Genetic improvement of wheat via alien gene transfer, an assessment

Plant Science 165 (2003) 1147 /1168 www.elsevier.com/locate/plantsci

Genetic improvement of wheat via alien gene transfer, an assessment Ashok Kumar Sahrawat, Dirk Becker, Stephanie Lu¨tticke, Horst Lo¨rz * Center for Applied Plant Molecular Biology (AMP II), University of Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany Received 23 January 2003; received in revised form 24 June 2003; accepted 2 July 2003

Abstract In recent years, with advent of the development of efficient plant regeneration systems in cereal crops, the field of recombinant DNA technology has opened up new avenues for genetic transformation of crop plants. Monocots particularly cereals were initially considered difficult to genetically engineer, primarily due to their recalcitrance to in vitro regeneration and their resistance to Agrobacterium . Continuous efforts and systematic screening of cultivars and tissues for regeneration potential, development of various DNA delivery methods and optimization of gene expression cassettes have led the development of reliable transformation protocols for the major cereals including wheat. Since the production of first fertile transgenic wheat plants in 1992s, microprojectile-mediated gene transfer has proved the most successful method for genetic transformation of wheat not only for the introduction of marker genes but also agronomically important genes for improving quality of wheat flour, transposon tagging, building resistance against fungal pathogen and insects, engineering male sterility, and resistance to drought stress. Despite tremendous successes in producing fertile transgenic wheat plants using various methods and approaches, elite cultivars of wheat still remain recalcitrant to transformation. Moreover, in comparison with other major cereals like rice and maize, the development of a high throughput wheat transformation systems has been slowed and severely affected by genotype effects on plant regeneration, low transformation efficiencies and problems with transgene inheritance and stability of expression. Majority of the researcher worldwide have used genetic engineering to tailor wheat for specific end-use by using immature embryos as the primary target tissue for the delivery of desired foreign genes. Hence, we have focused our attention to the work done on stable gene expression and transformation of wheat by employing microprojectile bombardment and Agrobacterium as a source of foreign DNA delivery into immature embryos. Recent advances in wheat transformation especially successes in genetic transformation of wheat with agronomically important genes and novel and innovative approaches for wheat transformation based on different selection schemes are also discussed. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alien gene transfer; Wheat transformation; Transgenic wheat; Genetic engineering; Immature embryos; Plant regeneration

1. Introduction Wheat is a member of the Triticeae group of cereals and indisputably one of the major food crops of the world and a foundation of human nutrition worldwide. In addition to its basic caloric value, wheat, with its high protein content, is the single most important source of plant protein in the human diet [1]. Traditionally, genetic improvement in wheat is generated by using extensive crossing program and then systematically selection of useful new combinations [2,3]. Although,

* Corresponding author. Fax:
/49-40-42816-229. E-mail address: [email protected] (H. Lo¨rz).

with the help of the genetic variation present in wheat, plant breeders have tried for decades to improve the yield of wheat by using conventional methods of breeding, but their efforts were reaching plateau especially with respect to yield. Growth rates of yields have slowed during the period between 1987 and 2001 [4]. Moreover, the world’s population will reach 8 billion by 2025 and it has been estimated that food and feed production must continue to rise annually by 1.2% to satisfy the demand of the world’s population [5]. Past success, therefore, does not guarantee a food abundant world in coming decades. Hence the genetic improvement of wheat has received considerable attention worldwide over the years with the purpose of increasing the grain yield, to minimize crop loss due to unfavorable

0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00323-6

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environmental conditions and development of resistance against various pests and pathogens [6,7]. The last decade has witnessed the extensive use of recombinant DNA technology for introduction of exogenous DNA into major cereal crops including wheat [8]. The technology is based on the delivery of defined foreign genes into plant cells, obtaining integration of the genes into plant genome and subsequently plant regeneration from transformed cells or tissues. Although, in vitro culture techniques exist for several decades but they are now playing a key role in the applicability of genetic engineering techniques for the improvement of plant species. The efficiency of transformation is greatly influenced by genotype, explant source and also medium composition. Therefore, most of the approaches to transform wheat have attempted to develop a genotype independent and cost effective procedure for the introduction of alien genes. The first transgenic wheat plants were produced by Vasil et al. [9], followed by Vasil et al. [10], Weeks et al. [11], Nehra et al. [12], Becker et al. [13] and Altpeter et al. [14] employing microprojectile bombardment as a method of DNA delivery. Subsequently, the development of methodology for the delivery of genes into intact plant tissues by bombardment of DNA-coated gold or tungsten particles has revolutionized the field of wheat transformation. In recent years, sincere efforts are being made to transform wheat genetically with different alien genes of agronomically importance [15 /21]. However, in majority of reports, genetic transformation with a single target gene has been used for the production of transgenic wheat expressing tolerance to herbicide, resistance to fungal and viral diseases [7,8]. As most agronomic traits are polygenic in nature, wheat genetic engineering will require the integration of multiple transgenes into the plant genome, while ensuring their stable inheritance and expression in succeeding generations. Therefore, recent developments in wheat genetic engineering are aimed to the integration of multiple transgenes into the plant genome and coordinated expression of these transgenes in transformed plants. In next decade, therefore, it is assumed that wheat transformation is going to play a very crucial role in complementing the conventional wheat breeding for generating novel cultivars with desirable characters [22]. Irrespective of methods and genotypes used in attempts to transform wheat, the best results have been obtained by direct bombardment of scutellum of immature embryos. Therefore, the present review is devoted to the achievements made in obtaining transgenic plants by employing microprojectile bombardment as a device for direct DNA delivery into the immature embryos of wheat. In recent years, Agrobacterium mediated transformation has emerged highly efficient alternative to direct gene delivery in a number of economically important crop plants including wheat

[23 /25]. Therefore, we have also focused our attention to the attempts made in obtaining transgenic wheat using Agrobacterium as a vehicle of DNA delivery.

2. Tissue culture and regeneration: a prerequisite for genetic transformation of wheat Genetic transformation of cereals including wheat largely depends on the ability of transformed tissues to proliferate on selection medium and subsequently regeneration of plants from transformed cells. Indeed, it is the totipotency of plant cells that underlies in the success of most plant transformation systems. In cereals, immature embryos are considered the most responsive explant in culture because of their ability to produce readily embryogenic callus and subsequently large number of plants. This ability of immature embryos makes them the more suitable primary explants for genetic transformation of wheat. Complete plantlets were first regenerated from immature embryos of wheat by Shimada [26]. Later, Shimada and Yamada [27] optimized various factors, such as age and size of the embryos and auxin concentration for callus induction and found that embryos isolated 14 days after anthesis induced high frequency embryogenic callus with high intensity of green spots (shoot primordia) formation. And subsequently, the scutellar tissue of embryos isolated 10 /15 days after anthesis has been widely exploited to induce fast-growing embryogenic and highly regenerable calli [28 /30]. Embryogenic tissues are, in general, very prolific and allow recovery of transformants that are, in most cases, non-chimeric because of the assumed single cell origin of somatic embryos [31,32]. The authors provided, for the first unequivocal demonstration of regeneration via somatic embryogenesis with supporting evidence from scanning electron microscopy. In a detailed study, Magnusson and Bornman [33] demonstrated that somatic embryos can be developed from the epithelium, procambium or ground tissue of the scutellum. However, epiblast of the immature embryos has been an explant of the choice for high frequency embryogenic callus induction [32,34]. In cereals, the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is the most widely used growth regulator for initiation of callus from cultured tissues [35,36]. Initiation or maintenance of callus on low level of 2,4-D has been found to trigger formation of somatic embryos [37]. Recently, Rasco-Gaunt et al. [38] have also reported that scutella of a winter wheat cultivar ‘Florida’ when cultured on 0.5 or 1.0 mg/l 2,4-D produced highest proportion of embryogenic to nonembryogenic tissue and shoot regeneration was more efficient in cultures induced on low level of 2,4-D. While it has been well established that 2,4-D induces somatic embryogenesis, other nutritional factors as well as

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hormones often cause precocious germination of embryos, one of the major factors which restrict development of embryogenic callus in cultured embryos. Various methods and procedures have been devised to suppress precocious germination for better recovery of embryogenic callus induction and enhancing the frequency of somatic embryogenesis. Papenfuss and Carman [39] reported that precocious germination of embryos can be checked effectively by adding Dicamba in culture medium. They showed that response could be improved further by incubating culture on medium containing L-tryptophan and 2,4-D. Later, Bapat et al. [40] used high concentration of 2,4-D in order to suppress precocious germination of cultured embryos. Suppression of precocious germination of cultured immature embryos by adding abscisic acid (1.9 mmol/l) in the callus induction medium was reported by Carman [41]. The effect of embryo orientation on the medium surface has been found to influence the formation and development of nodular embryogenic callus. Various studies have shown high frequency induction of embryogenic callus from the scutellum when embryos were cultured facing away from the medium and consequently the enhanced frequency of plant regeneration was reported [37,42,38]. In cereal tissue culture, it is well established that somatic embryogenesis and transformation frequency are influenced by the age of the explant and that younger embryos produce comparatively more somatic embryos and consequently more transgenic plants than the older explants [43,44]. Scutellum size has also been shown to influence culture response and subsequently transformation frequency in wheat [28,29,38]. Sears and Deckard [28] reported optimum callus initiation from embryos when the scutellum size was about 1 mm long. Similarly, in comparison to scutella smaller than 0.5 mm or larger then 1.5 mm, Rasco-Gaunt et al. [38] also obtained highest embyrogenesis and shoot regeneration from scutella in the 0.75 /1.0 mm size class. In addition with hormones, several other factors including level of sucrose in the callus and regeneration medium and macro and microelements have been reported to influence culture response and regeneration potential of immature embryos of wheat [45,43,47,38]. The sucrose content of the culture medium has been reported to affect the initiation of epiblast callus from immature embryos through an interaction with the growth regulators in the medium [32]. Galiba and Erdei [45] studied effects and concentrations of carbohydrates on callus growth and plant regeneration and found that 2% sucrose supported optimal callus growth and high number of shoots. Rasco-Gaunt et al. [38] increased embryogenesis in cultured immature embryos of wheat (cv. Florida) from 78 to 98% by increasing the sucrose content of induction medium from 3 to 6 or 9%. They further observed enhanced regeneration frequency from

