% percent of the transformed meristematic tissues.

4.1. The chimeric question: Are my transgenic plants genetically uniform?

This is one of the most exciting questions that plant biotechnology researchers ask to themselves after all the long extensive, intensive and difficult labour needed to transform most of the fruit species. Some of the transformed regenerants can be chimeras, a mix of transformed and non transformed cells in the tissues, in other words, non genetically uniform organisms (Hanke et al., 2007). Recently, Petri and collaborators (2011) described that most of the known and efficient regeneration methods for fruit trees are based on organogenesis, where new adventitious shoot formation is originated from a determined number of cells. So, it comes hard to detect non chimeric and stable transgenic lines without using a selectable marker gene. Very recently, different authors using marker free technology as alternative systems or with genetically programmed marker excision reported the appearance of chimeric transformants in apple, strawberry, lime, citrus or plum (Domínguez et al., 2004; Schaart et al., 2004; Ballester et al., 2007; Malnoy et al., 2011; Petri et al., 2011).

Strawberry is highly sensitive to kanamycin selection, and it was described that selection of transgenic regenerants in these sensitive tissues can be associated with chimeric shoots containing transgenic and non-transgenic sections (Husaini, 2010). It was observed that increasing antibiotic concentration gradually avoid chimerisms in strawberry (Mathews et al., 1998; Husaini et al., 2010). Even under this strictly methodology some authors pointed out the inactivation events on the selection agent must be performed through the transformed cells, so, non transformed cells can develop and grown (Petri & Burgos, 2005; Wang, 2011). A useful methodology was also proposed for the quick and low-cost identification of chimeras by Faize and collaborators (2010) in tobacco and in apricot based in quantitative real-time PCR even in early developmental stages, and also let to monitor their dissociation.

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5. Future perspectives and concluding remarks

Currently, most of the fruit genetic transformation protocols integrated the new genes randomly and in unpredictable copy numbers influencing negatively its expression. Also public concerns and reduced market acceptance of transgenic crops have promoted the development of alternative marker free technologies in fruit species. For those reasons development of protocols to obtain transgenic fruits without marker genes and the use of the own plant DNA resources, such as “cisgenic” fruit plants, are the big challenges. ZFNs have also been succesfuly used to drive the replacement of native DNA sequences with foreign DNA molecules and to mediate the integration of the targeted transgene into native genome sequences.

Most of the fruit transgenic plants are generated by introducing just one single new character (gene of interest), however, some authors proposed that multigene transfer technology (MGT) needs to be developed to obtain new traits related at the same time. The combination of multiple traits can be a highly interesting approach as it could be applied to achieve resistance to several biotic or abiotic stresses and traits related to fruit quality such as large fruit size, high coloration of the fruit epidermis, increase flesh firmness to improve ripening without the need of several rounds of introgressive backcrossing.

The future of fruit genetic transformation is required of genotype-independent protocols, accuracy molecular tools to drive the T-DNA insertion and its expression, and efficiency and highly-yield selection and regeneration in vitro culture methodology. But Agrobacterium-mediated transformation procedure is a high non linear complex biological process, and its complexity can be understood with the composition of many different and interacting elements governed by non-deterministic rules and influenced by external factors. In this sense, the emergent technology dedicated to meta-analysis can be really useful to increase our understanding of fruit genetic transformation, making possible to identify relationships among several factors and extracting useful information generating understable and reusable knowledge (Gago et al., 2011; Gallego et al., 2011; Perez-Pineiro et al., 2012) Under these perspectives, modeling any fruit transformation procedure (Agrobacterium-mediated, biolistics, electroporation etc.) including the genetic engineering, in vitro plant tissue culture and regeneration stages will be improved for the next years.

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Acknowledgments

The authors wish to thank to Dr. Mariana Landín and to Dr. María José Clemente for critical reading of this work. This work was supported by Regional Government of Xunta de Galicia: exp.2007/097 and PGIDIT02BTF30102PR. This study was also partially supported by # TBAG- 209T030 from TUBITAK—The Scientific and Technical Research Council of Turkey. PG thanks to Minister of Education of Spain for funding the sabbatical year at Faculty of Science, University of Utrecht, Netherlands.

