Introduction

In conventional transformation system, usually a single-target gene is transformed in a plant species [1, 2]. However, for improved and more effective disease resistance, more than one gene need to be pyramided (also called gene stacking) as the pathogens may overcome the single-gene resistance [3]. Gene stacking or gene pyramiding is the integration of multiple genes, each conferring resistance to single or a separate pest following their independent host pathways. Stacking multiple genes in a genotype is one of the promising tools for breeding higher and durable resistance, especially with the resistant genes that originate from the different gene clusters and represent different host resistance (HR) interactions between the resistance (R) genes and their Avr (effectors) proteins [4]. Employing traditional breeding for gene stacking may lead to linkage drag in gene stacking, while genetic engineering is an efficient and effective strategy to integrate multiple resistance genes into the existing variety. The area of genetically altered crops with stacked genes or traits is likely to rise in near future with combination of the new traits to fulfill needs of the consumers and the producers [5]. Pyramiding genes for disease resistance in crops has become possible because of availability of disease-resistant genes [6] with the improved gene manipulation tools [7].

This means that one may stack genes conferring resistance to insect pests, or one may also pyramid genes that may confer resistance to both the insects and weeds. For instance, transgenic cotton, Bollgard II, developed by Monsanto and registered in U.S. in 2002, expressed two Bt genes, Cry1A(c) and Cry2A(b)2 for conferring resistance to lepidopterans on cotton.

A number of efforts have been made to pyramid more than one transgene in plants for enhanced and effective disease resistance against one or more pathogens. Transgenic rice transformed with a polyprotein and two antimicrobial proteins, Dahlia merckii-antimicrobial protein (Dm-AMP1) and Raphanus sativus- antimicrobial protein (Rs-AFP2) exhibited higher protection to Rhizoctonia solani and Magnaporthe oryzae compared to non-transformed controls or the plants engineered with single-gene constructs [8]. Transgenic potatoes containing chitinase C gene (extracted from Streptomyces griseus) were re-transformed with wasabi defensin (WD) gene (extracted from Wasabia japonica). The transgenic plants stacked with the ChiC and the WD genes were found more resistant to Alternaria solani and Fusarium oxysporum than the wild-type and/or the transgenic plants expressing single gene (ChiC or WD) [9] (Table 1).

Table 1 Summary of disease resistance genes stacked in transgenic plants isolated from different organisms for enhanced resistance against phytopathogens
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Transgene Stacking for Insect Resistance

Currently, commercial transgenic crops are usually stacked with insect-resistant and herbicide-tolerant genes as both traits are highly valuable in the production of major crop plants such as corn and cotton [10]. Bt gene-stacking strategy introduces different insecticidal genes into plant, and proved to be an efficient way of delaying insect resistance to Bt toxin [11]. Thus, the Bt genes with their different modes of action were usually stacked together in the newly developed transgenic crops. Multiple insect-resistant genes and herbicide-tolerant genes were stacked in newly developed commercial transgenic crops. For example, a total of eight genes for insect resistance or herbicide tolerance were stacked in the Genuity® SmartStax™ corn released by Monsanto (USA).

Multiple insect-resistant genes stacking in the transgenic Bt crops have been employed to confer resistance to the insects and herbicides. The first transgenic Bt crop (cotton) with stacked genes, Cry1Ac and Cry2Ab2, registered for use in the U.S. in 2002, was Bollgard II. These stacked genes in the transgenic cotton have been very effective against pink bollworms (Pectinophora gossypiella) Steffey et al. [12]. These genes (Cry1Ac and Cry1C), also stacked in transgenic Bt broccoli, had the potential to delay resistance to diamondback moth (Plutella xylostella) more effectively than the transgenic plants with single-Bt gene [11]. Roundup Ready Flex, a GM cotton also developed by Monsanto, contains three transgenes, Cry1A(c), Cry2A(b)2 and EPSPS genes conferring resistance to insects and herbicide, glyphosate. An altered form of EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), an enzyme causing conversion of sugars to amino acids, was isolated from a soil bacterium which was not affected by glyphosate. Transgenic rice, transformed with Cry1Ac and Cry1Ig and altered glyphosate-tolerant EPSPS genes through single T-DNA, exhibited enhanced resistance to striped rice stem borer (Chilo suppressalis) and rice leafroller (Cnaphalocrocis medinalis), and to glyphosate as well [13]. The triple genes construct (Cry1Ac-Cry2Ab-EPSPS) was also expressed in Nicotiana benthamiana. The transgenic plants had higher resistance to armyworm (Spodoptera littoralis) and herbicides compared to non-transformed control [14]. Transgenic corn, with triple genes stacks, containing Cry3B(b)1 for protection against corn root worm (CRW), Cry1A(b) for imparting resistance to stalk-boring insect, and Roundup Ready® trait for herbicide tolerance were also produced (Table 1).