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72 to 90% in cultures induced at higher sucrose concentrations. Li et al. [46] published that the efficiency of somatic embryogenesis of isolated scutella from commercial wheat cultivars was significantly affected by the interaction of days of spike storage at 4 8C and sucrose concentrations in embryo induction medium. He et al. [43] reported enhancement in the frequency of white callus formation upon increasing the concentration of macroelements from half strength to full or double strength. Addition of L-tryptophan in the callus induction medium has also been found to enhance embryogenic callus formation significantly [48,49]. Ozias-Akins and Vasil [32] showed that a doublestrength MS medium with normal or double concentration of vitamins together with casein hydrolysate and other adjuvants supported a 30% increase in the induction of embryogenic callus. Antibiotics, cefotoxime and carbinicillin, which are used to eliminate Agrobacterium in co-cultivation experiments were found to stimulate callus growth and subsequently plant regeneration [50]. In addition, precocious germination of embryos was found to be suppressed with the addition of cefotoxime in the medium. The regeneration frequency of durum wheat was also improved by inclusion of cefotoxime in the medium [51]. Inclusion of AgNO3 in the medium was found to enhance shoot regeneration remarkably [52 /54]. AgNO3 is a potent inhibitor of ethylene, and therefore, the stimulatory effect of AgNO3 may be due to the inhibition of ethylene action, which is known to suppress shoot regeneration. Purnhauser [47] and Purnhauser and Gyulai [55] observed the beneficial effect of copper ion used a cupric sulphate on shoot regeneration in wheat callus derived from immature embryos of wheat. The stimulatory effect of copper my be due to the fact that copper is an important part of several enzymes and hence might play a key role on morphogenesis at an optimum concentration. Barro et al. [56] showed the positive effect of zeatin (5 or 10 mg/l) on shoot regeneration from cultured tissues of wheat. The variations in immature embryos for embryogenic and non-embryogenic callus induction among wheat genotypes have been found to be related with endogenous hormone level [57,58]. These authors analyzed the endogenous hormone concentrations of callus cultures derived from immature embryos of different genotypes of wheat and reported various degrees of competence. More recently, Jime´nez and Bangerth [59] have shown that endogenous hormone levels of the immature embryos of wheat critically influence the competence of immature embryos for embryogenic and non-embryogenic callus formation. They reported that embryogenic callus had higher concentrations of endogenous free IAA, in comparison to the concentrations found in nonembryogenic callus. Therefore, a better understanding of the relationship between endogenous hormone concentrations in the target explant tissues and the in vitro

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callus cultures may allow extending the range of genotypes to be used in genetic transformation experiments of wheat. Apart from explants, growth regulators and composition of media, genotype of the explant is considered one of the major factors which critically influence successful transformation of wheat. Strong genotype effects have been observed in callus induction and regeneration from immature embryos of wheat [35,36,60,56]. Extensive genotypic variation for tissue culture performance has been resulted in limited number of highly responsive cultivars for genetic transformation experiment of wheat. Recently, Varshney and Altpeter [61] investigated the tissue culture response of 38 European winter wheat cultivars and reported a great degree of variations in their response for callus induction and plant regeneration. They showed that all the cultivars tested formed callus on defined medium but the number of regenerated plants per explant differed significantly among genotypes. In the selection of spring wheat variety ‘Bobwhite’ for achieving rapid transformation, the importance of genotype is clearly evidenced [11]. Although, in terms of agronomical and grain quality ‘Bobwhite’ ranks inferior, but credits more successful transformation of wheat because of its good response in tissue culture [28,41,62]. In recent years, efforts are being made to extend the engineering of wheat to elite genotypes, which are either agronomically important breeding lines or currently commercial varieties [38,46,61,63 /68]. Undoubtedly, it is desirable to introduce transgenes into the ideal genetic background generally grown under large area of cultivation and more preferred by farmers and consumers.

3. Genetic transformation of wheat employing microprojectile bombardment Successful genetic manipulation requires the ability to deliver biologically active and functional DNA into plant cells followed by recovery of transgenic plants expressing a foreign gene. Success in genetic transformation of cereals was difficult to achieve and often limited to transient gene expression because of the lack of suitable regenerative systems and incapability of Agrobacterium to infect cereal tissues. This is the main reason that the method of introducing DNA into cells by physical means (microprojectile bombardment) was developed to overcome the biological limitations of Agrobacterium and difficulties associated with plant regeneration from protoplasts. The very first attempt to transform wheat was made by Lo¨rz et al. [68] in Triticum monococcum . They transferred the nptII gene with the nos promoter and ocs polyadenylation sequences into cell suspension-derived protoplasts using PEG. The difficulties associated with plant regeneration from protoplasts compelled researchers worldwide to

look for alternative target tissues with better regenerability. And therefore, attention was shifted from protoplasts to embryogenic suspension cells, immature embryos and embryogenic callus derived from scutellar tissue of immature embryos. Later, Wang et al. [69] used suspension cells of a T. monococcum cell line and reported transient expression of GUS and CAT by bombarding DNA into aggregates of suspension cells. For the first time, Lonsdale et al. [70] used wheat embryos as a target tissue for transformation. They bombarded embryos with tungsten particles carrying the uidA gene and showed blue patches in transformed embryos. However, there was no comparable report on Triticum aestivum until 1991s when Vasil et al. [71] obtained stable transformed callus lines using microprojectile bombardment of plated suspension cells. They transformed callus tissues with nptII , uidA and ESPS synthase gene, either on the same or different plasmid. In the same year, transient expression of two marker genes, cat and uidA , was also obtained by microprojectile bombardment of immature embryos of wheat [72]. Both GUS and CAT activities were detected through histochemical analysis and ELISA technique, respectively. In subsequent year, Vasil et al. [9] obtained first transgenic wheat plants and this report is considered as a milestone in wheat transformation. They generated transgenic wheat plants 12 /15 months after particle bombardment of plasmid vector pBARGUS into cells of type-C, long term regenerable embryogenic callus. The gene construct (pBARGUS) contained dual promoter, the Adh1 with Adh1 intron 1 to drive the uidA gene, a reporter gene encoding b-glucuronidase and, CaMV 35S with the Adh1 intron 1 to drive the bar gene, a selectable marker gene. Southern blot analysis of transgenic plants confirmed the presence of the bar gene. Following the report of Vasil and co-workers [9], several research groups made sincere attempts to refine or improve the protocol aiming to accelerate the pace and frequency of production of transgenic plants (Table 1). Further improvements in the procedure significantly reduced the time required for production of transgenic wheat plants from an initial 12/15 to 5 /7 months by bombardment of immature embryos and embryogenic calli [10], about 4 months by bombardment of immature embryos [11], about 3 months using isolated scutella and with improvement in regeneration system [12] and 55/ 66 days by bombardment of cultured immature embryos [14]. Table 1 clearly shows that most of the researchers have utilized immature embryos, isolated scutellum and calli derived from immature embryos as the primary target tissue for delivery of foreign DNA by particle bombardment. The first successful transformation of durum wheat was also reported by particle bombardment of isolated scutella [73]. Direct bombardment of immature embryos has thus emerged over the years as a

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Table 1 Wheat transformation mediated by particle bombardment and A. tumefaciens Transformation meth- Target tisods sue

Transformation efficiency

Promoter/reporter

Selectable marker gene

References

Particle Particle Particle Particle Particle Particle

EC IE IE EC IE IE

n.d n.d 0.21% 0.7 /2.5% 1.3% 0.15%

Adh1:gus Adh1:gus//Ubi1:gus , CaMV35S:gus ubi1:gus Adh1:gus , act1:gus act1:gus

[9] [10] [11] [12] [13] [78]

Particle gun Particle gun Particle gun Agrobacterium Particle gun Particle gun Particle gun Particle gun Particle gun Particle gun Agrobacterium Particle gun Particle gun Particle gun Partcle gun Particle gun Particle gun Particle gun Agrobacterium Particle gun Particle gun Particle gun Particle gun

SC IE IE IE, SC IE IE SC IE IE SC MS, IE IE IE, IF IE IE IE IE SC IE IE AC IE IE

2.6 /5.5% n.d 1.5% 1.12 /1.56% n.d 0.16 /1.71% n.d n.d 0.25 /1.2% 0.48% 1.2 /2.2% n.d 1.2 /3.1% 2.75 /3.56% n.d n.d n.d n.d 3.7 /5.9% n.d n.d 0.8 /5% 2.3%

CaMV35S:gus Adh1:gus ubi1:gus CaMV35S:gus act1:gus act1:gus ubi1:luc ubi1:uidA , act1:uidA act1:gus ubi1:luc CaMV35S:gus