References

  1. 1. AgarwalS.KanwarK. 2007 Comparison of genetic transformation in Morus alba L. via different regeneration systems. Plant Cell Rep 26, 177185
  2. 2. AkhondM. A. Y.MachrayG. C. 2009 Biotech crops: technologies, achievements and prospects. Euphytica 166, 4759 .
  3. 3. AlonsoJ.BarredoJ. L.DiezB.MelladoE.SaltoF.GarciaJ. L.CortesE. 1998 D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology, 144, 10951101
  4. 4. MAAsifZafarY.IqbalJ.MMIqbalRashidU.AliG. M.ArifA.NazirF. 2011 Enhanced expression of AtNHX1, in transgenic groundnut (Arachis hypogaea L.) Improves Salt and Drought Tolerence. Molecular Biotechnology 49, 250256 .
  5. 5. AtkinsonR. G.CandyC. J.GardnerR. C. 1990 Agrobacterium infection of five New Zealand fruit crops. Zealand of Crop and Horticultural Science, 18, 153156
  6. 6. BallesterA.CerveraM.PeñaL. 2007 Efficient production of transgenic Citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination. Plant Cell Reports, 26, 3945
  7. 7. BallesterA.CerveraM.PeñaL. 2008 Evaluation of selection strategies alternative to nptII in genetic transformation of citrus. Plant Cell Reports 27, 10051015
  8. 8. BallesterA.CerveraM.PenaL. 2010 Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants. Plant Cell Tissue and Organ Culture, 102, 329336
  9. 9. BarcelóM.El -MansouriI.MercadoJ. A.MAQuesadaAlfaroF. P. 1998 Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell Tissue and Organ Culture, 54, 2936
  10. 10. BeckerD.DugdaleB.SmithM. K.HardingR. M. J.DaleJ. L. 2000 Genetic transformation of Cavendish banana (Musa spp. AAA group) cv. ‘Grand Nain’ via microprojectile bombardment. Plant Cell Reports, 19, 229234
  11. 11. BellR. L.ScorzaR.SrinivasanC.WebbK. 1999 Transformation of ‘Beurre Bosc’ pear with the rolC Gene. J. Amer. Soc. Hort. Sci. 124(6), 570574
  12. 12. BenjaminI.KenigsbuchD.GalperinM.AbrametoJ. A.CohenY. 2009 Cisgenic melons over expressing glyoxylate-aminotransferase are resistant to downy mildew. European Journal of Plant Pathology, 125, 355365
  13. 13. BIORAD 2002 http://www.bio-rad.com.
  14. 14. BidneyD. 1999 Plant transformation method using Agrobacterium species adhered to microprojectiles. United States Patent, 5, 932, 782
  15. 15. BillintonN.KnightA. W. 2001 Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Analytical Biochemistry, 291, 175197
  16. 16. BolarJ. P.NorelliJ. L.WongK. W.HayesC. K.HarmanG. E.AldwinckleH. S. 2000 Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology, 90, 7277
  17. 17. BordasM.MontesinosC.DabauzaM.SalvadorA.RoigL. A.SerranoR.MorenV. 1997 Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt Tolerance. Transgenic Research, 6, 4150
  18. 18. BornhoffB. A.HarstM.ZyprianE.TöpferR. 2005 Transgenic plants of Vitis vinifera cv. Seyval blanc. Plant Cell Reports, 24, 433438 .
  19. 19. BoscariolR. L.AlmeidaW. A. B.DerbyshireM. T. V. C.Mourâo-FilhoF. A. A.MendesB. M. J. 2003 The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Reports, 22, 122128
  20. 20. BosselutN.GhelderCV.ClaverieM.VoisinR.OnestoJP.RossoMN.EsmenjaudD., 2011 Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants. Plant Cell Reports, 30(7), pp. 1313-1326
  21. 21. BroothaertsW.KeulemansJ.Van NerumI. 2004 Self-fertile apple resulting from S-RNase gene silencing. Plant Cell Reports, 22, 497501
  22. 22. CaiM.WeiJ.XianghuaL.CaiguoX.ShipingW. 2007 A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue specific gene expression in transgenic plants, Plant Biotechnology Journal, 5, 664674
  23. 23. CaiW. Q.GonsalvesC.TennantP.FerminG.SouzaM.SarinduN.JanF. J.ZhuH. Y.GonsalvesD. 1999 A protocol for efficient transformation and regeneration of Carica papaya L. In Vitro Cellular and Developmental Biology-Plant, 35(1), 61-69
  24. 24. CaoX.LiuQ.RowlandL. J.HammerschlagF. A. 1998 GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency. Plant Cell Reports, 18, 266270
  25. 25. ChalfieM.TuY.EuskirchenG.WardW.PrasherD. 1994 Green fluorescent protein as a marker for gene expression. Science, 263 802805
  26. 26. ChavarriM.GarcíaA. V.ZambranoA. Y.GutiérrezZ.DemeyJ. R. 2010 Insertion of Agrobacterium rhizogenes rolB gene in Mango. Interciencia, 35 (7), 521525 .
  27. 27. ChenG.YeC. M.HuangJ. C.YuM.LiB. J. 2001 Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gen. Plant Cell Reports, 20, 272277
  28. 28. ChevreauE.DupuisF.TaglioniJ. P.SouriceS.CournolR.DeswartesC.BersegeayA.DescombinJ.SiegwartM.LoridonK. 2011 Effect of ectopic expression of the eutypine detoxifying gene Vr-ERE in transgenic apple plants. Plant Cell Tissue and Organ Culture, 106, 161168
  29. 29. MDChiltonDrummondM. H.MerioD. J.SciakyD.MontoyaA. L.GordonM. P.NesterE. W. 1977 Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell, 11, 263271
  30. 30. ChristouP. 1992 Genetic transformation of crop plants using microprojectile bombardment. Plant Journal, 2, 275281
  31. 31. ChuY.FaustinelliP.RamosM. L.HajduchM.StevensonS.ThelenJ. J.MalekiS. J.ChengH.Ozias-AkinsP. 2008b Reduction of IgE binding and nonpromotion of Aspergillus flavus fungal growth by simultaneously silencing Ara h 2 and Ara h 6 in peanut. J. Agric. Food Chem. 56, 1122511233
  32. 32. ChuY.DengX. Y.FaustinelliP.Ozias-AkinsP. 2008a Bcl-xL transformed peanut (Arachis hypogaea L.) exhibits paraquat tolerance. Plant Cell Reports, 27, 8592
  33. 33. MSCostaMiguelC. M.MMOliveira 2006 An improved selection strategy and the use of acetosyringone in shoot induction medium increase almond transformation efficiency by 100-fold. Plant Cell, Tissue and Organ Culture, 85, 205209
  34. 34. Cruz-HernándezA.WitjaksonoLitz. R. E.GomezLim. M. 1998 Agrobacterium tumefaciens-mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos. Plant Cell Reports, 17, 497503
