Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata
© Yadav et al.; licensee Springer. 2012
Received: 11 September 2012
Accepted: 29 November 2012
Published: 10 December 2012
A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T0 and T1 plants. Rooted T0 and T1 shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.
KeywordsAgrobacterium mediated transformation Annexin Double cotyledonary node Vigna radiata
Grain legumes constitute an important dietary constituent for humans and animals. They associate with nitrogen fixing bacteria and play an important role in low input agricultural production systems; particularly small and marginal farm holdings. Greengram (Vigna radiata L. Wilczek) is an important grain legume grown widely in Southeast Asia, Africa, South Africa and Australia. The crop is grown mainly as a source of vegetable protein for its high protein content (about 25%), which makes it as an excellent supplement to cereal diets. The cultivation of this crop is gaining more popularity by virtue of its early maturity, nutritional value and easy digestibility. In India, it is cultivated mainly under limited and erratic rainfall conditions and on marginal and sub-marginal lands where numerous biotic and abiotic stresses limit its productivity (Jaiwal and Gulati 1995; Jaiwal and Singh 2003). Conventional breeding for enhancing biotic and abiotic stress tolerance in crop plants has several constraints and since the available genetic variability is low, transfer of alien genes of proven value offer possible viable option for crop improvement. Legumes in general are recalcitrant to tissue culture and are highly genotype specific (Somers et al. 2003).
Reproducible and efficient protocols for shoot regeneration have been established for greengram (Amutha et al. 2006; Kaviraj et al. 2006; Mahalaxmi et al. 2006; Mundhara and Rashid et al. 2006; Vijayan et al. 2006; Yadav et al. 2010a& 2010b). The immense potential of biotechnological tools for improving against biotic and abiotic stresses can be realized by supplementing the breeding programmes through introduction of alien genes of recognized relevance into elite germplasm of crop plants (Chandra and Pental, 2003; Somers et al. 2003; Popelka et al. 2004; Dita et al. 2006; Eapen 2008). An efficient regeneration and transformation protocol will be the key to success of genetic transformation. Though there are reports claiming successful transformation, owing to their highly recalcitrant nature in culture and very low frequency of regeneration especially after transformation, progress in development of transgenics for various legumes has been very slow (Chandra and Pental, 2003; Somers et al. 2003; Popelka et al. 2004; Dita et al. 2006). In the present study, we describe a reproducible and efficient Agrobacterium mediated genetic transformation protocol for greengram using double cotyledonary node (DCN) explants derived from three day old seedlings with binary vector pCAMBIA 2301 containing annexin gene. Ectopic expression of annexin has been shown to improve tolerance to various biotic and abiotic stresses in tobacco plants (Jami et al. 2008).
Annexins are Ca2+ and phospho-lipid binding proteins forming an evolutionary conserved multi-gene family expressed throughout plant kingdom. Annexins play a critical role in plant cell from regulation of Ca2+ dependent biochemical signalling processes to phospho-lipid metabolism. Gene expression of annexins in plants appears to be regulated by developmental and environmental signals and is known to be regulated by Ca2+ in stimulus response coupling in many plant cell–signalling pathways. Plant annexins from Medicago sativa and Arabidopsis thaliana have been implicated in oxidative stress response. We hypothesis that incorporation of annexin transgene will contribute to better tolerance to oxidative stress as the crop is predominantly grown in conditions which generate ROS. Annexin 1 from Brassica juncea was used in the present study.
Results and discussion
Transformation efficiency expressed in percentage equal to number of explants regenerated and showing PCR amplification/Total number of explants infected × 100
No. of explants infected
No. of explants regenerated on kanamycin
No. of hardened plants showing PCR amplification
Transformation efficiency (%)
Various parameters were optimized to establish a reproducible and efficient transformation protocol using at least 10 cotyledonary node explants in three replications for each experiment. The success of transformation was confirmed by transient and stable GUS expression as well as PCR using kanamycin and gene specific primers in transformed explants and regenerated putative plants. Axillary cells of the cotyledonary node explants are known to possess cells that are competent for regeneration and targeted gene delivery (Chandra and Pental 2003). Transformation of cotyledonary nodes leading to the recovery of transgenic plants has been reported earlier in mungbean (Jaiwal et al. 2001).
Optimizing kanamycin concentration to select transformants
Observed effects of different concentrations of kanamycin on DCN explant
Kanamycin conc (mg/L)
Green shoots regenerated
Green shoots regenerated
Green shoots regenerated
Pale green shoots were found
No shoots observed
Explants were bleached
Factors effecting genetic transformation
Experiments were designed to work out the most optimal conditions for transformation and carried out with a bacterial concentration of 0.5 O.D. with explants derived from 3-day old seedlings (Yadav et al. 2010a) and varying the parameter under study.
