GENETIC TRANSFORMATION AND REGENERATION OF COM- MON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM

ommon beans (Phaseolus vulgaris L.) are the most important food legume in the developing world, and an important human dietary constituent being a rich source of protein for millions of people (Colpaert et al., 2008; Kelly, 2010; Amugune et al., 2011; Kwapata et al., 2012). Immature pods of common beans are popular as a vegetable for human consumption in Africa, Asia and Latin America (Veltcheva et al., 2005; DelgadoSánchez et al., 2006; Varisai Mohamed et al., 2006; Gatica Arias et al., 2010; Kwapata et al., 2010). Therefore, there is considerable interest in the introduction of useful traits into beans. The lack of an efficient regeneration system for common bean is a major bottleneck to genetically transform this species. Plant biotechnology, and conventional breeding methods, could facilitate bean improvement since resistance to biotic and abiotic stress could be increased (Liu et al., 2005). Nevertheless, efficient in vitro culture system that results in efficient regeneration is an essential requirement for improvement of common bean through genetic transformation (Svetleva et al., 2003; Kwapata et al., 2010). In addition to genetic improvement, in vitro culture is an important tool for the recovery and conservation of germplasm (Delgado-Sánchez et al., 2006). With the development of transformation technology, genetic engineering has emerged as an alternative, potentially important approach to create new varieties.


GENETIC TRANSFORMATION AND REGENERATION OF COM-MON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM E. A. EISSA
Genetics Department, Faculty of Agriculture, Fayoum University, 63514 Fayoum, Egypt ommon beans (Phaseolus vulgaris L.) are the most important food legume in the developing world, and an important human dietary constituent being a rich source of protein for millions of people (Colpaert et al., 2008;Kelly, 2010;Amugune et al., 2011;Kwapata et al., 2012). Immature pods of common beans are popular as a vegetable for human consumption in Africa, Asia and Latin America Delgado-Sánchez et al., 2006;Varisai Mohamed et al., 2006;Gatica Arias et al., 2010;Kwapata et al., 2010). Therefore, there is considerable interest in the introduction of useful traits into beans. The lack of an efficient regeneration system for common bean is a major bottleneck to genetically transform this species. Plant biotechnology, and conventional breeding methods, could facilitate bean improvement since resistance to biotic and abiotic stress could be increased (Liu et al., 2005). Nevertheless, efficient in vitro culture system that results in efficient regeneration is an essential requirement for improvement of common bean through genetic transformation (Svetleva et al., 2003;Kwapata et al., 2010). In addition to genetic improvement, in vitro culture is an important tool for the recovery and conservation of germplasm (Delgado-Sánchez et al., 2006). With the development of transformation technology, genetic engineering has emerged as an alternative, potentially important approach to create new varieties.
Difficulties in obtaining plants from somatic cells in common beans have till now hampered the production of transgenic plants by the application of the most common methods available for the introduction of foreign DNA into cells. Regeneration from callus in P. vulgaris is hard to attain but it has been reported only by Dillen et al. (2000) and Arellano et al. (2009). Direct regeneration from explants via shoot organogenesis, is certainly recommendable for gene transfer technology. However, most large seeded legumes have proven to be recalcitrant to regeneration. In case of beans, shoot regeneration can be an important step in plant transformation procedures, and many attempts have been made with results showing that the genotype and the hormones both have great effect (Eissa et al., 1999 andGatica Arias et al., 2010;Kwapata et al., 2010). Though scientists have tried extensively, there is still little reproducible and efficient protocol for bean genetic transformation (Kwapata et al., 2012). Micropropagation of Vicia faba L. culti-C vars has been achieved by somatic embryogenesis and nodal segment explants raised seedlings (Hamdy and Hattori, 2006). Naz et al. (2007) reported the efficient plant regeneration from chick pea via direct regeneration that can be used in genetic transformation techniques.
Successful plant transformation requires specific knowledge of Agrobacterium-host compatibility (Godwin et al., 1992). Agrobacterium-mediated gene transfer can be one of the methods of choice to introduce gene/genes into common beans. The use of the naturally occurring compound acetosyringone in a preinoculation culture step has been shown to increase the level of virulence in Agrobacterium (Vernade et al., 1988) and lead to higher transformation rates (Becker et al., 1994;Lee et al., 2001).
Several protocols have been reported for shoot organogenesis from apical or axillary meristems in Phaseolus (McClean and Grafton, 1989;Franklin et al., 1991;Saxena, 1991 andMohamed et al., 1992;Eissa et al., 2002;Delgado-Sánchez et al., 2006;Varisai Mohamed et al., 2006) or through somatic embryogenesis (Zambre et al., 1998;Schryer et al., 2005). Although several protocols have been described for bean regeneration, an optimal in vitro culture system still remains a major challenge since this species from the Phaseolus genus are recalcitrant for regeneration . The best characterized and most commonly used trans-formation system for plants is the disarmed A. tumefaciens strains. Since long time, preliminary data regarding the capability of Phaseolus to establish a compatible reaction with Agrobacterium were reported by Allavena (1985) and McClean et al. (1988). Putative transformants have been reported in one common bean cultivar (Mariotti et al., 1989). While McClean et al. (1991) were able to introduce genes into beans using the Agrobacterium system; they were unable to regenerate transgenic plants. Early efforts to produce transgenic bean plants failed due to poor DNA delivery and regeneration systems. Franklin et al. (1993) produced transformed gusA positive callus from a kidney bean cultivar inoculated with Agrobacterium strain EHA101. So far, transformation of P. vulgaris has been achieved through direct gene transfer into the apical meristem region of seedlings of one cultivar (Russell et al., 1993). Embryonic axes and the apical meristems of P. vulgaris exposed using biolistic method, however, the frequency of transgenic plants they obtained was much lower 0.03% , and Aragão et al. (1999) attempted also to introduce a gene coding for methionine in bean via the same method.
To date only a few reports are available on attempts to transform common beans. Therefore, the objective of the present study was to optimize an in vitro plant transformation and regeneration systems for two commercial common beans varieties (P. vulgaris L.). Two Agrobacterium strains were used, one containing the neomycin phosphotransferase II (nptII) and β-glucoronidase (gusA) marker genes, and the other containing phosphinothricin acetyl transferase (bar) and βglucuronidase (gusA) marker genes. A marker gene and a selectable gene were used in co-transformation of regenerated common bean plants. According to these experiments engineered strains of A. tumefaciens were used to attempt transgenic plant recovery directly from common bean meristematic tissues. Using an optimized regeneration protocol via organogenesis from axillary and adventitious buds derived from nodes explants obtained kanamycin resistant, gusApositive shoots and plantlets, and herbicide resistant, gusA-positive shoots and plantlets. The achieved results have been utilized for establishing genetic transformation and regeneration method in order to improvement common beans varieties.