  35. 35. Cruz-HernandezA.GomezLim.MALitzR. E.(1997 (1997).Transformation of mango somatic embryos. Acta Horticulturae, 455 292298
  36. 36. DaniellH.KrishnanM.Mc FaddenB. F. 1991 Transient expression of β-glucuronidase in different cellular compartments following biolistic delivery of foreign DNA into wheat leaves and calli. Plant Cell Reports, 9, 615619
  37. 37. DavidsonS. N. 2008 Forbidden Fruit: Transgenic Papaya in Thailand. Plant Physiology, 147, 487493
  38. 38. De BlockM.Herrera-EstrellaL.Van MontaguM.SchellJ.ZambriskiP. 1984 Expression of foreign genes in regenerated plants and their progeny. EMBO Journal, 3, 16811689
  39. 39. DegenhardtJ.PoppeA.MontagJ.SzankowskiI. 2006 The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants. Plant Cell Reports, 25, 11491156
  40. 40. DominguezA.GuerriJ.CambraM.NavarroL.MorenoP.PenaL. 2000 Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Reports, 19, 427433
  41. 41. DruartP.DelporteF.BrazdaM.Ugarte-BallonC.daCâmara.MachadoA.Laimerda.CâmaraMachado. M.Jacquemin & Watillon, B. 1998 Genetic transformation of cherry trees. Acta Horticulturae, 468, 7176
  42. 42. DuttM.LiZ. T.DhekneyS. A.GrayD. J. 2007 Transgenic plants from shoot apical meristems of Vitis vinifera Thompson Seedless via Agrobacterium-mediated transformation. Plant Cell Reports, 26, 21012110
  43. 43. EbinumaH.SugitaK.MatsunagaE.EndoS.YamadaK. 1997 Selection of marker-free transgenic plants using the isopentyl transferase gene. Proceedings of National Academy of Science USA, 94, 21172121
  44. 44. FagoagaC.TadeoF. R.IglesiasD. J.HuertaL.LlisoI.VidalA. M.TalonM.NavarroL.Garcia-MartinezJ. L.PeñaL. 2007 Engineering of gibberellin levels in citrus by sense and antisense overexpression of a GA 20-oxidase gene modifies plant architecture. Journal of Experimental Botany, 58(6), 14071420
  45. 45. FagoagaC.LópezC.Hermoso deMendoza. A.MorenoP.NavarroL.FloresR.PeñaL. 2006 Post-transcriptional gene silencing of the p23 silencing supperssor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Molecular Biology, 60, 153165
  46. 46. FagoagaC.RodrigoI.ConejeroV.HinarejosC.TusetJ. J.ArnauJ.PinaJ. A.NavarroL.PeñaL. 2001 Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related PR-5. Molecular Breeding, 7, 175185
  47. 47. FaizeM.MalnoyM.DupuisF.ChevalierM.ParisiL.ChevreauE. 2003 Chitinases of Trichoderma atroviride induce scab resistance and some metabolic changes in two cultivars of apple. Phytopathology, 93, 14961504
  48. 48. FaizeM.FaizeL.BurgosL. 2010 Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation. BMC Biotechnology, 10, 53
  49. 49. FanJ.LiuX.XuS. X.XuQ.GuoW. W. 2011 T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange. Plant Cell Tissuse and Organ Culture, 107, 225232
  50. 50. FangG.GrumetR. 1993 Genetic engineerring of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Molecular Plant-Microbe Interactions 6, 358367
  51. 51. FebresV. J.LeeR. F.MooreG. A. 2008 Transgenic Resistance to Citrus Tristeza Virus in Grapefruit. Plant Cell Reports, 27(1), 93104
  52. 52. FebresV. J.NiblettC. L.LeeR. F.MooreG. A. 2003 Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with Citrus Tristeza Closterovirus genes. Plant Cell Reports, 21, 421428
  53. 53. MMFitchManshardtR. M.GonsalvesD.SlightomJ. L.SanfordJ. C. 1992 Virus resistant papaya derived from tissues bombarded with the coat protein gene of papaya ringspot virus. Bio/Technology, 10, 14661472
  54. 54. FraryA.EckJ. V. 2005 Organogenesis From Transformed Tomato Explants. In: Methods in molecular biology, 286 Transgenic plants: methods and protocols. L., Pena; NJ., Totowa (Eds.), 141-151, Humana Press Inc.
  55. 55. FuX.KhanE. U.HuS. S.FanQ. J.LiuJ. H. 2011 Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliate L. Raf). Environmental and Experimental Botany (in press)
  56. 56. FuchsM.GonsalvesG. 2007 Safety of virus-resistant transgenic plants two decades after their introduction: lesson from realistic field risk assessment studies. Annual Review of Phytopathology, 47, 173202
  57. 57. GagoJ. 2009 Biotechnology of Vitis vinifera L.: Modellization through Artificial Intelligence. Doctoral Thesis. University of Vigo (Spain) (in Spanish).
  58. 58. GagoJ.Grima-PettenatiJ.GallegoP. P. 2011 Vascular-specific expression of GUS and GFP reporter genes in transgenic grapevine (Vitis vinifera L. cv. Albariño) conferred by the EgCCR promoter of Eucalyptus gunnii. Plant Physiology and Biochemistry, 49, 413419
  59. 59. GallegoP. P.RodriguezR.de la TorreF.VillarB. 2002 Genetic transformation of Eucalyptus globulus. In: Sustainable Forestry Wood Products and Biotechnology. S. Espinel, Y. Barredo, & E. Ritter (Eds.), 163170 , DFA-AFA Press, Vitoria-Gasteiz, Spain
  60. 60. GallegoP. P.LandínM.GagoJ. 2011 Artiticial neural networks technology to model and predict plant biology process. In: Artificial Neural Networks- Methodological Advances and Biomedical Applications. K. Suzuki (Ed.), 197216 , Intech Open Access Publisher: Croatia
  61. 61. GallegoP. P.RodríguezR.de la TorreF.VillarB. 2009 Procedimiento para transformar material vegetal procedente de árboles adultos. Patent ES2299285 A1), Universidade de Vigo, Spain.
  62. 62. GalunE.BreimanA. 1998 Transgenic Plants. Imperial College Press, London, UK.
  63. 63. GelvinS. B. 1998 The introduction and expression of transgenes in plants. Current Opinion in Biotechnology, 9, 227232
  64. 64. GeorgeE. F.MAHallDe KlerkG. J. 2008 Adventitious regeneration. In: Plant Propagation by Tissue Culture, 3rd edn. E.F. George, M.A. Hall & G-J, De Klerk. (Eds.), 355401 , Dordrecht, Netherlands: Springer
  65. 65. GhorbelR.JuarezJ.NavarroL.PeñaL. 1999 Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theoretical and Applied Genetics, 99, 350358