Transformation Strategies for Stacking Transgenes in Plants

Transformation with the Genes of Interest on a Plasmid or Separate Plasmid

The transgenes with their appropriate promoters and terminators (transgenic cassettes) are placed on a single T-DNA and transformed into plants as a unit to a single locus. Jha and Chattoo [8] transformed rice with Agrobacterium tumefaciens harbouring Dm-AMP1, a linker peptide of the I. balsamina antimicrobial peptides (Ib-AMP) and Rs-AFP2 on a single plasmid, pFAJ3105 (Fig. 1d). The co-expression of the transgenes, Rs-AFP2 and Dm-AMP1 in the transformed plant provided more resistance to fungal pathogens than the singly transformed plants. Transgenic chickpea (Cicer arietinum) co-transformed with the two insecticidal genes, cry1Ab and cry1Ac, exhibited higher resistance to pod borer larvae of Helicoverpa armigera than the single-gene transgenic plants expressing one toxin [15].

Fig. 1
figure 1

Schematic representation of Tranformation for gene stacking. A-C, Sequential transformation of two disease resistance genes. a and b Marker-free transgenic potato plants were produced by MAT vector system. c The Marker-free transgenic potato plants were re-transformed with wasabi defensin gene [28]. dDm-AMP1 and Rs-AFP2 genes were connected by linker peptide on same plasmid between left and right borders [8]. e Three genes, Rpi-sto1, Rpi-vnt1.1 and Rpi-blb3 were transformed simultaneously in potato [19]. f In GAANTRY system more than three genes could be transformed simultaneously in plants [45]. GUS, beta-glucuronidase. hpt, hygromycin phophotransferase. nptII, neomycin phophotransferase. LP, linker peptide region isolated from the seeds of Impatiens balsamina. Dm-AMP1, Antimicrobial proteins from Dahlia merckii. Rs-AFP2, Antimicrobial proteins from Raphanus sativus. Rpi-sto1, Resistance gene for Phytophthora infestans from Solanum stoloniferum. Rpi-vnt1.1, Rpi from S. venturii. Rpi-blb3, Rpi from S. bulbocastanum. TBS transformation booster sequence, MYB CsMybA, Bar bialaphos resistance, GFP enhanced green fluorescent, Luc firefly luciferase, Sul1 sulfadiazine resistance

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Transgenic rice co-transformed, simultaneously, with three genes, the cry1Ac and cry2A genes and the lectin gene from snowdrop (Galanthus nivalis agglutinin; gna) exhibited higher resistance to the important insect pests of rice: rice leafroller (Cnaphalocrocis medinalis), brown planthopper (Nilaparvata lugens) and yellow stem borer (Scirpophaga incertulas) [16]. Walnut rootstock genotype was co-transformed to stack resistance genes for crown gall and nematodes infection using A. tumefaciens binary vector, pDE00.0201 carrying the iaaM (tryptophan 2-monooxygenase), ipt (isopentenyl transferase), GUS (β-glucuronidase), and nptII genes, and A. rhizogenes vector, pGR-Pv010 carrying Pv010 (from Pratylenchus vulnus) and GFP genes. The genes, iaaM, ipt and pv010 were silenced using the RNAi. Silencing of these genes in the transgenic lines caused complete suppression of the crown gall and 32% fewer nematodes than the control lines [17]. Transgenic potato were generated by transforming with a gene construct containing both the cisgenic late blight (Phytophthora infestans (Pi) R genes, the Rpi-vnt1.1 (Solanum venturii) and the Rpi-sto1 (S. stoloniferum), but with no selection marker gene, nptII, using Agrobacterium-mediated transformation. The transformed cells or events were screened by PCR analysis in the regenerated shoots. The transgenic marker-free potato exhibited broadspectrum and durable resistance to the late blight infection [18]. In another attempt, Zhu et al. [19] transformed potato susceptible to late blight by introducing the three genes, Rpi-sto1, Rpi-vnt1.1 and Rpi-blb3 (S. bulbocastanum). The transgenic plants stacked with the triple Rpi genes were found highly resistant to late blight (Table 1 and Fig. 1e).