Plant materials and surface sterilization method of seeds
Two commercial common beans (Phaseolus vulgaris L.) varieties "Fönix" and "Maxidor" seeds were used as source of plant materials. The seeds are kindly supplied by the Genetics and Horticultural Plant Breeding, Faculty of Horticultural Sciences, Corvinus University, Budapest, Hungary, to whom my thanks are due. For surface-sterilization method, seeds were selected based on a healthy and similar size. Seeds were soaked for 4 min in 75% ethanol, disinfected with 20% solution of commercial bleach containing (5.25% sodium hypochlorite) with four drops of tween 20 solution for 20 min and rinsed three times with sterile double-distilled water.

In vitro seeds germination method and explants preparation
Surface sterilized seeds of the common beans were then germinated in test tubes on MS (Murashige and Skoog, 1962) agar medium. The pH of the media was adjusted to 5.7 with KOH and supplemented with 30 g/l sucrose and 7 g/l agar. Media were dispensed into glass tubes or bottle glasses, autoclaved at 120C for 20 min. The explant cotyledonary nodes containing a portion of the hypocotyl, epicotyl and cotyledons cultures were initiated and prepared from excised three-days-old seedlings. Approximately, 2/3 of each cotyledon was removed from the explant, leaving the cotyledon fragments attached to the nodal region. Full strength MS medium, supplemented with 1 mg/l BA (6-benzyladenine) and 0.1 mg/l NAA (α-naphthalene acetic acid) were used for shoot induction from the explant nodes. Phenolics compound acetosyringone was added after autoclaving when media were cooled to below 60C. The presence of acetosyringone during co-cultivation generally enhanced the virulence of Agrobacterium strains.
Axenic seeds explant cultures for transformations and regenerations were incubated on the media at 24-26C under 16/8 hours photoperiod at white light in growth cabinet. The early events of multiple shoot formation were examined using scanning electron microscopy.
The binary vector (pEHA101::pTd33) encodes a plant expressible neomycin phosphotransferase II (nptII) gene as selectable marker and βglucuronidase (gusA) sequence coding region as a reporter gene. The protein product of nptII gene phosphorrylates kanamycin and renders the drug non toxic to plant cells. The binary vector (pRGG bar H1) contains bar gene as a selection marker, encodes a modifying enzyme phosphinothricin acetyl transferase (PAT), which confers resistance to herbicide phosphinothricin (PPT), bialaphos as selectable marker and gusA as a reporter gene ( Table 1). The bacterial cultures of each Agrobacterium strain were grown in solid Luria Bertani (LB) medium (10 g/l bacto-tryptone, 5 g/l bacto-yeast extract, 10 g/l NaCL) with appropriate antibiotic kanamycin at concentration of 50 mg/l. The cultures of each bacterial strain were incubated over-night at 28ºC.