  66. 66. GhorbelR.LópezC.FagoagaC.MorenoP.NavarroL.FloresR.PeñaL. 2001 Transgenic Citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms. Molecular Plant Pathology 2, 2736
  67. 67. GittinsJ. R.PellnyT. K.BiricoltiS.HilesE. R.PasseyA. J.JamesD. J. 2003 Transgene expression in the vegetative tissues of apple driven by the vascular-specific rolC and CoYMV promoters. Transgenic Research, 12, 391402
  68. 68. GonsalvesC.XueB.YepesM.FuchsM.LingK.NambaS. 1994 Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections, Journal of American Society for Horticultural Science, 119, 345355 .
  69. 69. GonsalvesD. 1998 Control of papaya ringspot virus in papaya: a case study. Annual Review of Phytopathology, 36, 415437
  70. 70. GouldS. J.SubramaniS. 1988 Firefly luciferase as a tool in molecular and cell biology. Analytical Biochemistry, 175, 513
  71. 71. Guzmán-GonzálezS.Valádez-RamírezP.Robles-BerberR. E.Silva-RosalesL.Cabrera-PonceJ. L. 2006 Biolistic genetic transformation of Carica papaya L. using HeliosTM gene gun. HortScience, 41, 10531056
  72. 72. HaldrupA.PetersenS. G.OkkelsF. T. 1998 Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Reports, 18, 7681
  73. 73. HankeM. V.ReidelM.ReimS.FlachowskyH. 2007 Analysis of tissue uniformity in transgenic apple plants. Acta Horticulturae, 738, 301306
  74. 74. HaoJ.NiuY.YangB.GaoF.ZhangL.WangJ.HasiA. 2011 Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway. Biotechnology Letters, 33, 5561
  75. 75. HebertD.KikkertJ. R.SmithF. D.ReischB. I. 1993 Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Reports, 12, 585589
  76. 76. HenselG.HimmelbachA.ChenW.DouchkovD. K.KumlehnJ. 2011 Transgene expression systems in the Triticeae cereals. Journal of Plant Physiology, 168, 3044
  77. 77. Heslop-HarrisonJ. S. 2005 Introduction. In: Biotechnology of Fruit and Nut Crops (Ed. RE Litz). CAB International, London, UK. p. xix.
  78. 78. HieiY.OhtaS.KomariT.KumashiroT. 1994 Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271282
  79. 79. HigginsC. M.DietzgenR. G.AkinM.SudarsonoH.ChenK.XuZ. 1999 Biological and molecular variability of peanut stripe potyvirus. Curr Topics Virol, 1, 126
  80. 80. HigginsC. M.HallR. M.MitterN.CruickshankA.DietzgenR. G. 2004 Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences Transgenic Research, 13, 5967
  81. 81. HoleforsA.XueZ. T.WelanderM. 1998 Transformation of the apple rootstock M26 with the rolA gene and its influence on growth. Plant Science, 136, 6978
  82. 82. HondaC.KusabaS.NishijimaT.MoriguchiT. 2011 Transformation of kiwifruit using ipt gene alters tree architecture. Plant Cell Tissuse and Organ Culture 107, 4550
  83. 83. HorschR. B.FryJ. E.HoffmanN. L.EichholtzD.RogersD.FraleyR. T. 1985 A simple and general method for transferring genes into plants. Science, 227, 12291231
  84. 84. HraškaM.HeřmanováV.RakouskýS.ĈurnV. 2008 Sample topography and position within plant body influence the detection of the intensity of green fluorescent protein fluorescence in the leaves of transgenic tobacco plants. Plant Cell Reports, 27, 6777
  85. 85. HuangY. C.ChiangC. H.LiC. M.YuT. A. 2011 Transgenic watermelon lines expressing the nucleocapsid gene of Watermelon silver mottle virus and the role of thiamine in reducing hyperhydricity in regenerated shoots. Plant Cell Tissuse and Organ Culture 106, 2129
  86. 86. HusainiA. M. 2010 Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants. Plant Cell Reports, 29, 97110
  87. 87. IshidaY.SatioH.OhtaS.HieiY.KomariT.KumashiroT. 1996 High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology, 14, 745750
  88. 88. IsmailR. M.El -DomyatiF. M.EEWagihASSadikAbdelsalamA. Z. E. 2011 Construction of banana bunchy top nanovirus-DNA-3 encoding the coat protein gene and its introducing into banana plants cv. Williams. Journal of Genetic Engineering and Biotechnology, 9 (1), 3541
  89. 89. JDJamesPasseyA. J.BarbaraD. J.BevanM. 1989 Genetic transformation of apple (Malus pumila Mill) using a disarmed Ti-binary vector. Plant Cell Reports, 7, 658661
  90. 90. JeffersonR. A. 1987 Assaying chimeric genes in plants: the gus gene fusion system. Plant Molecular Biology Reporter, 5, 387405
  91. 91. JoersboM.DonaldsonI.KreibergJ.GuldagerPetersen. S.BrunstedtJ.OkkelsF. T. 1998 Analysis of mannose selection used for transformation of sugar beet. Molecular Breeding, 4, 111117
  92. 92. JoersboM.JoregensenK.BrunstedtJ. 2003 A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose: galactose-1-phosphate uridyltransferase gene as selective gene. Molecular Breeding, 11, 315323
  93. 93. JoersboM.PetersenS. G.OkkelsF. T. 1999 Parameters interacting with mannose selection employed for the production of transgenic sugar beet. Physiologia Plantarum, 105, 109115
  94. 94. JoshiS. G. 2010 Towards durable resistance to apple scab using cisgenes. Wageningen, The Netherlands: Wageningen University, PhD Thesis
  95. 95. KayimM.KocN. M.TorM. 1996 Gene Transfer into citrus (Citrus limon L.) nucellar cells by particle bombardment and expression of gus activity. Turkish Journal of Agriculture and Forestry, 20, 349352
  96. 96. KhandelwalA.RenukaradhyaG. J.RajasekharM.SitaG. L.MSShaila 2011 Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep. Veterinary Immunology and Immunopathology, 140, 291296
  97. 97. KikkertJ. R.Hebert-SouleD.WallaceP. G.MJStriemReischB. I. 1996 Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Reports, 15, 311316
  98. 98. KikkertJ. R.HumistonG. A.RoyM. K.SanfordJ. C. 1999 Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants. In Vitro Cellular & Developmental Biology-Plant, 35, 4350