CaMV35S:bar CaMV:bar ubi1:bar CaMV35S:bar , act1:nptII CaMV35S:bar CaMV35S:GOX , CaMV35S:CP4 ubi1:bar , ubi1:hpt CaMV35S:bar ubi1:bar CaMV35S:nptII CaMV35S:bar act1:bar ubi1:bar ubi1:Bar , CaMV:neo ubi:bar//ubi:aphA//ubi:hpt ubi1:bar CaMV35S:npt act 1:ubi1:bar ubi:bar act:bar act1:nptII act1:bar ubi:bar , CaMV35S:neo ubi1:bar ubi1:nptII ubi1:bar ubi:bar , CaMV35S:hpt act:sgfp , CaMV35S:nptII ubi:nptII , nos:nptII

[79] [75] [14] [23] [202] [138] [96] [121] [64] [99] [203] [191] [54] [93] [190] [102,103] [203] [65] [204] [144] [140] [111] [205]

Particle gun Particle gun Particle gun Particle gun Particle gun Agrobacterium Agrobacterium Particle gun Particle gun Particle gun Particle gun

IF IE IE IE IE Sn-cell IE IE SC, IE IE IE

n.d 0.7 /5% 20% 1.2% 0.2 /2.0% 1.8% 1.2 /3.9% 0.3 /7.4% 0.13 /4.93% n.d 0 /4.6%

ubi:bar ubi1:pmi ubi:pat// , ubi:GST-27 ubi1:bar CaMV35S:bar ubi1:bar H2B:mopat ubi1:bar ubi1:bar ubi1:bar

[141] [44] [124] [129] [61] [24] [25] [131] [112] [100] [206]

gun gun gun gun gun gun

ubi:gus ubi:gus act1:uidA CaMV35S/ Bperu and C1):act1 ubi:uidA , act:uidA ubi1:uidA ubi1:gus , LMWG1D1:gus ubi:gus//act:gus , act:gus act1/gfp CaMV35S CaMV35S:2-5A snthetase gene// Pamt:RNaseL Glu-1D-1:gus ubi:uidA

CaMV35S:gfp ubi:gus H2B:gus ubi1:uidA , CaMV35S:gfp ubi1:luc

Abbreviations: EC, embryogenic callus; IE, immature embryos; SC, scutllem derived calli; Sn-cells, suspension cells; MS, mature seeds derived calli; n.d, not determined; ubi1 , maize ubiquitin promoter with its first intron; bar and pat , phosphinothricin acetyl transferase; gus or uidA , ßglucuronidase gene; CaMV35S , cauliflower mosaic virus promotor; nptII , neomycin phosphotransferase II; hpt , hygromycin phosphotransferase gene; act1 , rice actin promotor; R, anthocyanin synthase gene; gfp , green fluorescent protein; pmi or manA , phosphomannose isomerase; CP4 and GOX , glyphosate oxidoreductase genes; C1/Lc , anthocyanin-biosynthesis regulatory genes; Adh1 , maize alcohol dehydrogenase gene promotor; H2B , maize histone gene promotor; GST-27 , maize gluthione S -transferase gene; Bperu and CI , regulatory elements of anthocyanin biosynthesis.

reproducible method for the routine production of transgenic wheat plants [7,22]. Most of the attempts to increase transformation frequency have largely been focused on increasing embryogenic capacity of the target explant, bombardment conditions (amount of DNA, pressure, distance etc.), type of selection and time allowed for each stage of the procedure [13,14,46,54]. The influences of different parameters such as the quantity of plasmid DNA, spermidine concentration,

amount of gold particles, acceleration pressure, bombardment distance, the osmotic condition of tissues and the type of auxin on transient GUS expression were investigated in detail by Rasco-Gaunt et al. [54]. Although no clear correlation between transient expression and stable transformation was observed, but some of the parameters investigated were found to influence transient GUS expression. Nevertheless, the transgenes introduced in immature embryos by biolistic approach

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display a considerable degree of stability in integration and expression in subsequent generations [74 /76]. Genotype and age of the donor plants are considered key factors in order to achieve successful transformation of wheat [44,61,65]. Iser et al. [65] and Varshney and Altpeter [61] observed significant variability both in regeneration and transformation frequency between German wheat cultivars. Pastori et al. [44] transformed two elite wheat varieties by particle bombardment of scutella isolated from immature embryos and observed a strong correlation between transformation frequency and the age of the wheat donor plant. They were able to increase mean transformation frequency from 0.7 to 5% by using immature embryos from young plants. Rasco-Gaunt et al. [38] transformed ten European wheat varieties at efficiencies ranging from 1 to 17% (mean 4% across varieties) by optimizing particle bombardment and tissue culture procedure. Among parameters tested, authors found that selection of transformed scutella at high sucrose level (9%) enhanced somatic embryogenesis, shoot induction and consequently stable transformation efficiency. An osmotic treatment of target tissues for stable transformation results in plasmolysis of cells and restrict damages by preventing extrusion of the protoplasm from bombarded cells [77 /80].

4. Genetic transformation of wheat mediated by Agrobacterium tumefaciens Biotechnological strategies for crop improvement demand efficient procedures for routine introduction of low copy number of defined foreign genes into plant genome. The use of Agrobacterium vectors for genetic transformation confers advantages, over direct DNA delivery techniques, which include a high frequency of stable genomic integration and single/low copy number of the intact transgene. Infact, these characteristics of Agrobacterium -mediated transformation have encouraged researcher to develop efficient protocols for genetic transformation of economically important cereals employing Agrobacterium as a vehicle of delivery of foreign DNA. The early studies of Agrobacterium -mediated transformation of cereals including wheat were somewhat controversial [81,82]. In 1993s, situation, however, begun to change when Chan et al. [83] produced a few transgenic rice plants by inoculating immature embryos with A. tumefaciens . Although they showed the inheritance of transferred DNA to progeny plants but their analysis was based only on one transformed plant. For the first time, Hiei et al. [84] demonstrated irrefutable evidences for genetic transformation of Japonica rice mediated by A. tumefaciens and developed a method for efficient production of fertile transgenic rice plants. Their evidence was based on molecular and genetic

analysis of large number of transgenic plants and also on the analysis of the sequence of T-DNA junction in transgenic plants. In subsequent years, Rashid et al. [85] and Ishida et al. [86] developed efficient methods for Agrobacterium -mediated transformation of rice and maize, respectively. Indeed, in majority of successful reports, super binary vectors have played a key role in the development of protocols for genetic transformation of cereals including wheat mediated by Agrobacterium [25,84,86,87]. The virB , virC and virG genes of the pTiBo542 plasmid of the supervirulent strains A281 were used to construct super binary vector. In this vector systems, the virulence of Agrobacterium strains harboring a disarmed Ti-plasmid is increased by the extra copies of virB , virC and virG . In genetic transformation of cereals, another break through came in 1997, when Tingay et al. [88] used non-super virulent strain and reported successful transformation of barley. A phenolic compound ‘acetosyringone’ which is known to induce expression of virulence (vir ) genes located on the Ti-plasmid, played a major role in the success of Tingay and co-workers. Following the success of Agrobacterium -mediated genetic transformation of rice [84,89], maize [86] and barley [88], Cheng and co-workers [23] developed an efficient method for A. tumefaciens -mediated transformation of wheat using freshly isolated immature embryos, pre-cultured immature embryos and embryogenic calli as target tissues. They used a disarmed nopaline A. tumefaciens strain C58 (ABI) carrying the ‘ordinary’ binary vector pMON18365 containing the b-glucuronidase gene (uidA ) with an intron as reporter gene, and a selectable marker gene, neomycin phosphotransferase II gene (nptII ). About 35% of the transgenic plants selected on G418 showed a single copy of the transgene and one to five copies of the transgene were integrated into the wheat genome without rearrangement. Cheng and co-workers also reported the importance of acetosyringone in successful transformation of wheat and showed that efficiency of T-DNA delivery into the target was significantly decreased in the absence of acetosyringone. In addition, the presence of a surfactant during inoculation of tissue with Agrobacterium was found an important parameter for the efficient delivery of T-DNA into wheat. In subsequent year, McCormac et al. [90] reported transformation of wheat following inoculation of immature embryos with both A. tumefaciens and Agrobacterium rhizogenes and demonstrated that anthocyanin-biosythesis regulatory genes (C1/Lc) can be used as a visual marker. Inoculation and cocultivation of immature embryos with A. tumefaciens strains EHA101 (a ‘supervirulent’ strain) and LBA4404 and A. rhizogenes strain (LBA9402 and Ar2626) harboring the CI/Lc genes containing vector (pBECKS.red) led to the spontaneous development of deeply red pigmented wheat cells. Non-random distribution of