Re-transformation

Re-transformation of a transgenic plant can be employed to stack or pyramid transgenes into one plant line. For example, Singla-Pareek et al. [20] re-transformed a transgenic tobacco containing glyoxalase I (gly-I) with the glyoxalase II (gly-II) gene which showed enhanced salinity tolerance. [21] re-transformed transgenic potato containing dermaseptin (from Phyllomedusa sauvagii) with double gene construct, the AP24 osmotin (from Nicotiana tabacum) and the lysozyme (from Gallus gallus). Increased level of resistance to Erwinia carotovora was found in the transgenic lines expressing the dermaseptin and lysozyme sequences. These transgenes also exhibited enhanced resistance against F. solani, Phytophthora infestans and Rhizoctonia solani, depicting that stacking of transgenes is an effective strategy to get higher resistance to the fungal and bacterial pathogens.

The disadvantages of using the consecutive transformation strategy include the need for unique selection agents and markers, which are limited and the multiple genomic locations of transgene insertion. It is difficult to obtain offspring with transgenes all localized together in the progeny following genetic segregation. After each transformation, transgenic lines have to be screened for position effects, which render this method less than optimal and practical. Marker-free transformation in which the selection marker gene is excised from the transgenic plants can be an alternate and environment-friendly option to re-transform the transgenic plants with another gene. Site-specific recombination systems (Cre-Lox, FLP-FRT and R-RS recombination) have been used for deletion and integration of DNA sequence at specific sites within genome [22,23,24,25]. When selection marker-free transgenic tobacco containing the ChiC gene was re-transformed with WD gene, the transgenic plants co-expressing both the genes were found significantly with higher resistance to F. oxysporum f.sp. nicotianae (Fon) than the corresponding isogenic lines expressing single gene [26]. Previously, using the multi-auto-transformation (MAT) vector system [27], we produced transgenic potato (free of the selection marker) containing transgene, ChiC, [28]. The marker-free potato was re-transformed with WD gene to stack the two antifungal genes, ChiC and WD (Fig. 1a–c). The transgenic plants expressing both the transgenes were found more resistant to F. oxysporum and A. solani than the wild-type and the single-gene transgenic lines [9].

The marker-free transgenic rice containing the rice chi11 gene was re-transformed with AP24 (tobacco osmotin) using the A. tumefaciens harbouring cointegrate vector, pGV2260::pSSJ1 (a single-copy) and the binary vector, pBin19DnptII-ap24 (a multi-copy) in the same cell. The transgenic plants expressing the stacked genes were found highly resistant to Rhizoctonia solani [29].

Stacked Genes with Linker Peptide

Uncoordinated expression is considered a major constraint in the co-expression of the transgenes, even if the transgenes are linked physically [30]. Multiple copies of transgenes in genome of the transgenic plants may also lead to silencing of the transgenes [31]. To cover these limitations, gene sequences for different proteins can be introduced in an open reading frame using the short linkers. The linker peptides, subsequently, are cleaved in protein units by proteinase from the host cell when passing through the endomembrane system [32].

Jha and Chatto prepared a gene construct, consisting of two antimicrobial proteins, Rs-AFP2 and Dm-AMP1 linked by Ib-AMP linker peptide (16 amino acids) extracted from seeds of Impatiens balsamina (Fig. 1d). Transgenic rice was produced with single-protein gene and the cleavable chimeric polyprotein gene constructs using Agrobacterium-mediated transformation. It was found that the transgenic rice showed increased resistance to rice blast fungus (90% higher) and Rhizoctonia bacteria (79% higher) than the wild-type rice [8].

Francoise and his co-workers expressed a chimeric polyprotein, consisting of two AMPs linked by the LP4, in transgenic Arabidopsis. The chimeric proteins conferred antifungal activity under in vitro condition [32]. 2A, a linker peptide, isolated from the virus causing foot-and-mouth disease, has widely been used in potato, tobacco, tomato, and other crops for gene fusions [33,34,35]. Researchers have also used 2A for transgene stacking in staple food crops [36, 37]. 2A has also been used as linker peptide between carotene desaturase gene (isolated from Pantoea) and phytoene synthase gene (isolated from Capsicum) to make a fusion vector construct and was introduced into rice for high carotenoid contents in “Golden Rice” [38].