Explants inoculation with Agrobacterium strains
The procedure used to obtain common bean plants resistant to kanamycin or phosphinothricin began with the germinating "Fönix" and "Maxidor" seeds on MS medium for three days. Explants (cotyledonary nodes) of common beans were inoculated with Agrobacterium strain EHA101 or A281. Hence, for each variety, 500 explants were cultured, cocultivated with each strain. Freshly streaked an overnight cultures of each Agrobacterium strain were suspended in MS medium (pH 5.2) or sterile distilled water. 100 µM Acetosyringone were added to the co-cultivation medium plus 1 mg/l BA and 0.1 mg/l NAA to increase the infection rate of Agrobacterium on the regenerated buds and shoots including differentiating tissues around the nodal region. Cotyledonary nodes with lateral meristems explant were excised, and the nodal region was punctured numerous times wounded and inoculated below and above the point of excision with Agrobacterium strains EHA101 (pEHA101::pTd33) or A281 (pRGG bar H1). Either by immersion in a diluted overnight bacterial suspension for three hours or by repetitive puncturing of meristems with a needle or a surgical blade previously dipped or stabbed into the diluted bacterial culture. The actively dividing cells resulting from wound-induced cell division were conceived to show more transformation. Newly synthesized cell wall was found to be essential for the production attachment of Agrobacterium pre-ceding transformation. The active DNA replication of rapidly dividing cells was also proposed to be very important for the integration of plasmid DNA. The explant nodes were cultured on solidified cocultivation media for three days. The explant nodes were then washed in sterile water, blotted dry on sterile paper towels, and cultured on MS regeneration media without the use of any chemical selections for 24 hours, to allow expression of the resistance gene. The regeneration media are containing 1 mg/l BA, 0.1 mg/l NAA, 500 mg/l carbenicillin and 800 mg/l cefotaxime, and pH 5.7.

Sensitivity of cotyledonary node explants to kanamycin, and bialaphos (phosphinothricin)
An effective concentration of kanamycin, and bialaphos for the selection of transformants were assessed by preliminary tests. For both bean varieties, cotyledonary node explants were cultured on shoot regeneration MS media, plus various concentrations of kanamycin (0, 50, 75, 100, 150 and 200 mg/l), and bialaphos (0, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/l). Kanamycin and bialaphos was filter sterilized and added to the media after autoclaving. Kanamycin at a concentration of 100 mg/l, and bialaphos at a concentration of 4 mg/l were used for the selection of transformed shoots and plants for both bean varieties.

Stable genetic transformation, kanamycin, and bialaphos resistance
Co-cultivated nodes with strain EHA101, or A281 were transferred at first to selection media plus only 50 mg/l kanamycin, or 1 mg/l bialaphos, and 500 mg/l carbenicillin and 800 mg/l cefotaxime to eliminate Agrobacterium overgrowth for one week. Nodes were then transferred to selection media plus 100 mg/l kanamycin, or 2 mg/l bialaphos. The transformed explant nodes were transferred three times at three day intervals to fresh MS media plus 500 mg/l carbenicillin and 800 mg/l cefotaxime to eliminate Agrobacterium overgrowth of the target regions. The explants of both bean varieties co-cultivated with strain EHA101 were then transferred to MS medium plus 200 mg/l kanamycin and 500 mg/l carbenicillin. The kanamycin concentration was increased incrementally over three months period to 300 mg/l. To prevent the escape of nontransformed buds and shoots regenerated from the explants of both two varieties cocultivated with strain A281, 2 mg/l bialaphos was added to regeneration medium as a selection agent. The selection of stable transgenic shoots for both bean varieties was based on the use of 4 mg/l bialaphos selection for the bar herbicide resistance marker gene, and stable transgenic green shoots continued on this medium. Putative transformed green shoots were transferred onto kanamycin, or bialaphos containing media to verify their ability to root.

Histochemical staining of transformed tissues
Segments from putative transgenic green shoot and plant tissues for both bean varieties selected on kanamycin, or bialaphos transformed with the (pEHA101::pTd33), and (pRGG bar H1) were tested and assessed for expression of the gusA reporter gene by the histochemical assay using X-gluc. This substrate works very well, giving a blue precipitate at the site of enzyme activity. All of the procedures using the gusA reporter gene system are based on the work of Jefferson (1987). The vials samples were then incubated in a thermostat at 37C dark incubator for overnight, next morning the samples are checked.