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red pigment through out the embryo and callus pieces was observed. In the same year, Guo et al. [91] investigated various factors and reported that acetosyringone and Agrobacterium strain were vital for achieving high frequency of transient GUS expression in transformed tissue of wheat. Agrobacterium strain EHA101 carrying an ordinary binary vector ‘pIG121Hm’ was found superior than LBA4404 (pTOK233) and GV3101 (pPCV6NFHGusInt) for higher frequency of GUS expression in wheat cells. Xia et al. [92] further refined the protocol and produced fertile transgenic wheat plants at the rate of 3.7 /5.9% by using A. tumefaciens strain AGLI carrying plasmid pUNN-2 containing the selectable marker NPTII driven by the Ubi1 promoter. Selection of transformed immature embryos and embryogenic calli was carried out on a medium containing paromomycin (an analogue of kanamycin). The difference in the competence of Agrobacterium strains for the successful T-DNA transfer to wheat cells was also reported by Uze´ et al [93]. Higher frequency of GUS expression after co-cultivation of wheat tissues was observed with A. rhizogenes strain LBA9402 carrying a binary vector ‘pBin9UG’ than EHA105, C58CI and LBA4404 all carrying the same binary vector. McCormac et al. [90] compared the T-DNA transfer efficiency in wheat between two A. rhizogenes strain (LBA9402 and Ar2626) and two A. tumefaciens strain (LBA4404 and EHA101) and found that only EHA101 (a supervirulent strain) facilitated TDNA delivery successfully into wheat tissue. One possible reasons for the difference in the effectiveness of these strains between species-to-species, variety to variety and even explant to explant might be due to the difference in the plant cell receptors involved in Agrobacterium attachment to the target tissues. For the first time, Weir et al. [24] reported Agrobacterium -mediated transformation of four varieties of wheat using GFP (green fluorescent protein) as visual marker. They compared several Agrobacterium strains for efficient T-DNA delivery into target tissues (suspension cells, scutella and scutella-derived callus) and reported high level of transient GFP activity and stable transformed callus line with Agrobacterium strain AGL0 harboring plasmid ‘pTO134’ containing a gfp gene with an enhanced CaMV 35S promotor and a bar gene with a 35S promoter. In addition with Agrobacterium strain and plasmid vector, authors found that initiation and co-culture period critically influenced the recovery of stable callus and subsequently plant regeneration in wheat. Weir and co-workers recovered transgenic wheat plants at a transformation frequency of 1.8%. Recently, Khanna and Daggard [25] produced fertile transgenic wheat plants after infection and cocultivation of immature embryo-derived calli of spring wheat cultivar (Veery5) with Agrobacterium strain LBA4404 carrying either binary vector pHK22 or

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superbinary vector pHK21. In this report, authors have clearly demonstrated the superiority of super binary vector over binary vector by producing 17 stable transformants after infecting 658 calli with LBA4404 harboring super binary vector pHK21. While not even a single stable plant was recovered out of 587 calli infected with LBA4404 carrying binary vector pHK22. Interestingly, they were able to increase the overall transformation frequency from 1.2 to 3.9% with the addition of 0.1 M spermidine in regeneration medium. Therefore, it is evident from their study that in wheat, recovery of transformants can be increased tremendously by using super binary vector and optimizing the level of polyamine in the regeneration medium. Despite successes in transforming wheat by using Agrobacterium , induced cellular necrosis after pathogen infection is still considered a drawback in routine application of Agrobacterium for genetic transformation of wheat. Induced cellular necrosis is typical of the resistance mechanism termed the hypersensitive response and it is associated with limiting further ingress of the pathogen [94]. Recently, Parrott et al. [95] found that immature embryos inoculated with Agrobacterium increased production of H2O2, browned and displayed altered cell wall composition and higher level of cellular necrosis leading to cell death. Interestingly, Parrott and co-workers found that the reduction of the O2 tension from 7.4 to 2.1 mM during interaction of Agrobacterium with tissues significantly reduced the extent of embryo and root cell death. Apart from reducing necrosis and cell death in transformed tissue, this technique could also be valuable for achieving maximum growth of embryogenic tissues and subsequently high frequency plant regeneration because low oxygen tension has been reported to promote the growth of embryogenic tissues [41]. In near future, it can be expected that Agrobacterium will be employed as a reliable, efficient and economical vector for the introduction of agronomically important genes.

5. Reporter and selectable marker genes for wheat transformation Irrespective of methods used for the delivery of foreign DNA into target tissue of wheat, the choice of markers, reporter, promoters and introns greatly influences the final outcome. In addition, better selection of transformed cells minimize the risk of escapes and allows better chance for the recovery of transformed cells and subsequently transgenic plants. The reporter genes produce a visible effect and therefore, allows detection of expression of foreign DNA within the transformed tissue at an early and pre-integration stage. A such visual reporter gene of choice has been the uidA gene [96], which encodes the histochemically-detectable

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b-glucuronidase enzyme (GUS) and is commonly used in wheat transformation experiments [9,10,13,14,37,97]. The histochemical assay is required 5-bromo-4-chloro-3indolyl b-D-glucuronic acid (X-Gluc) as a substrate and produces a product that utilizes oxidative dimerization with potassium ferricyanide and ferrocyanide to produce the bright blue color [96]. Although, the GUS reporter gene has successfully been used in majority of reports on wheat transformation, but one of the major limitations of GUS reporter gene system is the destructive nature of its assay. Therefore, in recent years, vital reporter genes encoding for anthocyanin biosynthesis, green fluorescent protein and firefly luciferase have successfully been utilized in wheat transformation to study the fate of introduced gene in living cells [90,98]. The luciferase gene (luc ) from the firefly Photinus pyranus catalyses the luciferin substrate, which subsequently emits a yellow /green light that can be easily detected with a light imager [98,99]. This marker has advantages over GUS as the assay for luciferase activity is not lethal to the assayed tissue and therefore, fascilitate rapid detection of the fate of introduced transgenes in individual transformed tissues. Recently, Bourdon et al. [100] reported that homozygous transgenic wheat plants, which showed high luciferase activity in T1 generation did not maintain the high level of expression in the next generation. On the contrary, all T1 plants expressing low level of luciferase activity conserved their expression levels in T2 generation. This may be due to the fact that complex integration patterns induced by bombardment of wheat embryos could lead to instability in the genome and thereby affecting both inheritance and expression levels of the transgene in the T2 generation. It is also possible that the rearrangements of the genome taking place during meiosis might have created a different genetic background less favorable to high levels of transgene expression. The study of Bourdon et al. [100] suggests that a high expression level of the transgene might be disadvantageous for the plants in maintaining the same level of expression through out their progenies. Another set of vital markers, anthocyanin markers, which allows visualization of transgenic tissue from the beginning and throughout development without sacrificing the transformed tissue have successfully been used for wheat transformation [90,101 /103]. Anthocyanin biosynthesis can be activated in cereals including wheat by introduction of regulatory genes B and C1 from maize [104]. Direct visual markers C1/Lc [105] when co-introduced into cereal cells, interact with endogenous pathway to cause massive accumulation of anthocyanin [90,106]. The C1 gene is responsible for the production of anthocyanin pigmentation in the aleurone and the embryos of maize and the Lc gene is a member of the maize R gene family which is involved in anthocyanin pigmentation of the aleurone, anthers and coleoptile

[105]. Transformed cells show red pigmentation under stereo-microscope. As a visual marker protein, the synthetic version of green fluorescent protein (GFP) of the jellyfish Aequorea victoria has gained popularity over other reporter proteins because it allows efficient expression within plant cells and permit non-lethal destruction under specific excitation wave length [107,108]. The gfp gene encodes a self-catalyzing fluorescent chromophore that emits green fluorescence when exposed to ultraviolet light and bright green fluorescence can be observed with a standard fluorescent microscope [109]. In recent years, this non-destructive reporter gene is being utilized in the optimization and improvement of wheat transformation [24,90,110 /112]. In future, vital marker proteins may be exploited not only for better tracing of introduced transgene in plant cells but also in the assessment of the effectiveness of delivery procedures at an early stage of the integration of transgenes into the target tissues. In contrast to reporter genes, selectable marker genes allow survival of the transformed cells on a selection medium enriched with a selective agent. The selection regimes for transformed cells that carry introduced gene sequence are based on the expression of a gene termed as the selectable marker producing an enzyme that confers resistance to a cytotoxic substance often an antibiotic or a herbicide (Table 2). In order to see the expression of foreign genes the selectable markers commonly employed are bar gene [113], which confer resistance to herbicides bialaphos and Basta, nptII (neomycin phosphotransferase II) gene [114], which confer resistance to aminoglycoside antibiotics (kanamycin, G4118 and geneticin) and hpt (hygromycin phosphotransferase) gene which confers resistance to antibiotic hygromycin B [70]. Ortiz et al. [79] developed a highly efficient protocol for generating stable wheat transformants using hygromycin resistance as a selectable marker and reported the superiority of hpt gene over bar gene in selecting transformed cells of wheat. Transgenic wheat plants were produced with the frequency of 5.5% using hygromycin phosphotransferase as selectable marker, while the frequency was only 2.6 with bar gene. However, in addition with high transformation frequency (5.5%), authors also reported high frequency ( /60%) of escapes. One of the possible reasons for this high escape frequency may be due to the fact that authors did not supply hygromycin in regeneration medium. Hagio et al. [115] produced hygromycin resistance transgenic barley plants without any escape when regeneration step was carried out on a regeneration medium containing hygromycin. In a detailed study, Witrzens et al. [64] compared hpt , bar and nptII as selectable marker genes for wheat transformation and reported that they were unable to recover transgenic wheat plants using the hpt gene. In comparison, they obtained more transgenic wheat plants using nptII gene

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Table 2 Selectable marker genes commonly used in wheat transformation Selectable marker gene

Encoded enzyme

Selective agent

Mode of action

pat , bar Hpt NptII

Phosphinothricin acetyltransferase Hygromycin phosphotransferease Neomycin phosphotransferase II

Inhibits glutamine synthase Binds 30S ribosomal subunit, inhibits translation Binds 30S ribosomal subunit, inhibits translation