  99. 99. KleinT. M.WolfE. D.WuR.et al. 1987 High velocity microprojectiles for delivering nucleic acids into living cells. Nature, 327, 7073
  100. 100. KleinT. M.JonesT. J. 1999 Methods of genetic transformation: the gene gun. In: Molecular Improvement of Cereal Crops: Advances in Cellular and Molecular Biology of Plants, 5 I.K. Vasil, (Ed.), 21-42, Dordrecht, The Netherlands: Kluwer Academic Publishers
  101. 101. KoK.NorelliJ. L.ReynoirdJ. P.Boresjza-WysockaE.BrownS. K.AldwinckleH. S. 2000 Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnology Letters, 22, 373381
  102. 102. KokkoH. I.KärenlampiS. O. 1998 Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens. Plant Cell Reports, 17, 822826
  103. 103. KrausJ. 2010 Concepts of Marker Genes for Plants. In: Genetic Modification of Plants. F., Kempken, C., Jung (Eds.), 39-60, Biotechnology in Agriculture and Forestry, Volume 64, 1, Springer-Verlag, Berlin-Heidelberg
  104. 104. KrensF. A.SchaartJ. G.GroenwoldR.WalravenA. E. J.HesselinkT.ThissenJ. T. N. M. 2011 Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines. Transgenic Research, 20, 11131123
  105. 105. LauJ. M.KorbanS. S. 2010 Transgenic apple expressing an antigenic protein of the human respiratory syncytial virus. Journal of Plant Physiology, 167, 920927
  106. 106. LeeY. K.KimI. J. 2011 Modulation of fruit softening by antisense suppression of endo-b-1,4-glucanase in strawberry. Molecular Breeding, 27, 375383
  107. 107. LeymanB.AvonceN.RamonM.Van DijckP.IturriagaG.TheveleinJ. M. 2006 Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation. Journal of Biotechnology, 121, 309317
  108. 108. LiZ. T.DhekneyS.DuttM.Van AmanM.TattersallJ.KelleyK. T.GrayD. J. 2006 Optimizing Agrobacterium-mediated transformation of grapevine. In Vitro Cellular and Developmental Biology-Plant, 42, 220227
  109. 109. LindseyK. 1992 Genetic manipulation of crop plants. Journal of Biotechnology 26, 128
  110. 110. LiuZ.ParkB. J.KannoA.KameyaT. 2005 The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with leagene. Molecular Breeding, 16, 189197
  111. 111. López-NogueraS.PetriC.BurgosL. 2009 Combining a regeneration-promoting ipt gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes. Plant Cell Reports, 28, 17811790
  112. 112. Lopez-PerezA. J.VelascoL.Pazos-NavarroM.DabauzaM. 2008 Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low agrobacterium density. Plant Cell Tissue and Organ Culture, 94, 189199
  113. 113. MaddumageR.FungR. M. W.DingH.SimonsJ. L.AllanA. C. 2002 Efficient transformation of suspension culturederived apple protoplasts. Plant Cell Tissue and Organ Culture, 70, 7782
  114. 114. MaghulyF.LeopoldS.MachadoA.BorrotoFernández. E.MAKhanGambinoG.GribannoI.SchartlA.LaimerM. 2006 Molecular characterization of grapevine plants transformed with GFLV resistance genes: II. Plant Cell Reports, 25, 546553
  115. 115. MalnoyM.EEBoresjzaNorelliJ. L.MAFlaishmanGidoniD.AldwinckleH. S. 2010 Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree Genetics and Genome, 6, 423433
  116. 116. ManimaranP.RamkumarG.SakthivelK.SundaramR. M.MSMadhavBalachandranS. M. 2011 Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: Present status and future prospects. Biotechnology Advances, 29, 703714 .
  117. 117. ManteS.MorgensP. H.ScorzaR.CordtsJ. M.CallahanA. M. 1991 Agrobacterium-mediated transformation of plum (Prunus domestica L) hypocotyls slices and regeneration of transgenic plants. Bio-Technology, 9, 853857
  118. 118. MathewsH.DeweyV.WagnerW.BestwickR. K. 1998 Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols. Transgenic Research, 7, 123129
  119. 119. MathewsH.LitzR. E.WildeD. H.MerkelS.WetzsteinH. Y. 1992 Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos. In Vitro Cellullar and Developmental Biology- Plant, 28, 172178
  120. 120. MathewsH.LitzR. E.WildeH. D.WetzsteinH. Y. 1993 Genetic transformation of mango. Acta Horticulturae, 341, 9397
  121. 121. MaximovaS. N.DandekarA. M.MJGuiltinan 1998 Investigation of Agrobacterium-mediated transformation of apple using gren fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting. Plant Molecular Biology, 37, 549559
  122. 122. Mc CabeD.ChristouP. 1993 Direct DNA transfer using electrical discharge particle acceleration (ACCELL™ technology). Plant Cell Tissue and Organ Culture, 33, 227236
  123. 123. MercadoJ. A.TrainottiL.Jiménez-BermúdezL.Santiago-DoménechN.PoséS.DonolliR.BarcelóM.CasadoroG.Pliego-AlfaroF.MAQuesada 2010 Evaluation of the role of the endo-β-(1,4)-glucanase gene FaEG3 in strawberry fruit softening. Postharvest Biology and Technology, 55, 814
  124. 124. MezzettiB.PandolfiniT.NavacchiO.LandiL. 2002 Genetic transformation of Vitis vinifera via organogenesis. BMC Biotechnology, 2, 1827
  125. 125. MikiB.Mc HughS. 2004 Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology, 107, 193232
  126. 126. NakanoM.HoshinoY.MiiM. 1994 Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenes- mediated transformation of embryogenic calli. Journal of Experimental Botany, 45, 649656
  127. 127. NaqviS.FarréG.SanahujaG.CapellT.ZhuC.ChristouP. 2009 When more is better: multigene engineering in plants. Trends in Plant Science, 15, 4856
  128. 128. CANewell 2000 Plant transformation technology; developments and applications. Molecular Biotechnology, 16, 5365
  129. 129. NiralaN. K.DasD. K.SrivastavaP. S.SoporyS. K.UpadhyayaK. C. 2010 Expression of a rice chitinase gene enhances antifungal potential in transgenic grapevine (Vitis vinifera L.). Vitis, 49, 181187
  130. 130. OECD. 1999 Concensus document on general information concerning the genes and their enzymes that confer tolerance to phosphinothricin herbicide. Series on Harmonization of Regulatory Oversight in Biotechnology, 11