RESULTS AND DISCUSSION
The results obtained in this investigation showed that full strength MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA were the optimal for shoot induction from meristematic ring of cotyledonary nodes explant in both bean varieties "Fönix" and "Maxidor" (Fig. 1). Early events on the development of highly regenerable tissue culture procedure from the meristematic ring on nodal explants were examined by scanning electron microscope (SEM) in both bean varieties after 48 hours cultured on MS regeneration medium, which showed organogenesis and bud formation from the meristematic cells in the cotyledonary nodes explant (Fig. 2). The results indicated that the shoots were formed from actively dividing cells located at the axillaries bud regions. A method was designed to optimize rapid and high frequency direct shoot regeneration of the commercial important common beans. Therefore, this method for regeneration was suitable for indirect Agrobacterium-mediated transformation protocol and used in transformation and regeneration experiments in both common bean varieties. Agrobacterium strain EHA101 (pEHA101::pTd33) and A281 (pRGG bar H1) attachment to cotyledonary nodes explant in both common bean varieties were examined by scanning electron microscope during Agrobacterium-mediated transformation. Scanning electron microscope studies demonstrated that cells of A. tumefaciens strains attach to cells of cotyledonary nodes in both common bean varieties (Fig.  3). The development of two common beans varieties "Fönix" and "Maxidor" plants that are able to grow in the presence of high concentrations of kanamycin and that express the gene product required, for resistance to the drug, or in the presence of high concentrations of phosphinothricin and that express the gene product required, for resistance to the herbicide were reported.
Transformation and successful regeneration procedures were based on a direct shoot organogenesis protocol previously described by Zhang et al. (1997) and Eissa et al. (2002). The natural ability of A. tumefaciens to introduce DNA into plant cells is being widely exploited for the genetic transformation of plants (van Wordragen and Dons, 1992). This system is, therefore, considered to be suitable for the transformation of common beans. It was tried to improve the Agrobacterium infection by the application of acetosyringone which is a phenolic compound produced by the wounding of plant tissues and which induces the transfer of T-DNA from Agrobacterium to the plant genome (Godwin et al., 1991;Lee et al., 1995). Agrobacterium infection can readily deliver DNA in Phaseolus (McClean et al., 1991). The lack of in vitro regeneration capacity in P. vulgaris has prompted investigators to devise regeneration independent approaches, and one such approach has met with success (Russell et al., 1993). Apical meristems of seedlings were submitted to particle bombardment, and after stimulation of axillary shoot proliferation, transgenes plants were recovered at 0.03% of shoots. Using the same strategy, Aragão et al. (1996) obtained transformants of cv. Olathe. Aragão and Rech (1997) hypothesized that a good ability for axillary bud formation, and proper apex morphology the features render a genotype suitable for transformation through meristem bombardment. Success with bean transformation has been very limited; the few successful transgenic bean varieties have been developed by physical methods that circumvent problems with plant regeneration (Kelly, 2010). In the present study, the nptII gene coding was tested by selection of kanamycin stable transgenic shoots. The transgenic character of kanamycin-resistant shoots was confirmed by detection of βglucuronidase activity in resistant tissues. Thus, these studies provide strong confirmation for a kanamycin resistant gene transferred to plant cells. Similar results were achieved by Jaiwal et al. (2001) who regenerated transformed shoots directly from cotyledonary node explants of mungbean cultured on medium contain-ing 75 mg/l kanamycin after co-cultivation with LBA4404 (pTOK233). Dillen et al. (1995) delivered DNA to intact embryonic axes of common bean through electroporation and expression of the gusA reporter gene was observed in tissues.
In the present research, addition of 100 µM acetosyringone to freshly cut cotyledonary nodes tissues could increase the transformation efficiency. The increase in the transformation efficiency of freshly cut tissues in the presence of acetosyringone suggests that the synthesis of vir inducers during incubation, and the accumulation of vir-inducing compounds in the wounded and preincubated plant tissues are an important factors contributing to the increased competence of the incubated common bean cotyledonary node tissues to Agrobacterium-mediated transformation. Stimulation of plant cell division and activation of the DNA replication machinery during the incubation period may play an important role in the integration of plasmid DNA leading to stable transformation (Baron and Zambryski, 1995). The results of the current work showed that the use of cotyledonary node explants is more efficient compared for genetic transformation of a commercial important P. vulgaris common bean varieties.