EPSPS manA , pmi

5-Enolpyruvylshikimate-3-phosphate synthase Mannose-6-phosphate isomerase

Phosphinothricin, bialaphos Hygromycin Kanamycin, G418, paromycin, geneticin Glyphosate Mannose

Cah

Cynamide hydratase

Cyanamide

GOX CP4

Glyphosate oxidoreductase enolpyrovylshikimate-5-phosphate synthase

Glyphosate

than with bar gene. Nevertheless, bar gene, which encodes the enzyme phosphinothricin acetyltransferase (PAT), is widely used as a selectable marker for genetic transformation of wheat (Table 1). The activity of PAT provides the plant with the ability to detoxify phosphinothricin (PPT), the active compound in bialaphos and Basta† (Hoechst, Germany). PPT is a glutamate analogue that irreversibly inhibits glutamine synthetase activity, the key enzyme for ammonium assimilation and regulation of nitrogen metabolism in plants. The inhibition of glutamine synthetase results in the death of untransformed tissues and plants due to the accumulation of ammonium [116,117]. However, the major drawback in using PAT as a selectable marker is the regeneration of large number of plantlets popularly called as ‘escapes’ [11 /14,64,79]. To address this serious problem which not only check transformation frequency but utilizes resources and labor, there have been several studies aimed at improving PPT-based selection for early detection of non-transformed plants [118,119]. A multiwell ammonium-evolution bioassay was introduced by De Block et al. [118]. This assay allows both qualitative and quantitative assessment of PAT expression in transgenic plants and works on the basis that leaf tissues from non-transformed plants start accumulating ammonium after treatment with PPT and eventually lead to death. While leaf tissues from transgenic plants carrying the bar gene assimilate ammonium and survived. Recently, Rasco-Gaunt et al. [120] further improved ammonium-multiwell assay and reported that improved assay allows quick quantitative and qualitative evaluation of the expression of the enzyme phosphinothricin acetyl transferase and results can obtain within a day. In most of the cases, however, these escapes can be monitored and eliminated by molecular analysis like PCR at early stage or by spraying plants with Basta† . In comparison to phosphinothricin or bialaphos, fewer escapes (20 /50%) and similar transformation frequency (1 /2%) were observed on selection medium containing G418 [12,64,121]. Two

Inhibits aromatic acid biosynthesis (EPSPS) Unique ability to grow on mannose as a sole carbon source Unique ability to grow on cyanamide containing media, converts cyanamide into urea Degradation of glyphosate to non-toxic aminomethyl phosphoric acid

others herbicide resistance genes, cp4 and gox have also been employed successfully as selectable markers for wheat transformation [78]. Although the recovery of transgenic plants with glyphosate was low (0.15%) in comparison with bialaphos (10 /15%) or G418 (1 /3%) under the same experimental conditions, but no escapes were obtained using glyphosate. The CP4 gene was isolated from a glyphosate-tolerant strain (CP4) of Agrobacterium sp. [122,123] and code for the enzyme enolpyrovylshikimate-5-phosphate synthase (EPSPS). The GOX gene encoding for a glyphosate oxidoreductase was cloned from Achromobacter sp. [122]. Both CP4 and GOX detoxify glyphosate by converting it to aminomethyl phosphonic acid, which is non-toxic to plant cells. When either of these genes are used, they confer resistance to glyphosate, an active ingredient in the herbicide Roundup† . In recent years, a new selectable marker gene which encodes for the enzyme mannose-6-phosphate isomerase (PMI) has been employed for achieving wheat transformation successfully [124]. PMI is an Escherichia coli glycolytic pathway enzyme that catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate [125]. This system utilize the phosphomannose isomerase (manA ) gene as the selectable marker gene and mannose as the selective agent [126,127]. The selection of transformed tissues is based on the fact that PMI in plant cells makes mannose-6-phosphate available as a carbon source by converting it to fructose6-phosphate and therefore, plants harboring manA gene survive, and non-transformed tissues eventually lead to death. Wright et al. [124] exploited this ability of PMI for wheat transformation and reported a 20% transformation rate without any escapes. Another novel selectable marker gene, cah (cyanamide hydratase), which was isolated from Myrothecium verrucaria (a soil fungus) has also been successfully used for selecting transformed wheat cells [128]. Weeks and co-workers produced transgenic wheat plants with a 0.2% transformation frequency employing cah as a selectable marker

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gene. In comparison to other selectable markers, cyanamide hydratase is being considered a unique marker because of its ability to convert cyanamide (a chemical used in manufacturing plastics) into urea, a useful nitrogen compound, which can be used as a fertilizer source. Recently, the potentiality of the maize gst27 gene (gluthione S -transferase subunit) as a selectable marker for wheat transformation has been suggested by Milligan et al. [129]. They transformed wheat via particle bombardment of immature embryos with plasmid construct ‘pCAT0’ containing a maize gst-27 gene under the control of maize ubiquitin promoter and pPUN construct containing bar gene for selection of transformed cells. This study resulted in six independent transgenic lines showing stable expression of GST-27. T1 progeny of transgenic lines were germinated on medium enriched with chloroacetanilide herbicide alachlor and tolerance to this herbicide was found to be correlated with GST-27. T2 homozygous plants were found resistance not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thicarbamate herbicide EPTC. After getting these results, they suggested that maize GST-27 could be exploited as a selectable marker gene in wheat transformation. In this study, the degree of protection was not reported as great as that afforded by PAT to bialaphos, but GST system has yet to be optimized before jumping to any conclusion. It has already been confirmed that maize GST-27 has glutathione peroxidase activity [130] and therefore, there is also a possibility to exploit this additional activity of GST-27 in generating wheat transgenic lines tolerance to abiotic stresses and pathogen attack. Recently, Rasco-Gaunt et al. [131] produced transgenic wheat plants by using novel selectable marker genes, namely mopat and popat (synthetic version of the pat selectable marker) driven by H2B, a novel constitutive promoter from the maize histone. They were able to recover stable herbicide transgenic wheat plants using an H2B:mopat construct and phosphinothricin selection.

6. Promoters for driving transgene in wheat Efficient expression of a gene in target tissue critically dependent on the selection of an appropriate promoter. Several promoters have been used to control the expression of selectable marker and reporter genes in transgenic wheat. These include heterologous promoters such as those of shrunken gene of maize [132], copia LTR from Drosophila [133], the cauliflower mosaic virus (CaMV) 35S RNA transcript (35S) promoter [133,134] and mannopine synthase 2 gene [135]. However, because level of the expression of introduced genes was quite low in transformed tissues, Last et al. [136] constructed the modified maize alcohol dehydrogenase 1 promoter (Emu) in order to achieve high level expres-

sion of introduced genes in cereal cells. This promoter contains a truncated maize Adh1 promoter with six copies of ‘anaerobic responsive element’ from the maize adh1 gene and four repeats of the ocs enhancer elements from the octopine synthase gene. Based on the premise that actin is a universal component of the plant cell cytoskeleton, McElroy et al. [137] used the 5? region of the rice actin 1 gene (Act1) in a chimeric construct with GUS and observed high level of GUS expression in transient assays of transformed rice and maize cells. Subsequently the actin promoter was also employed for transformation of wheat [138 /140]. Comparatively, new promoters that have been most effective and gained popularity over the years in driving interested genes efficiently in wheat cells are the maize ubiquitin promoter (Ubi1) with its first intron and the CaMV 35S (cauliflower mosaic virus) promoter (Table 1). These are constitutive promoters and allow expression of transgenes in a number of tissues throughout the plant. Weeks et al. [11] found that the ubiquitin promoter supported high levels of expression of the marker genes bar and uidA in transformed wheat tissues. Other promoters which have been successfully used in genetic transformation of wheat are the wheat high molecular weight (HMW) glutenin gene promoter [15,17,74,141] the wheat granule bound starch synthase gene promoter [142], tapetum specific promoter from rice and corn [143], stilbene synthase gene promoter from grapes [16] and rice sucrose synthase gene promoter [144]. Recently, Rasco-Gaunt et al. [131] produced herbicide resistance transgenic wheat plants at efficiencies ranging from 0.3 to 7.4% using a novel constitutive H2B promoter (H2B:mopat construct) from the maize histone h2b gene. These studies indicate that for specific application like improvement in the qualitative and quantitative traits of bread wheat, a specific promoter is required in order to express the desired transgene in desired tissues. And therefore, due to the limited availability of tissue specific promoter, continuous hunting for identification and isolation of tissue specific promoter sequences is required. The importance of the presence of introns have been shown in a number of systems to promote expression of genes several fold [145 /150]. Callis et al. [145] observed that placement of a maize intron in the 5? noncoding region of the chimeric gene resulted in 50 /100-fold stimulation of expression. A chimeric gene construct containing maize intron sequences (adh1) was found to enhance transient expression of CAT in protoplasts isolated from wheat caryopsis [146]. Oard et al. [147] observed a 30 /185-fold increased CAT activity by inserting intron 6 of a maize adh gene between the promoter of CaMV 35S and CAT sequences. RascoGaunt et al. [131] transformed wheat and reported that the use of introns of the adh1 or ubi1 genes was necessary for the recovery of transgenic wheat plants.

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In the same study, they observed that transformation efficiencies with the ubi1 intron were between 1.4 and 16 fold higher than with the adh1 intron. A number of mechanisms have been proposed in order to explain the function of intron in improving gene expression [151,152]. Liu and Mertz [152] reported that the presence or absence of cis -acting sequences in genes actually determine the need for introns for proper expression. It has also been proposed that in the absence of an intron, cis -acting sequences in genes act to impair expression of genes [153]. And therefore, introns can be considered an important factor for enhancing gene expression and restoring the normal expression of genes.