  131. 131. OliveiraM. L. P.FebresV. J.CostaM. G. C.MooreG. A.OtoniW. C. 2009 High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Reports, 28, 387395
  132. 132. MMOliveiraBorrosaJ. G.MartinsM.MSPais 1994 In Y. P. S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, 29 189210 . Berlin: Springer
  133. 133. MMOliveiraBarrosoJ.MSPais 1991 Direct gene transfer into kiwifruit protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS based method. Plant Molecular Biology, 17, 235242
  134. 134. PadillaI. M. G.BurgosL. 2010 Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Reports, 29, 12031213
  135. 135. PadillaI. M. G.GolisA.GentileA.DamianoC.ScorzaR. 2006 Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes. Plant Cell Tissue and Organ Culture, 84, 309314
  136. 136. ParadelaM. R.De PazP.GallegoP. P. 2006 Comparation of two kiwifruit transformation methods, Proceedings of 11th International Association for Plant Tissue Culture & Biotechnology Congress. Biotechnology and Sustainable Agriculture 2006 and Beyond, 76 P-1121), Beijing China, September 27-30, 2006
  137. 137. PaszkowskiJ.ShillitoR. D.SaulM.MandakV.HohnT.HohnB.et al. 1984 Direct gene transfer to plants. EMBO Journal, 3, 27172722
  138. 138. PaszkowskiJ.WithamS. A. 2001 Gene silencing and methylation processes. Current Opinion in Plant Biology, 4, 123129
  139. 139. PawlowskiW. P.SomersD. A. 1996 Transgene inheritance in plants genetically engineered by microprojectile bombardment. Molecular Biotechnology, 6, 1730
  140. 140. Pérez-ClementeR. M.Pérez-SanjuánA.García-FérrizL.BeltránJ. P.CañasL. A. 2004 Transgenic peach plants (Prunus persica L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an in vivo marker. Molecular Breeding, 14, 419427
  141. 141. Pérez-PiñeiroP.GagoJ.LandínP.GallegoP. P. 2012 Agrobacterium-mediated transformation of wheat: general overview and new approaches to model and identify the key factors involved. In: Transgenic Plants. Y., Ozden Çiftçi (Ed.), XX_XX, Intech Open Access Publisher: Croatia (in press)
  142. 142. PetolinoJ. 2002 Direct DNA delivery into intact cells and tissues. In: Transgenic Plants and Crops. G. Khachatourians, A. McHughen, R. Scorza, W-K. Nip &Y. Hui, (Eds.), 137141 , Mercel Dekker Inc.; New York
  143. 143. PetriC.BurgosL. 2005 Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Research, 14, 1526
  144. 144. PetriC.AlburquerqueN.García-CastilloS.EgeaJ.BurgosL. 2004 Factors affecting gene transfer efficiency to apricot leaves during early Agrobacterium-mediated transformation steps. Journal of Horticultural Science and Biotechnology, 79, 704712
  145. 145. PetriC.HilyJ. M.VannC.DardickC.ScorzaR. 2011 A high-throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica). Annals of Applied Biology, 159, 302315
  146. 146. PetriC.WangH.AlburquerqueN.FaizeM.BurgosL. 2008 Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants. Plant Cell Reports, 27, 13171324
  147. 147. PurohitS. D.RaghuvanshiS.TyagiA. K. 2007 Biolistic-mediated DNA delivery and transient expression of GUS in hypocotyls of Feronia limonia L.- A fruit tree. Indian Journal of Biotechnology, 504507
  148. 148. RadchukV. V.KorkhovoyV. I. 2005 The rolB gene promotes rooting in vitro and increases fresh root weight in vivo of transformed apple scion cultivar ‘Florina’. Plant Cell Tissue and Organ Culture, 81, 203212
  149. 149. RameshS.KaiserB. N.FranksT. K.CollinsG. G.SedgleyM. 2006 Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols. Plant Cell Reports, 25, 821828
  150. 150. RaoA. Q.BakhshA.KianiS.ShahzadK.AAShahidHusnainT.RiazuddinS. 2009 The myth of plant transformation. Biotechnology Advances, 27, 753763
  151. 151. RaquelM. H.MMOliveira 1996 Kiwifruit leaf protoplasts competent for plant regeneration and direct DNA transfer. Plant Science, 121, 107114
  152. 152. ReedJ.PrivalleL.PowellM. L.MeghjiM.DawsonJ.DunderE.SuttieJ.WenckA.LaunisK.KramerC.ChangY. F.HansenG.WrightM. 2001 Phosphomannose isomerase: an efficient selectable marker for plant transformation In Vitro Cellular and Developmental Biology-Plant, 37, 127132
  153. 153. RemyS.BuyensA.CammueB. P. A.SwennenR.SagiL. 2000 Production of transgenic banana plants expressing antifungal proteins. International Symposium on Banana in the Subtropics. Acta Horticulturae, 490, 219277
  154. 154. RommensC. M.MAHaringSwordsK.DaviesH. V.BelknapW. R. 2007 The intragenic approach as a new extension to traditional plant breeding. Trends in Plant Science, 12, 397403
  155. 155. RuginiE.PellegrineschiA.MecucciniM.MariottiD. 1991 Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes. Plant Cell Reports, 6, 291295
  156. 156. SagiL.PanisB.RemyS.SchoofsH.SmetK.SwennenR.GammueB. P. A. 1995 Genetic transformation of banana and plantain (Musa spp.) via particle bombardment. Biotechnology, 13, 481485
  157. 157. SagiL.RemyS.SwennenR. 1998 Genetic transformation for the improvement of bananas- a critical assessment. In: INIBAP Annual Report. 3336 . Montpellier: FRA.
  158. 158. SakuanrungsirikulS.SarinduN.PrasartseeV.ChaikiatiyosS.SiriyanR.SriwatanakulM.LekananonP.KitprasertC.BoonsongP.KosiyachindaP.et al. 2005 Update on the development of virus-resistant papaya: virus-resistant transgenic papaya for people in rural communities of Thailand. Food Nutr Bull, 26, 422426
  159. 159. SanfordJ. C.KleinT. M.WolfE. D.AllenN. 1987 Delivery of substances into cells and tissues using a particle bombardment process. Journal of Paniculate Science and Technology, 6, 559563
  160. 160. Sanford JC. 1988 The Biolistic Process. Trends in Biotechnology 6, 299302
  161. 161. SanfordJ. C. 1990 Biolistic plant transformation. Physiologia Plantarum 79, 206209