Sensitivity of cotyledonary node explants to kanamycin, and herbicide
A gene coding for the enzyme neomycin phosphotransferase II, or phosphinothricin acetyl transferase is ex-pressed constitutively in host plant cells to which it is transferred during transform induction and confers kanamycin, or herbicide phosphinothricin (bialaphos) resistance and transcripts on transformed plant cells, and confers on transformed cells the ability to grow in the presence of normally lethal levels of the antibiotic kanamycin, or herbicide (bialaphos). Kanamycin and phosphinothricin a widely used markers for plant transformation that can be phytotoxic and inhibit untransformed tissues. These genes can be used as selectable markers in plants. The development of efficient transformation protocols for common beans will open up the possibility of transferring an enormous range of useful genes into this crop, including gene for herbicide resistance. Kanamycin at a concentration of 50 mg/l, or phosphinothricin at a concentration of 1 mg/l were sufficient to completely kill any morphogenetic event in both bean varieties "Fönix" and "Maxidor". All the cotyledonary node explants of both varieties produced an average of 14.58 and 27.10 buds and shoots, respectively, in 100% of the cultures on kanamycin-free or bialaphos-free MS media. Kanamycin at a concentration of 100 mg/l, or phosphinothricin (bialaphos) at a concentration of 4 mg/l was used for the selection of transformed plants in both bean varieties (Tables 2 and 3). These results showed that kanamycin, and phosphinothricin are effective selection markers for common beans. Influence of carbenicillin and cefotaxime on shoot initiation and on subsequent growth was also checked by culturing explants on shoot regeneration medium containing 500 mg/l carbenicillin and 800 mg/l cefotaxime. This concentration had no effect on shoot initiation and subsequent growth, but effectively controlled the Agrobacterium growth.