7. Agronomically important genes introduced into wheat Recent advances in transformation technology have resulted in the routine production of transgenic wheat plants for the introduction of not only marker genes but also agronomically important genes for quality improvement, male sterility, transposon tagging, resistance to drought stress, resistance against fungal pathogen and insect resistance (Table 3). However, unlike rice and maize, only a few agronomically useful genes have been introduced in wheat. The first report of an agronomically trait (the coat protein of barley yellow mosaic virus) transferred to wheat was published only in 1996s [154]. However, no data concerning the biological activity of the recombinant protein was presented. Wheat is attacked by a number of viral, bacterial and fungal pathogens and also by insect and nematode pest which severely check grain yield. 5/10% losses of total wheat production is reported just because of fungal pathogen [155]. Therefore, most of the work on genetic engineering of wheat for resistance against biotic stress has focused on developing protection against fungal pathogen [156]. Since the first attempt in engineering wheat with an agronomically important trait, success of transformation has progressed to a level where the transfer of agronomically useful genes such as ribosome-inactivating proteins (RIP) that inhibit protein synthesis and PR protein genes can now be introduced into wheat [22]. In most of the reports, chitinases, a subgroup of PR proteins with a role in plant defense, catalyze the degradation of chitin, which is component of the cell wall of many filamentous fungi has been successfully integrated into wheat genome for the engineering of resistance against fungal pathogen. Chen et al. [53] introduced a rice chitinase gene (chi11 ) under the control of constitutive CaMV 35S promoter into wheat employing particle bombardment and reported constitutive expression of the rice chi11 gene in T0 plants. However, instability of chitinase gene in sexual progeny was found and consequently plants were not challenged with pathogen. In subsequent

1157

year, Chen et al. [157] transformed ‘Bobwhite’, a spring variety of wheat with a plasmid construct containing rice chi11 driven by CaMV 35S and rice thaumatin like protein (tlp ) gene driven by maize ubi 1 promoter and reported stable expression of the tlp gene in three generations. Leckband and Lo¨rz [16] produced transgenic wheat plants expressing the stilbene synthase gene (vst1 ) and reported the expression of foreign phytoalexin resveratrol in transgenic plants. Stable expression of a chimeric stilbene synthase (sts) gene in trangenic lines of two commercial German spring wheat cultivars ‘Combi’ and ‘Hanno’ was also reported by Fettig and Hess [158]. Expression of the sts gene was proved by RT-PCR, and for the first time Fettig and Hess used HPLC and mass spectrometry for detection of the stilbene synthase product resveratrol in transgenic plants. However, both studies did not present any data regarding the resistance of phytoalexin resveratol-expressing transgenic wheat lines against wheat pathogens. Resveratrol (3,5,4?-trihydroxystilbene) which is synthesized by the catalysis of resveratrol synthases not only plays a role in the defense against fungal diseases in some plant species like groundnut or grapevine but also has been found to be linked with reduced human pathological processes such as atherosclerosis and carcinogenesis [159,160]. The findings that peanut seeds are a source of resveratrol (3,5,4?-trihydroxystilbene) have attracted attention on the role of resveratrol as a phytochemical with human health benefits. Recently, Chung et al. [161] have reported that resveratrol synthesis is also induced by biotic and abiotic factors through the regulation of resveratrol synthases transcription and that stress hormone such as salicylic acid and ethylene are involved in the resveratrol synthases gene expression in peanut. These findings indicate that there are enormous possibilities for creating resistance in wheat against abiotic stresses and fungal pathogen by introducing stilbene synthase gene in elite cultivars of wheat. Bliffeld et al. [139] introduced barley seed class II chitinase gene (pr3) driven by maize ubi promoter along with either a ribosome-inactivating protein (rip ) gene or b-1,3-glucanase gene in Bobwhite cultivar of wheat using particle bombardment. Stable expression of transgenes was observed in successive three generations and transgenic plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis . The authors also reported the adverse effect of a ribosome inactivating protein on plant regeneration. Bieri et al. [19] introduced a barley ribosome-inactivating protein (RIP30) into wheat mediated by particle bombardment and reported the expression of RIP30 in transgenic wheat plants which resulted in moderate protection against E. graminis . Towards enhancing fungal resistance in transgenic wheat, Oldach et al. [162] expressed three cDNAs encoding the antifungal protein AFP from the fungus Aspergillus giganteus , a

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Table 3 Agronomically important genes transferred into wheat by particle bombardment Target tissue

Source of the gene

Gene

Selectable marker

Phenotype

References

IE IE

Barley yellow mosaic virus T. aestivum L.

bar bar

T. aestivum L.

EC IE

Bacillus amyloliguefaciens T. aestivum L.

No data on phenotype Acumulation of glutenin subunit 1Ax1 Accumulation of hybrid glutenin subunit Nuclear male sterility Increased dough elasticity

[154] [74]

IE

IE

Vitis vinifera

Coat protein (cp) High molecular weight glutenin subunit (1Ax1) High molecukar weight glutenin hybrid subunits (Dy10:Dx5) Barnase High molecular weight glutenin subunits Dx5, 1Ax1 Stilbene synthase (Vst1)

[16]

IE

T. aestivum L.

bar

IE EC IE

Oryza sativa Hordeum vulgare L. O. sativa

IE

Zea mays

High molecular weight glutenin hybrid subunits (Dy10:Dx5) Rice chitinase Class II chitinase (chiII) Thaumatin-like protein (tlp), chitinase (chi11) Transposase (Ac)

IE

T. aestivum L.

No data on resitance to fungus diseases Accumulation of hybrid glutenin subunit No data on phenotype Resistance to fungus (E. graminis ) Resistance to fungus (F. graminearum ) Synthesis of an active transposase protein in transgenic Ac line Increased dough strength

IE

T. aestivum L.

[17]

IE

H. vulgare L.

IE

Agglutinin (gna)

bar

IE

Galanthus-nivalis agglutinin (GNA) H. vulgare L.

Chimeric stilbene synthase gene (sts)

bar

IE

Wheat streak mosaic virus

Replicase gene (NIb)

bar

IE

H. vulgare L.

HVA1

bar

IE

Monoclonal antibody T84.66 Single chain Fv antibody (ScFvT84.66)

bar , hpt

EC IE

U. maydis infecting virus A. niger

Antifungal protein (KP4) Phytase-encoding gene (PhyA)

bar bar

IE

H. vulgare L.

Ribosome-inactivating protein (RIP)

bar

IE

Tritordeum, tomato, oat

IE

T. aestivum L.

S -adenosyl methionine decarboxylase bar gene (SAMDC), arginine decarboxylase gene (ADC) High molecular weight glutenin subunits (1Ax1, 1Dx5)

Increased dough strength and stability Resistance to angoumois grain moth (S. cerealella ) Decreased fecundity of aphids (Sitobin avanae ) Production of phytoalexin resveratrol, no data on resistance to fungus diseases Resistance to wheat steak mosaic virus (WSMV) Improved biomass productivity and water use efficiency Production of functional recombinant antibody in the leaves Resistance against stinking smut Accumulation of phytage in transgenic seeds Moderate resistance to fungal pathogen E. graminis No data on phenotype

IE IE

Bacterial ribonulease III, wheat streak mosaic virus A. giganteus, H. vulgare

IE

T. aestivum L.

IE

T. aestivum L.

IE

T. aestivum L. (soft wheat)

EC

F. sporotrichioides

IPS

Vigna aconitifolia

bar bar bar pat

bar bar bar , hpt bar

High molecular weight glutenin subunit (1Dx5) High molecular weight glutenin subunits bar (1Axx1, 1Dx5) Trypsin inhibitor (CMe) bar

Bacterial ribonulease III (rnc70), coat bar protein (cp) Antifungal protein afp from A. giganteus , bar a barley class II chitinase and rip I FKBP73 WFKBP77 bar High molecular weight glutenin subunits bar (1Ax1, 1Dx5) Protein puroindoline (PinB-D1a) bar Fusarium sporotrichioides gene (FsTRI101) D1-pyrroline-5-carboxylate synthetase (P5CS)

bar nptII

[172] [184] [15]

[207] [53] [139] [157] [187] [173]

[18] [144] [158]

[167] [184] [187] [164] [188] [19] [38]

Flours with lower mixing time, peak [174] resistance and sedimentation volumes No data on phenotype [20] Inhanced fungal resistance

[162]

Alteration in grain weight and composition in transgenic seeds No data on phenotype

[208]

Increased friabilin levels and decreased kernel hardness Increased resistance to FHB (F. graminearum ) Increased tolerance to salt

[183]

[97]

[170] [209]

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Table 3 (Continued ) Target tissue

Source of the gene

Gene

Selectable marker

Phenotype

References

IPS

Wheat streak mosaic virus

Coat protein gene (CP)

bar

Various degree of resistance to wheat streak mosaic virus

[21]

Abbreviations: IE, immature embryos; SC, scutllem derived calli; EC, embryogenic callus; IPS, indirect pollen system (in this system Agrobacterium suspension is pipetted on spikelets just before anthesis); bar , phosphinothricin acetyl transferase; nptII , neomycin phosphotransferase II; hpt , hygromycin phosphotransferase; Dy10, a high molecular weight glutenin subunit (HMW-GS) gene sequence.