  162. 162. SautterC. 1993 Development of a microtargeting device for particle bombardment of plant meristems. Plant Cell Tissue and Organ Culture, 33, 251257
  163. 163. SchaartJ.G.KrensF. A.PelgromK. T.MendesO.RouwendalG. J. 2004 Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnology Journal, 2, 233240
  164. 164. SchoutenH. J.KrensF. A.JacobsenE. 2006a Do cisgenic plants warrant less stringent oversight? Nature Biotechnology, 24, 753
  165. 165. SchoutenH. J.KrensF. A.JacobsenE. 2006b Cisgenic plants are similar to traditionally bred plants. EMBO Reports, 7, 750753
  166. 166. SchubertD.WillimsD. (2006).’Cisgenic’ as a product designation. Nature Biotechnology, 24 13271329
  167. 167. SchuermanP. L.DandekarA. M. 1993 Transformation of temperate woody crops- Progress and Potentials. Scientia Horticulturae, 55, 101124
  168. 168. ScorzaR.CordtsJ. M.GrayD. J.GonsalvesD.EmershadR. I.RammingD. W. 1996 Producing transgenic ‘Thompson Seedless’ grape (Vitis vinifera L.) plants. Journal of American Society for Horticultural Science, 121, 616619
  169. 169. ScorzaR.LevyL.DamsteegtV.YepesL. M.CordtsJ.HadidiA.SlightomJ.GonsalvesD. 1995 Transformation of plum with the papaya ringspot virus coat protein gene and reaction of transgenic plants to plum pox virus. Journal of American Society for Horticultural Science, 120, 943952
  170. 170. SerresR.StangE.Mc CabeD.RussellD.MahrD.Mc CownB. 1992 Gene transfer using electric discharge particle bombardment and recovery of transformed cranberry plants. Journal of American Society for Horticultural Science, 117, 174180
  171. 171. SichererS. H.Munoz-FurlongA.SampsonH. A. 2003 Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: a 5-year followup study. Journal of Allergy and Clinical Immunology, 112, 12031207
  172. 172. SinghS. 1992 Fruit crops of wasteland. Scientific Publishers, Jodhpur, India.
  173. 173. SingsitC.MJAdangLynchR.AndersonW. F.WangA.CardineauG.Ozias-AkinsP. 1997 Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer. Transgenic Research, 6, 169176
  174. 174. SmolkaA.WelanderM.OlssonP.HoleforsA.ZhuL. H. 2009 Involvement of the ARRO-1 gene in adventitious root formation in apple. Plant Science, 177, 710715
  175. 175. SongG. Q.SinkK. C. 2004 Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.). Plant Cell Reports, 23, 475484
  176. 176. StalkerH. T.CESimpson 1995 Genetic resources in Arachis. In: Advances in Peanut Science, Pattee HE., & Stalker HT, 1453 . American Peanut Research and Educational Society, Stillwater, OK, USA
  177. 177. StewartC. N. 2001 The utility of green fluorescent protein in transgenic plants. Plant Cell Reports, 20, 376382
  178. 178. SubramanyamK.SubramanyamK.SailajaK. V.SrinivasuluM.LakshmideviK. 2011 Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration. Plant Cell Reports, 30, 425436
  179. 179. SunQ.ZhaoY.SunH.HammondR. W.DavisR. E.XinL. 2011 High-efficiency and stable genetic transformation of pear (Pyrus communis L.) leaf segments and regeneration of transgenic plants. Acta Physiologiae Plantarum, 33, 383390
  180. 180. SundarI. K.SakthivelN. 2008 Advances in selectable marker genes for plant transformation. Journal of Plant Physiology, 165, 16981716
  181. 181. SzankowskiI.WaidmannS.DegenhardtJ.PatocchiA.ParisR.Silfverberg-DilworthE.BrogginiG.GesslerC. 2009 Highly scab-resistant transgenic apple lines achieved by introgression of HcrVf2 controlled by different native promoter lengths. Tree Genetics and Genomes, 5, 349358
  182. 182. TaylorN. J.FauquetC. M. 2002 Microparticle Bombardment as a Tool in Plant Science and Agricultural Biotechnology. DNA and Cell Biology, 21, 963977
  183. 183. TennantP. F.GonsalvesC.LingK. S.MMFitchManshardtR.SlightomL. J.GonsalvesD. 1994 Differential protection against papaya ringspot virus isolates in coat protein gene transgenic papaya and classically cross-protected papaya. Phytopathology, 84, 13591366
  184. 184. TerakamiS.MatsudaN.YamamotoT.SugayaS.GemmaH.SoejimaJ. 2007 Agrobacterium-mediated transformation of the dwarf pomegranate (Punica granatum L. var. nana). Plant Cell Reports, 26, 12431251
  185. 185. ThompsonC. J.MovvaN. R.TizardR.CrameriR.DaviesJ. E.LauwereysM.BottermanJ. 1987 Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO Journal, 6, 25192523
  186. 186. TianN.WangJ.XuZ. Q. 2011 Overexpression of Na+/H+ antiporter gene AtNHX1 from Arabidopsis thaliana improves the salt tolerance of kiwifruit (Actinidia deliciosa). South African Journal of Botany, 77, 160169
  187. 187. TianL.CanliF. A.WangX.Sibbald 2009 2009. Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker. Scientia Horticulturae, 119, 339343
  188. 188. TrickH. N.FinerJ. J. 1998 Sonication-assisted Agrobacterium-mediated transformation of soybean Glycine max L. Merrill embryogenic suspension culture tissue. Plant Cell Reports, 17, 482488
  189. 189. TrickH. N.FinerJ. J. 1997 SAAT: sonication-assisted Agrobacterium mediated transformation. Transgenic Research, 6, 329336
  190. 190. TripathiS.SuzukiJ. Y.CarrJ. B.Mc QuateG. T.FerreiraS. A.ManshardtR. M.PitzK. Y.MMWallGonsalvesD. 2011 Nutritional composition of Rainbow papaya, the first commercialized transgenic fruit crop. Journal of Food Composition and Analysis, 24, 140147
  191. 191. TripathiL. 2003 Genetic engineering for improvement of Musa production in Africa. African Journal of Biotechnology, 2, 503508
  192. 192. TripathiS.SuzukiJ. Y.FerreiraS. A.GonsalvesD. 2008 Papaya ringspot virus-P: characteristics, pathogenicity, sequence variability and control. Molecular Plant Pathology, 9, 269280
  193. 193. TwymanR. M.StögerE.KohliA.CapellT.ChristouP. 2002 Selectable and screenable markers for rice transformation. Molecular Methods of Plant Analysis, 22, 117