Kanamycin selection
The aim of the present work was to start the elaboration of a general transformation and regeneration system from P. vulgaris L. using A. tumefaciens gene transfer procedure. In order to test the efficiency of the nptII gene as selection marker for obtaining transformants, cotyledonary nodes explant of two common beans varieties "Fönix" and "Maxidor" were co-cultivated with Agrobacterium EHA101 containing nptII gene as selectable marker and gusA reporter gene. The availability of simple selection assay becomes extremely useful such as selection method may facilitate the identification of the transgenic plants. The cocultivated explants were incubated for 24 hours without drug selection to allow expression of the resistance gene. Explants were cultured on regeneration media with 50 and 75 mg/l kanamycin as selection agent for regeneration. Stable transformants were then selected by culturing the explants on medium containing 100 mg/l kanamycin (Fig. 4). The percentage of kanamycin resistant explants that was obtained per co-cultivation with Agrobacterium EHA101 is listed in Table  (2). The percentage of transformed cotyledonary nodes explant by strain EHA101 cultured on selective MS regeneration medium containing 100 mg/l kanamycin was 15 and 20% in "Fönix" and "Maxidor" varieties respectively ( Table  2). As shown in Table (2), co-cultivation with cotyledonary node explants results in high percentage of kanamycin resistant shoots. On the contrary, following several transfers on MS media containing kanamycin, most of the shoots obtained from co-inoculation turned yellow, browned and died (Fig. 4). Transformed shoots was selected on 100 mg/l kanamycin. Selection pressure was applied when excised shoots were rooted on MS medium containing 50 mg/l kanamycin. Subsequent selection on media with kanamycin resulted in the recovery of shoots and plants resistant to the kanamycin. Kanamycinresistant shoots and plants appeared 2-3 weeks following plating on solid medium. The presence of the DNA insert in the bean genome was shown by its resistance to kanamycin.
The results obtained here in the present work are comparable with the results obtained by other workers in common beans, mungbean, soybean, pea, pigeonpea, chickpea, lentil, strawberry, tobacco, lettuce, eggplant, cabbage, potato, and other plants with little differences due to plant species and genotypes. The transformed state of regenerated P. vulgaris L. plantlets after cutting the shoot apexes co-cultivated with Agrobacterium was assessed by kanamycin resistance test (Mariotti et al., 1989). Becker et al. (1994) reported that 50 and 100 mg/l kanamycin, allowed growth of cotyledonary nodes explant of different cultivars of P. vulgaris transformed with Agrobacterium A281 for about two and one week. Apical meristems of P. vulgaris cv. Jalo bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type showed a high frequency of tumor formation, 50 to 70% . The hypocotyl and epicotyl of two-week-old plantlets were stabbed using a syringe needle dipped in the Agrobacterium strain A281 solution contains oncogenes binary vector or harbors nptII and gusA marker genes to determine the susceptibility of 16 bean cultivars, four resulted in high frequencies of tumor formation (Karakaya and Ӧzcan, 2001). Amugune et al. (2011) evaluated the potential of two common bean varieties to Agrobacterium-mediated transformation. Germinated embryos for 1-2 days, were inoculated with Agrobacterium LBA4404 (pBI121), EHA105 (pCAMBIA1201) and EHA105 (pCAMBIA1301), harboring gusA plasmids, co-cultivated for 3-4 days on MS medium or with 10 µM BAP and cultured on regeneration and selection medium consisting of 10 µM BAP and 50 mg/l kanamycin. Transformed shoots were obtained in 40 weeks old Mwitemania plantlets from explants infected with Agrobacterium LBA4404 (pBI121). Soybean cotyledonary nodes inoculated with Agrobacterium, 50 mg/l kanamycin used for selection and plant regeneration (Trick et al., 1997). Nodus explants seedlings of P. sativum were transformed using Agrobacterium carrying nptII gene as selectable marker, transformed tissue via organogenesis was selected on 50 mg/l kanamycin (De Kathen and Jacobsen, 1990). Cotyledonary node explants of pigeonpea were transformed by co-cultivation with Agrobacterium LBA4404 carrying the gusA gene and the nptII gene, cultured on shoot regeneration MS medium with 2 mg/l BAP and 50 mg/l kanamycin for selection (Geetha et al., 1999). Embryogenic axis from germinated seedlings of chickpea infected with LBA4404 (pASKαAI1) Agrobacterium, and cultured on shoot induction MS medium with 1.0 mg/l BAP and 100 mg/l kanamycin for selection (Ignacimuthu and Prakash, 2006). A shoot regeneration system from leaf disks of strawberry inoculated with Agrobacterium contains nptII and gusA genes cultured on medium containing 50 mg/l kanamycin was described by Nehra et al. (1990). In tobacco, leaves were wounded by microprojectile prior to inoculate with Agrobacterium strain LBA4404 containing nptII gene, and transferred to regeneration medium with 100 mg/l kanamycin for selection (Bidney et al., 1992). High frequency of transformation, based on kanamycin resistance in selection medium containing 100 mg/l kanamycin was obtained from lettuce cotyledon explants co-cultivated with A. tumefaciens vectors (Torres et al., 1993). When leaves explant of eggplant were co-cultivated with A. tumefaciens containing nptII and gusA genes, transgenic buds and shoots were observed on the selection medium with 50 mg/l kanamycin (Billings et al., 1997). Petiole segments explants of Cyclamen persicum Mill. infected by Agrobacterium contains a vector includes the nptII gene as selection marker cultured on MS medium with 100 mg/l kanamycin for regener-ation were reported by Aida et al. (1999). Transgenic plants were obtained from B. oleracea L. and B. rapa L. by Agrobacterium strain EHA101 contains nptII and gusA genes, green shoots were obtained on medium containing 50 mg/l kanamycin (Kuginuki and Tsukazaki, 2001). Gene transfer by Agrobacterium is the established method of choice for the genetic transformation of most plant species. It is perceived to have several advantages over other forms of transformation, including the ability to transfer large segments of DNA and with fewer copies of inserted genes at higher efficiencies (Wu et al., 2003). Although plant tissue can be transformed using different direct DNA methods, the foreign genes tend to integrate as multiple copies. Sarker et al. (2003) and Hassan et al. (2007) reported that decapitated embryo of lentil was more effective in formation of multiple shoots on MS medium with 50 mg/l kanamycin following Agrobacterium LBA4404 harboring binary plasmid pBI121, containing the nptII and gusA genes infection and selection. Two regeneration systems were developed in cowpea, from shoot apices of 3-5-day-old seedlings (Mao et al., 2006), and via embryonic axes explants (Yusuf et al., 2008). Harini and Sathyanarayana (2009) reported the induction of somatic embryos in Mucuna pruriens belongs to the family Fabaceae from different explants cultured on MS medium with 11.31 µM 2,4-D produced embryogenic callus. Optimizing factors for T-DNA delivery with transformation efficiency more than 20% resulted in kanamycin-resistant transgenic shoots of Thlaspi caerulescens L. using Agrobacterium EHA105 harboring pBI121 with the nptII and the gusA genes were described by Safavi and Asgari (2011). Transformed potato were obtained by co-cultivating leaf explants with Agrobacterium contains the nptII gene as selectable in MS regeneration medium plus 50 mg/l kanamycin (González et al., 2008).