barley class II chitinase and a barley type I RIP under the control of maize ubiquitin1 gene promoter. This study showed not only the stable integration and inheritance of the chitinase and the rip1 gene from barley and the fungal afp gene up to T3 generations, but also significant reduction of formation of powdery mildew or leaf rust colonies on transgenic wheat leaves expressing AFP or chitinaseII when compared with control plants. However, the same effect was not attributed by RIP-expressing transgenic wheat lines. Bieri et al. [163] found a most significant rate of protection against powdery mildrew when transgenic wheat plants express the barley RIP. Apoplastic Barnase expression was less efficient and lines harboring barley chitinase and b-1,3-glucanase showed phenotypes from increased resistance to increased susceptibility. Combination of the three barley proteins failed to increase protection. An increased endogenous resistance against Tilletia tritici was reported by Clausen et al. [164] with the introduction of virally encoded antifungal protein gene (KP4) of Ustilago maydis into wheat. Bioassays conducted for scab (Fusarium graminearum ) resistance indicated a delayed scab infection in the rice thaumatin like protein expression plants. Resistance of trangenic wheat to Alternaria triticiana pathogen because of the expression of endogenous TLP induced by the introduction of a barley tlp antisense gene has recently been reported by Pellegrineschi et al. [155]. Zhang et al. [20] introduced a mutant bacterial ribonuclease III gene (rnc70 ) in wheat using particle bombardment and reported that transgenic lines showed a high level of resistance to Barley Stripe Mosaic Virus infection in three progeny of transgenic plants. As an alternative to the scheme based on utilization of Bt-endotoxin for creating insect resistant plants, plant derived or engineered inhibitors have been considered important tools for the building of resistance against insects in cereal including wheat [165]. Altpeter et al. [18] transformed immature embryos of wheat with trypsin inhibitor gene (itr1) and reported increased insect resistance in transgenic wheat lines expressing trypsin inhibitor CMe. They demonstrated functional integrity of the transgenic inhibitor on ribonucleic acid, protein and enzyme activity in transgenic wheat plants and their sexual progenies. In comparison to control, a significant reduction in the survival rate of the storage pest

Sitotroga cerealella was also reported after rearing of larvae on transgenic wheat seeds expressing trypsin inhibitor CMe. It is very well known that in comparison to the pests of lepidopteran order, the content of proteases in the gut of sap-sucking insects like aphids is very low, and therefore, rules out the same strategy which worked in creating resistance against storage pest S. cerealella by introducing a trypsin inhibitor gene. Towards this goal, Down et al. [166] introduced snowdrop lectin in potato and reported that transgenic plants that expressed snowdrop lectin inhibited the development and fecundity of aphids. After 3 years of this report, Sto¨ger and co-worker [144] produced transgenic wheat plants expressing snowdrop lectin (Galanthus nivalis agglutinin) gene (gna ) and observed that transgenic plants expressing snowdrop lectin at levels above 4% of the total extracted protein significantly decreased the fecundity, but not the survival of grain aphids (Sitobion avenae ). Transgenic wheat lines expressing resistance to wheat streak mosaic virus have recently been achieved after introduction of viral coat protein gene and the replicase (NIb ) gene of wheat mosaic virus (WSMV) into wheat via particle bombardment [21,167]. Transgenic wheat lines produced in this study showed various degree of resistance to WSMV. Fusarium head blight (FHB) disease occurs in cereals throughout the world and causes necrosis of florets which results in moderate to severe reductions in grain yield [156]. The primary causal agents of FHB are F. graminearum and Fusarium culmorum which produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastic (non-ruminant) animals [168]. Enhanced trichothecene in tobacco was demonstrated by Muhitch et al. [169] by expressing a trichothecene acetyltransferase (TRI101) gene from Fusarium sporotrichioides . Following this report, recently, Okubara et al. [170] transformed Bobwhite cultivar of wheat with the FsTRI101 gene driven by maize ubiquitin promoter via particle bombardment of immature embryos and reported partial protection against the spread of F. graminearum in inoculated wheat spikes. In addition to its basic calorific value, wheat with its high protein content is also an important source of plant protein in human diet. Amongst the cereals, the flour of bread wheat, T. aestivum , has a superior capability of

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forming leavened bread. This superiority of bread wheat flour is largely because of the structure and composition of its seed storage proteins, which upon hydration form a gluten complex, an insoluble, but highly hydrated, visco-elastic mess that endows the wheat dough with its unique property. Gluten protein networks are made up by the polymeric glutenins and the monomeric gliadins, which confirms the visco-elasticity properties of the dough and necessary for producing bread, pasta, cakes, cookies and other wheat products [171]. Therefore, the alteration of HMW glutenin composition in the wheat grain by direct manipulation of HMW-glutenin genes (HMW-GS) using genetic engineering techniques is being considered an important step in improving the quality of wheat flour [15,74,172 /174]. For the first time, a major step towards improving bread wheat quality was taken by Blechl and Anderson [172], when they demonstrated the possibility to genetically engineer wheat with a complex trait with the introduction of a hybrid Dy10:Dx5 HMW-GS gene under the control of its native endosperm-specific promoter. In the same year, Altpeter et al. [74] introduced natural HMW-GS 1Ax1 gene (known to be associated with superior bread making quality) in wheat cultivar ‘Bobwhite’ which lacks the subunit in the wild type by particle bombardment of immature embryos. Both groups reported an increased expression levels in total HMW glutenins and significant alteration in the overall dough properties of transgenic seeds. The introduced HMW-GS 1Ax1 gene under the control of HMW-GS promoter was expressed at high levels and stability was maintained for several generations [74]. These studies also demonstrated the importance of tissue-specific promoters for the expression of transgenic proteins in the endosperm of wheat. Evaluation of baking properties and gluten composition of 1Ax1 expressing R4 seeds of field grown wheat lines showed quantitative differences in gliadins, flour yield and single kernel characteristics between transgenic lines and the ‘Bobwhite’ control. A first large scale baking and mixing test revealed improved mixing time, loaf volume and water absorbance in some transgenic lines [175]. Transformation of wheat with one or two HMWGS subunit genes has been shown to results in stepwise increase in dough elasticity [15]. In contrast, Rooke et al. [173] reported that transgenic bread wheat plants expressing HMW-GS subunit 1Dx5, showed an increased dough strength and stability. He et al. [17] further demonstrated that genetic engineering approach can be used to modify the quality of pasta making durum wheat with the introduction of high molecularweight glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5) into wheat genome. Authors showed that expression of the additional subunits resulted in increased dough strength and stability. Alvarez et al. [176] obtained six independent transgenic lines of a commercial spring bread wheat cultivar (ProInta Federal)

expressing HMW-GS subunits 1Ax1 and 1Dx5 and observed varying expression of HMW-GS subunits in transgenic lines. One transgenic line over-expressed the 1Dx5 subunit without changes in the other endosperm proteins, two expressed the 1Ax1 subunit, and two lines expressed both transgenes with silencing of all endogenous HMW-GS genes. The prospect for changing glutenin composition by expressing modified HMW-GS in transgenic plants came from the study of Shimoni et al. [177], when they expressed a recombinant protein with x- and y-type HMW-GS subunit in transgenic wheat and reported alteration in gluten polymer composition. Apart from increasing protein quality and quantity of wheat grain, efforts are also being made to modify the endosperm texture of wheat grain by genetic engineering. Wheat grain hardness texture is one of the parameters that determine the end-product quality. Soft wheats have softer endosperm texture, requires less energy to mill, and yield smaller particles with less starch damage upon milling than to hard wheat [178]. In addition, soft wheats make superior cakes, while hard wheats make superior bread. One of the major locus called Hardness (Ha ), which is located on the short arm of chromosome 5D in wheat, has been reported to control most of the variability of kernel hardness in this species [179]. Three genes, puroindoline A (pinA ), puroindoline B (pinB ) and grain softness protein (gsp1a ) have been identified that are closely linked to Ha [180,181]. For the first time, Greenwell and Schofield [182] showed that puroindolines may be involved in grain hardness. However, later, the role of puroindoline in endosperm texture has been controversial. Recently, Beecher et al. [183] demonstrated that the pinB-D1b alteration, common in hard textured wheats, can be complemented by the expression of wild-type PinB-D1a in transformed wheat plants. They reported that transgenic wheat seeds expressing wild-type PinB were soft in phenotype, having greatly increased level of protein friabilin (a protein which is found in larger amounts in soft wheat starch) levels and greatly decreased kernel hardness and damaged starch. This study not only demonstrated that texture of endosperm of wheat can be converted from hard to soft by expressing wild type PinB sequences, but also proved that PinB is a functional part of the Ha locus. The development of suitable hybridization systems for crop plants including wheat requires a high degree of male sterility to avoid self fertilization. Genetic engineering of nuclear male sterility in wheat was undertaken by De Block et al. [143]. They engineered nuclear male sterility in wheat by introducing the barnase gene driven by tapetum specific promoters from corn and rice. Transgenic wheat plants obtained in this study showed stable expression of the barnase gene and consequently total male sterility was found in transgenic plants. Expression of barnase gene at specific stage of