Advantages of Stacking Transgenes

Pyramiding more than one transgene in crops may offer broader and more effective disease resistance and other agronomic characters that farmers need for higher yield and quality products. Gene stacking has the potential to pyramid transgenes for control of insect pests, fungal, bacterial and viral pathogens, weeds and abiotic stresses. The Bt gene technology has demonstrated well the multi-gene insect resistance for stronger and durable resistance against different types of insect pests as it is likely that the pest may not overcome the multiple insecticidal proteins [39]. Similarly, transgene stacking for conferring resistance to the commonly used herbicides has also been reported for the different herbicidal mode of action [40]. For example, the glyphosate-resistant gene, epsps was stacked with the pat (phosphinothricin N-acetyltransferase) gene for increased resistance to the herbicide, glufosinate, and/or with the dmo (bacterial dicamba monooxygenase) gene for higher resistance to the herbicide, dicamba [40].

The pyramided transgenic plants, as reported, exhibited stronger activation of the PR genes in response to pathogen infection than the single-gene-expressed plants [41]. The higher and stronger expression of the endogenous PR genes could be the result of synergistic effect of the stacked genes. Along with the PR genes, expression of other genes, such as allene oxide synthase (AOS), phenylalanine ammonia-lyase (OsPAL), and genes for jasmonic acid (JA) and SA (salicylic acid)-dependent signaling pathways, chitin-induced phytoalexins encoding gene from rice (OsMAPK6), and a rice homolog of the Arabidopsis NPRl (OsNHI), was also found stronger in the pyramided transgenic rice compared to the single-gene transgenics [41].

Gene stacking has been one of the effective approaches for metabolic engineering of the plants as most of the metabolic processes, the biochemical pathways and complex traits involve several interacting genes [42]. For example, all the signaling pathway for biosynthesis of the provitamin A (β-carotene) was genetically engineered in rice endosperm by stacking three β-carotene biosynthesis genes; the phytoene synthase (psy) isolated from daffodil (Narcissus pseudonarcissus, the phytoene desaturase (crtI) originated from Erwinia uredovora and the lycopene beta-cyclase (from N. pseudonarcissus) into rice [43]. Biosynthesis of provitamin-A was found improved in the endosperm of transgenic rice with the stacked transgenes. A modified flower color was developed in roses (biotech rose) by pyramiding two genes in pathway of the anthocyanin biosynthesis that altered pigmentation of the flower, imparting the biotech roses novel shades of blue coloration [44]. Genetic manipulation for down or up-regulation of flavonoid and anthocyanin pathway has lead to the development of changed color varieties in roses and other cut-flower plants [44].

Limitations

Stacking genetically modified traits may offer durable and effective multiple insect pests or pathogens resistance or multiple metabolic engineering for improving nutritional food quality and quantity. However, the advancement of the GM traits is still difficult because of some major hurdles. Some of the traits like yield, nutritional value, or quality of yield products need several genes to alter the several interconnected pathways regulating the complex traits. Very few genetically modified crops transformed with three or more stacked genes have yet obtained the regulatory approval such as multiple virus resistance in the squash. In addition, re- or co-transformation of the multiple transgenes driven by same promoter may result in transgene silencing.

A recently introduced system for multi-gene transformation is reported by [45] in Arabidopsis. The GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) system can be used for flexible and in vivo stacking of multifaceted genes within a T-DNA of Agrobacterium plasmid. They evaluated the system in Arabidopsis by introducing 10 genes-stack T-DNA consisting of eight transcriptional units; sul1 (sulfadiazine resistance), luc (firefly luciferase), eGFP (enhanced Green Fluorescent Protein), bar (bialaphos resistance), GUS (b-glucuronidase), CsMybA (Citrus sinensis anthocyanin-promoting Myb genes), tdTomato (Tandem dimeric orange fluorescent protein) and nptII genes (Fig. 1f). Many of the transgenic lines expressed all eight of the transgenic traits with varying level of expression. The GAANTRY system could be further evaluated in other plant species for multi-gene stacking and stable transformation.

Conclusions and Future Prospects

Transgene stacking has been used as one of the effective strategies of conferring disease resistance by incorporating more than one gene in transgenic crops. Introduction of traits controlled by single gene such as insect resistance or the herbicide tolerance has been proved well in agriculture, improvement of the multi-gene traits such as yield, nutritional quality and stress resistance will need integration of several genes and more sophisticated techniques. The recently introduced system, the GAANTRY system may enable the researchers to address some of these challenging tasks. In addition, therapeutic proteins can be expressed in transgenic plants as edible vaccines using multigene transformation technology.