Herbicide phosphinothricin, bialaphos selection
Phosphinothricin is a widely used marker for plant transformation that can be phytotoxic and inhibit untransformed tissues. In order to test the efficiency of the bar gene as selection marker for obtaining transformants, nodes explant of both bean varieties were co-cultivated with strain A281 containing bar gene as selectable marker and gusA reporter gene. The co-cultivated explants were incubated for 24 hours without herbicide selection to allow expression of the resistance gene. Cotyledonary nodes which were cocultivated with Agrobacterium strain A281 for three days were grown at first on regeneration media containing 1 and 2 mg/l herbicide bialaphos, phosphinothricin as selection agent and 500 mg/l cefotaxime. Stable transformants were then selected by culturing the explants on medium containing 4 mg/l phosphinothricin (Fig. 5a). The percentage of herbicide resistant explants that was obtained per co-cultivation with Agrobacterium A281 is listed in Table (3). The percentage of transformed cotyledonary nodes explant by strain A281 cultured on selective MS regeneration medium containing 4 mg/l phosphinothricin was 9 and 10% in "Fönix" and "Maxidor" varieties, respectively (Table 3). Transformed tissue was selected on 1 mg/l phosphinothricin. Selection pressure was applied when excised shoots were rooted on MS medium containing 1 mg/l phosphinothricin. Subsequent selection on media with phosphinothricin resulted in the recovery of shoots resistant to the herbicide. Herbicide resistant shoots and plants appeared 2-3 weeks following plating on solid medium. As shown in Table (3), cocultivation with cotyledonary node explants results in high percentage of herbicide resistant shoots. On the contrary, following several transfers on MS media containing herbicide, most of the shoots obtained from co-inoculation turned yellow, browned and died (Fig. 5a). Herbicide at a concentration of 4 mg/l was chosen for selecting transformed tissues, buds, shoots and plants of both common bean varieties. The presence of the DNA insert in the bean genome was shown by its resistance to phosphinothricin. In the present research, the development of two commercial common beans varieties "Fönix" and "Maxidor" plants that are able to grow in the presence of high concentrations of herbicide phosphinothricin (bialaphos) and that express the gene product required, for resistance to the herbicide were reported. Putative plant transformation and successful regeneration procedures were based on a direct shoot organogenesis protocol.
The results obtained in the present investigation are agreement with the results obtained by other workers in common beans, soybean, tobacco, wheat, rice, bentgrass, tomato, and other plants with little differences due to plant species and genotypes. Becker et al. (1994) reported that, cotyledonary nodes explant of different cultivars of P. vulgaris transformed with Agrobacterium A281 allowed growth on medium with 1 mg/l phosphinotricin, and the explants died within two weeks at 5 and 10 mg/l phosphinotricin. The results of Lee et al. (2001) indicated that to prevent the escape of chimeric or nontransformed buds and shoots, 2 mg/l glufosinate was added to shoot induction medium as a selection agent from cotyledonary node explants of P. vulgaris using Agrobacterium EHA101 containing bar gene. Rech et al. (2008) describes a method for recovery of transgenic common bean plants by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques, and the average frequency of transgenic plants is 2.7. Aragão and Faria (2009) used particle bombardment protocol to insert bar gene for herbicide resistance and virus resistance (RNAi) into the pinto bean cv. Olathe. Transgenes included the bar herbicide (glufosinate ammonium) resistance selectable marker used for selection of transgenic cultures which confirmed herbicide resistant common bean plants and gusA color marker which visually confirmed transgenic events (Kwapata et al., 2012).
Soybean cotyledon explants inoculated with A. tumefaciens pTiT37-SE conferring glyphosate tolerance, about 6% shoots were transgenic based on selected glyphosate tolerance (Hinchee et al., 1988). Öktem (1998) used Agrobacterium LBA4404 containing the bar gene to develop transgenic tobacco plants resistant to herbicide gluphosinate using the leaf disc. The result of Mihály et al. (2002) showed that all the transformed wheat hybrid embryos by selectable bar gene germinated onto 5 mg/l bialaphos medium expressed resistance, while the control showed sensitivity. Wu et al. (2003) reported that selection was carried out with 2-4 mg/l phosphinotricin for wheat immature embryos co-cultivated with Agrobacterium strain AGL1 harboring the bar gene, and transformation efficiencies ranged between 0.3% and 3.3% only. A procedure was developed to produce rice tolerant to the herbicide phosphinothricin by in vitro selection (Toldi et al., 2000). Chai et al. (2002) produced transgenic bentgrass plants via biolistic system; 5 mg/l phosphinothricin inhibited the growth of non-transformed plantlets but did not affect the growth of transformed. Plasmid JS101 containing the bar herbicide resistance gene was used as a selectable marker, 1 mg/l zeatin and 0.1 mg/l IAA were enhanced shoot regeneration in tomato (Hussain et al., 2008).