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anther development results in the destruction of tapetum and consequently failure of normal pollen development leads to pollen sterility. This system utilizes the ribonuclease-inhibitor barstar gene to restore the fertility of male sterile plants. For the first time, the same group demonstrated that complicated gene integration can be avoided by incubating tissue on medium containing niacinamide before particle bombardment. The authors suggested that the enzyme poly (ADP-ribose) polymerase (PARP), which play a crucial role in the processes of cell division and recombination, is inhibited by niacinamide, and therefore, resulting in simple integration pattern of the transgene. Drought is one of the major abiotic stresses that severely check crop productivity. Traditional approaches in creating resistance to crops are limited because of the complexity of stress tolerance traits as most of them are quantitatively linked traits (QTLs). In recent years, introduction of a gene by genetic engineering is being considered as a method of choice for the improvement of stress tolerance in wheat. The genes encoding for late embryogenesis abundant (LEA) protein which generally accumulate during seed desiccation, and also in drought conditions of the plants have attracted researcher for engineering wheat against drought. Sivamani et al. [184] introduced a barley HVA1 gene encoding for a LEA protein into a spring wheat cultivar by particle bombardment and reported that most of the transgenic lines tested showed improvement in higher water use efficiency values and improved biomass productivity than non-transgenic wheat plants when grown under soil water deficit conditions. This study shows that growth characteristics of wheat plants can be improved tremendously by introducing barley HVA1, a member of group 3 LEA protein in response to soil water deficit. Transposon mutagenesis has been widely exploited in various organism to isolate genes that encode unidentified products and commonly utilize the mutagenic potential of the transposable elements [185]. A transposon tagging system in wheat was developed by introducing the Ac transposase gene under the CaMV 35S promoter into cultured wheat embryos by particle bombardment [186]. For the development of tagging system, Takumi and co-workers bombarded immature embryos with the plasmid containing the maize dissociator (Ds ) element located between the rice Act1 promoter and the GUS gene. The expression of GUS was observed after the excision of Ds element. Southern and Northern analysis of transgenic plants showed stable expression and inheritance of the Ac transposase gene. Wheat is also being considered an ideal system for the production of novel compounds due to its excellent storage properties and the existence of an efficient processing industry. The production of recombinant

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antibodies (ScFvT84.66) in wheat has recently been reported with the expression of a medically important, single chain Fv recombinant antibody against the carcino-embryonic antigen (CEA) using a biolistic approach [187]. Around 30 mg/g of functional recombinant antibody was detected in the leaves and seeds. This is the first report which showed the possibilities of exploiting transgenic wheat plants as a bioreactor. Apart from human consumption, wheat is widely used as an animal feed for non-ruminants in several developed countries. The phytase of Aspergillus niger is used as a supplement in animal feeds to improve the digestibility and the bioavailability of phosphate and minerals. Brinch-Pedersen et al. [188] transformed wheat with the phyA gene from A. niger encoding for the phytase enzyme and reported stable expression in transgenic wheat. The inheritance of the phyA gene was monitored through three generations and transgenic seeds exhibited 4-fold increase of phytase activity. Therefore, there is an enormous possibility to produce Aspergillus phytase in wheat grains to be used as a feed for non-ruminant animals.

8. Conclusion and future prospective Although production of fertile transgenic plants of wheat is no longer a dream now, but genotype-dependent response and low transformation frequency are still major hurdle in utilizing the powerful recombinant DNA technology for genetic improvement of this important crop with agronomically important genes. In the majority of the published reports, the transformation frequency lies between 0.15 and 2.34% and therefore, the question, how to increase the efficiency of wheat transformation is still open. In order to enhance the transformation frequency, further investigation is required not only for the refinement of existing selection strategies but also developing novel schemes for the effective selection of transformed cells. In majority of the reports, plasmid construct uidA:bar has been used for transformation of wheat and tends to be the most consistent and favorable selection scheme (Table 1). However, apart from concerns about using herbicide and antibiotic gene in the genetic material, regeneration of high frequency of ‘escape’ plants is a major drawback for the use of uidA:bar gene construct. Recently, Wright et al. [22] have shown the superiority of PMI selection scheme over uidA:bar scheme by obtaining almost double the transformation frequency (20%) without any escape. The non-toxic sugar can not be metabolized unless the converting enzymes mannose-6-phosphate isomerase has been transformed into the plants. Therefore, new selection schemes which are based on utilization of manA gene for selecting transformed cells should be further exploited for increasing transformation

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frequency and also to resolve the existing concerns of using uidA:bar in selecting transformed cells. A single or few copies of the transgene with stable expression is desirable in most of the experiments aimed to obtain desirable product of the transgene. Transformants generated by biolistics are often characterized by multiple insertions of the delivered DNA [189]. These insertions are often linked and/or organized in tandem. Comparatively, these complicated integration patterns are less frequent if Agrobacterium is used as the delivery system. For the first time, De Block et al. [143] demonstrated that biolistic method can also be employed for generating transgenic plants with low copy numbers of the transgene by producing large number of transgenic wheat plants containing one to three copies of the transgene. They were able to prevent the high copy number and tandem integration of the transgene by incubating the tissue for several days on niacinamidecontaining medium before bombardment. By using such type of approach, researcher can control the integration of high copy number of transgenes in wheat even in most widely used biolistic-mediated transformation. Recently, Srivastava et al. [190] used the Cre/lox system to reduce the copy number of DNA inserts in the wheat genome after particle bombardment of target tissue with a transformation vector that consists of a transgene flanked by recombination sites in inverted orientations. This strategy of transforming wheat with Cre/lox system results in recombination between outermost sites, thereby resolving the integrated molecules into a single copy in the transgenic plants. Genetic transformation of wheat mediated by Agrobacterium has already been proved a method of choice mostly because of the unique ability of Agrobacterium to introduce low copy number of the transgenes. And therefore, the development of transformation systems which utilize application of niacinamide, the site-specific recombination (the Cre/ lox) system and Agrobacterium as a vehicle for the delivery of foreign DNA into the target tissue would facilitate the integration of single or low copy numbers of the transgene in wheat genome and consequently would be helpful in minimizing the problem of gene silencing in the progeny of transgenic wheat. For production of commercial cultivars of wheat, transgenes must be inherited in a predictable manner in successive generations and the expression must be stable. In recent year, however, the phenomenon of gene silencing leading to poor or non-expression of transgene in the progenies of wheat transgenic plants has frequently been reported [176,191]. Apart from homology-dependent (trans)gene silencing which result from inhibition of transcription initiation (transcriptional gene silencing, TGS), the degradation of the newly synthesized transcript in the cytoplasm (post transcriptional gene silencing, PTGS) results in silencing of transgene(s) in the successive generation of wheat

transgenic plants. It has already been proved that the presence of multiple copies of a transgene is often associated with dramatic inhibition of accumulation of product of the transgene or when inserted genes contain sequence homology to an endogenous gene [192 /194]. The use of specific sequences such as matrix attachment regions (MARs) could also be helpful in stabilizing transgene in the progeny of regenerated plants. Stable expression of ribosome-inactivating protein (RIP) over four generations due to MARs that flanked the transgene was demonstrated by Bieri et al. [19]. Recently, Sharp et al. [195] have found that those transgenic wheat lines which showed resistance against wheat streak mosaic virus (WSMV) in green house did not express resistance against WSMV under field conditions. Therefore, it is also necessary to generate large number of independent transgenic lines to evaluate them both under green house and field conditions in order to check not only the inheritance of gene of interest but also to get the ‘true’ effect of the gene. Another challenge in plant transformation of wheat is the removal of the marker gene that is usually transferred with the gene of interest to allow putative transgenic lines to be selected. Transgenic plants without the incorporation of marker genes for antibiotic or herbicide resistance would be more acceptable to the consumers. Elimination of marker gene by placing it between two directly orientated recognition sites of a recombinase (Cre/lox or FLP/FRT system) followed by the expression of recombinase resulting in recombination between flanking recognition site has already been proved a method for efficiently removing marker genes from transgenic plant genome [196 /198]. Recently, Hoa et al. [199] also reported excision of a seletable marker gene, hpt from transgenic rice plants following Cre/lox site-specific recombination systems. Another method based on intrachromosomal recombination between bacteriophage attachment P (attP) regions has also been developed and applied to remove selectable marker genes from tobacco transgenes [200]. This method has significant advantage over previous recombinase method in a way that it does not require additional recombinase gene. The twin-T-DNA strategy, which is based on the segregation of the selectable marker gene from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which are located adjacent to each other with no intervening region has been exploited in eliminating selection marker in rice [201] and barley [202]. Such a tools, therefore, can be applied for the excision of marker gene from the genome of transgenic wheat. Although it is clear now that Agrobacterium can transfer its T-DNA to wheat cells efficiently provided tissues containing ‘competent cells’ are infected in the presence of a inducer of virulence genes, but necrosis and subsequently cell death of tissues after pathogen

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infection are still major bottleneck in routine application of Agrobacterium for genetic transformation of wheat. Hence there is a need to focus attention on investigating parameters that would improve wheat transformation minimizing necrosis and cell death of transformed tissues. Interested avenues to pursue are the use and optimization of antioxidants during co-culture of tissues with Agrobacterium in order to check or minimize the production of reactive oxygen species referred to as an oxidative burst, modification of the potential Agrobacterium elicitors that signal plant cell death and optimization of oxygen tension during the plant bacterium interaction, a parameter which has recently been reported to reduce the browning and ultimately cell death of the transformed tissues [22]. With the recent advances made in wheat transformation especially in transferring agronomically important genes in wheat genome, we can anticipate that coming years will undoubtedly witness an increasing application of biotechnology for the genetic improvement of elite cultivars of wheat.

Acknowledgements A.K. Sahrawat wishes to thank the Alexander von Humbolt Foundation of Germany for the award of long-term Alexander von Humboldt Research Fellowship. The authors thank Dr Manfred Gahrtz for helpful discussions.

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