  194. 194. UematsuC.MuraseM.IchikawaH.ImamuraJ. 1991 Agrobacterium-mediated transformation and regeneration of kiwi fruit. Plant Cell Reports, 10, 286290
  195. 195. UrtubiaC.DeviaJ.CastroA.ZamoraP.AguirreC.TapiaE.BarbaP.Dell’OrtoP.MoynihanM. R.PetriC.ScorzaR.PrietoH. 2008 Agrobacterium-mediated genetic transformation of Prunus salicina. Plant Cell Reports, 27, 13331340
  196. 196. van LeeuwenW.HagendoornM. J. M.RuttinkT.van PoeckeR.van der PlasL. H. W.van der KrolA. R. 2000 The use of the luciferase reporter system for in planta gene expression studies. Plant Molecular Biology Reports, 18, 143a143t
  197. 197. VanblaereT.SzankowskiI.SchaartJ.SchoutenH.FlachowskyH.BrogginiG. A. L.GesslerC. 2011 The development of a cisgenic apple plant. Journal of Biotechnology, 154, 304311
  198. 198. VarshneyR. K.KailashC.BansalK. C.AggarwalP. K.DattaS. K.CraufurdP. Q. 2011 Agricultural biotechnology for crop improvement in a variable climate: hope or hype? Trends in Plant Science, 16, 363371
  199. 199. VeluthambiK.GuptaA. K.SharmaA. 2003 The current status of plant transformation technologies. Current Science, 84, 368380
  200. 200. VidalJ. R.KikkertJ. R.WallaceP. G.ReischB. I. 2003 High-efficiency biolistic co-transformation and regeneration of ‘Chardonnay’ (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes. Plant Cell Reports, 22, 252260
  201. 201. VidalJ. R.KikkertJ. R.MAMalnoyWallaceP. G.BarnardJ.ReischB. I. 2006 Evaluation of transgenic Chardonnay (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew. Transgenic Research, 15, 6982
  202. 202. VillarB.OllerJ. J.TeulieresC.BoudetA. M.GallegoP. P. 1999 In planta transformation of adult clones of Eucalyptus globulus sp using an hypervirulent Agrobacterium tumefaciens strain. In: Application of Biotechnology to Forest Genetic. Espinel, S. & Ritter, E. (Eds.), 2225 , DFA-AFA Press, Vitoria-Gasteiz, Spain.
  203. 203. VishnevetskyJ.White JrT. L.PalmateerA. J.FlaishmanM.CohenY.EladY.VelchevaM.HananiaU.SaharN.DganiO.PerlA. 2011 Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain. Transgenic Research, 20, 6172
  204. 204. WangT.RanY.AtkinsonR. G.GleaveA. P.CohenD. 2006 Transformation of Actinidia eriantha: A potential species for functional genomics studies in Actinidia. Plant Cell Reports, 25, 425431
  205. 205. WangH. 2011 Development of a genetic transformation protocol genotype-independent in apricot. Doctoral Thesis. CEBAS-CSIC, University of Murcia (Spain) (in Spanish).
  206. 206. WangQ.LiP.HananiaU.SaharN.MawassiM.GafnyR.SelaI.TanneE.PerlA. 2005 Improvement of Agrobacterium-mediated transformation efficiency and transgenic plant regeneration of Vitis vinifera L. by optimizing selection regimes and utilizing cryopreserved cell suspensions. Plant Science, 168, 565571
  207. 207. WeinthalD.TovkachA.ZeeviV.TzfiraT. 2010 Genome editing in plant cells by zinc finger nucleases. Trends in Plant Science, 15, 308321
  208. 208. WohllebenW.ArnoldW.BroerI.HillemanD.StrauchE.PuhlerA. 1988 Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene,70, 2537
  209. 209. WuH. W.YuT. A.RajaJ. A. J.WangH. C.YehS. D. 2009 Generation of transgenic oriental melon resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method. Plant Cell Reports, 28, 10531064
  210. 210. XuS. X.CaiX. D.TanB.GuoW. W. 2011 Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy. Plant Cell Tissue and Organ Culture, 104, 199207
  211. 211. XueB.LingK. S.ReidC. L.KrastanovaS.SekiyaM.MomolE. A.SuleS.MozsarJ.GonsalvesD.BurrT. J. 1999 Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens. In Vitro Cellular and Developmental Biology- Plant, 35, 226231
  212. 212. YamamotoT.IketaniH.IekiH.NishizawaY.NotsukaK.HibiT.HayashiT.Matsuta 2000ishizawa, Y.; Notsuka, K.; Hibi T, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Reports, 19, 639646
  213. 213. YanchevaS. D.ShlizermanL. A.GolubowiczS.YablovizZ.PerlA.HananiaU.MAFlaishman 2006 The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus communis L.). Plant Cell Reports, 25 183189
  214. 214. YanchevaS. D.GolubowiczS.YablowiczZ.PerlA.MAFlaishman 2005 Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants. Plant Science, 168, 14331441
  215. 215. YangC.ChenS.DuanG. 2011 Transgenic peanut (Arachis hypogaea L.) expressing the urease subunit B gene of Helicobacter pylori. Curr Microbiol, 63, 387391
  216. 216. YaoJ. L.WuJ. H.GleaveA. P.MorrisB. A. M. 1996 Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos. Plant Science, 113, 175183
  217. 217. YeX. J.BrownS. K.ScorzaR.CordtsJ. M.SanfordJ. C. 1994 Genetic transformation of peach tissues by particle bombardment. Journal of the American Society of Horticultural Science, 119, 367373
  218. 218. YipM. K.LeeS. W.SuK. C.LinY. H.ChenT. Y.FengT. Y. 2011 An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt. Plant Biotechnology Reports, 5, 245254
  219. 219. ZeldinE..JuryT. P.SerresR. A.Mc CownB. H. 2002 Tolerance to the herbicide glufosinate in transgenic cranberry (Vaccinium macrocarpon Ait.) and enhancement of tolerance in progeny. Journal of American Society for Horticultural Science, 127, 502507
  220. 220. ZhuY. J.AgbayaniR.Mc CaffertyH.AlbertH. H.MooreP. H. 2005 Effective selection of transgenic papaya plants with the PMI/Man selection system. Plant Cell Reports, 24, 426432
  221. 221. ZhuY. J.AgbayaniR.MooreP. H. 2004 Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation Plant Cell Reports, 22, 660667
  222. 222. ZhuL. H.HoleforsA.AhlmanA.XueZ. T.WelanderM. 2001 Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Science, 160, 433439

Notes

Written By

Hülya Akdemir, Jorge Gago, Pedro Pablo Gallego and Yelda Ozden Çiftçi

Submitted: 29 September 2011 Published: 07 March 2012