GUS analysis
Tissues which developed on nontransformed explants showed no detectable gusA activity when stained with X-gluc, while tissues on transformed explants stained blue (Fig. 5b). This suggests that the gusA gene be expressed in common bean tissues, after co-cultivation with EHA101, or A281 100% of kanamycin resistance, or of phosphinothricin resistant of the nodes explant showed gusA activity (Tables 2 and 3). This procedure exhibits distinct advantages over these previously reported, and the highly regenerable cotyledonary nodes rapidly produce transgenic shoots without an intermediate callus phase. The results of this study revealed that, in a binary vector system, cotyledonary node explants is effective at delivering DNA into common bean. Ideally, transgenic plant production involves Agrobacterium-mediated gene transfer, followed by selection of transformed tissues and regeneration from them. Integration of foreign DNA was confirmed by selective analysis and GUS-test. Shoots formed roots in half strength MS medium with 0.1 mg/l indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse.

Conclusion
In the present investigation, transgenic plants with nptII gene or bar gene in two varieties of the common bean were achieved. In view of the present results, there is a possibility of achieving high levels of kanamycin resistance, or herbicide phosphinothricin resistance in beans by using Agrobacterium-mediated strain EHA101 (pEHA101::pTd33), or A281 (pRGG bar H1), and phenolics compound acetosyringone. Traits such as herbicide resistance cannot be introduced from other species of plants or from distantly related species (soybean) as this would require genetic engineering. The development of efficient transformation protocols for common beans will open up the possibility of transferring an enormous range of useful genes into this crop, including genes for herbicide and pest resistance. This protocol should help achieve Agrobacterium-mediated genetic transformation to improve this important food legume.

SUMMARY
Agrobacterium-mediated transformation and regeneration systems has been achieved for two varieties "Fönix" and "Maxidor" of common beans. Transformation ability of cotyledonary nodes explant were tested by A. tumefaciens EHA101 (pEHA101::pTd33) with the nptII and gusA genes, or A281 (pRGG bar H1) with the bar and gusA genes. Transgenic for both bean varieties with nptII gene, or bar gene were produced through this approach. Among different selection methods, 100 mg/l kanamycin, or 4 mg/l herbicide phosphinothricin turned to be optimal, resulting in the highest transformation efficiency. Transgenic common bean plants demonstrated enhanced growth ability under antibiotic kanamycin, or herbicide phosphinothricin stress conditions. The increased resistance was also reflected by delayed development of damage symptoms caused by kanamycin, or phosphinothricin stress. Cotyledonary nodes were found to be effective in formation of multiple shoots cultured on se-lection MS medium supplemented with 1 mg/l BA, 0.1 mg/l NAA and plus 100 mg/l kanamycin, or 4 mg/l phosphinothricin. Stable expression of the gusA was observed in various parts of the transformed tissues. Optimal transformation conditions were obtained for both bean varieties by co-cultivating cotyledonary node explants with Agrobacterium in MS regeneration medium plus 100 µM acetosyringone for 3 days. The nodal region was punctured, wounded and infected with Agrobacterium strain by overnight immersion in bacterial suspension plus 100 µM acetosyringone. This wounding pattern permits Agrobacterium to penetrate deeper and more completely throughout the tissue, and increasing the probability of infecting plant cells. After inoculation for 3 hours and co-cultivation for 3 days, the explant nodes were cultured on MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA, pH 5.7. The explant nodes were transferred three times at three day intervals to fresh MS medium plus 500 mg/l carbenicillin and 800 mg/l cefotaxime to eliminate Agrobacterium overgrowth of the target region. After cocultivation for 24 hours on MS medium, the explants nodes were transferred to a selective medium containing 100 mg/l kanamycin, or 4 mg/l phosphinothricin. Gene expression of kanamycin resistance (nptII), or herbicide phosphinothricin resistance (bar) and gusA in transformed buds and shoots for both bean varieties was demonstrated by selection test and gusA histochemical analysis. Shoots and buds of transformed explants continued on selective media. Putative transformed green shoots were transferred onto kanamycin, or phosphinothricin containing medium to verify their ability to root. Expression of the β-glucuronidase reporter gene was verified by histochemical staining.  Percentage of transformed explants (explants giving rise to one or more kanamycin resistant shoot compared to the total number of explants. Maxidor 4 500 50 10 10 Percentage of transformed explants (explants giving rise to one or more phosphinothricin resistant shoot compared to the total number of explants.