Effect of optical density (O.D.) of Agrobacterium culture
Explants from 3-day old seedlings were co-cultured with Agrobacterium culture of varying optical density (O.D.560) between 0.5-1.5, keeping pH 5.8 and kanamycin concentration 100 mg/l. An O.D. of 0.8 was observed to be the give the best transformation response. This O.D. value might be representing the most active log phase of Agrobacterium growth and thus very effective for transformation. Similar results were also reported by Jaiwal et al. (19982001). Contrary to present report, a decline in transformation efficiency with increase in bacterial cell density has been reported in blackgram (Saini and Jaiwal 2007) which could be due to variation with regard to plant species, explanted tissue, duration of co-cultivation and mode of regeneration.
Concentration of bacterial cells
Different concentrations of the bacterial cells tested included 106 to 109 cells cm-3, transformation frequency (data not shown) increased with increase in concentration of Agrobacterium cells up to 108 cells cm-3. Similar results have been obtained in most of the grain legumes earlier (Bean et al. 1997).
Infection time of 10, 15, 20 and 30 min was tested and 15 min was observed to deliver the best response. Injuries inflicted with the help of hypodermic needle, enhanced the frequency of transient GUS expression, at the cotyledonary node attachment site. Wounding of the tissue before co-cultivation allows better bacterial penetration into the tissues facilitating the accessibility of plant cells for Agrobacterium or possibly stimulated the production of potent vir gene inducers like phenolic substances such as acetosyringone and hydroxyl-acetosyringone (Stachel et al. 1985) and enhanced the cell capability for transformation (Binns and Thomashow 1988). Wounding the plant material has been shown to increase transformation frequency (Bidney et al. 1992). Mechanical injury of the meristematic region probably induces meristem reorganizations promoting formation of large transgenic sectors and enhanced recovery of transformants.
Co-cultivation duration also affected the transformation efficiency. Extending the co-cultivation time up to three days increased the transient transformation frequency and subsequent increase in co-cultivation time decreased the transformation frequency resulting in bacterial over growth. Co-cultivation period of 2 d has been found to be optimum in Antirrhinum majus (Holford et al. 1992), Vigna unguiculata (Muthukumar et al. 1996), Vigna radiata (Jaiwal et al. 2001), Cajanus cajan (Mohan and Krishnamurthy 2003), Glycine max (Li et al. 2004) and Nicotiana tabacum (Uranbey et al. 2007).
Age of explants
Explants excised from 2- and 3- day-old seedlings were tested for their suitability to achieve higher transformation. Study indicated 3-day old seedlings produced best results. This could be due to differences in the regenerative capacity of the two explants, which in turn is regulated by levels of endogenous hormones.
Different levels acetosyringone concentrations were tested and acetosyringone at 100 μM was found to give the best regeneration response. Transformation studies in other plant species have indicated that acetosyringone at an appropriate concentration enhances efficiency of transformation (Srivastava et al. 2009).
Of the various infection media tested, MSB5 gave the highest regeneration response. Other important factors affecting transformation efficiency studied included pre-culture of explants on regeneration medium prior to inoculation. Pre-conditioning of explants for a period of 2–3 days gave the best results. Pre-culture of explants on regeneration medium prior to inoculation and co-cultivation with Agrobacterium has been reported to enhance efficiency of transformation in Vigna unguiculata (Muthukumar et al. 1996) and Cajanus cajan (Geetha et al. 1999). These optimized transformation conditions were used for development of transgenics in greengram. Selection of regenerants was carried out on kanamycin medium combined with previously standardized regeneration procedure.
Regeneration of transgenic plants
Explants after transformation were regenerated on shoot bud induction medium which contained 100 mg/l kanamycin and 250 mg/l cefotaxime. To achieve the best shoot proliferation response, the concentration of kanamycin was reduced to half at second sub-culture stage. Rooting (90%) was achieved successfully on ½ MSB5 medium. The entire cycle was completed in about 80 days. Rooted transgenic plantlets were efficiently hardened and upon transfer to pots attained sexual maturity and produced viable seeds.
Confirmation of putative transformants by Southern Blotting
It is summarized that the pre-cultured and mechanically injured three day old DCN explants of Vigna radiata were successfully transformed by using bacterial concentration 108 cells cm-3. The protocol opens window to genetically enhance the greengram genotypes with genes of proven agronomic importance to attain sustainable production under fragile crop growing environments. Direct shoot regeneration method described here also minimizes the possibility of somaclonal variation besides the applicability of method is season independent by way of using seedling explants. The annexin transgenics of greengram developed are being evaluated for physiological and biochemical traits for improved drought stress tolerance.
Seeds of a popular cultivar of greengram, ML 267 were obtained from Agricultural Research Station, Lam, and Andhra Pradesh, India. Healthy and uniform seeds were surface sterilized and double cotyledonary node (DCN) explants excised and cultured for efficient shoot regeneration as described by Yadav et al. 2010a.
Agrobacterium strain and gene construct
Kanamycin kill curve for selection of transformants
Prior to transformation, an effective concentration of kanamycin was determined for selection of transformants by culturing untransformed DCN explants on shoot bud induction medium containing various concentrations of kanamycin (0–150 mg/l).
Explant preparation and optimization of conditions for transformation
Selected seeds were rinsed with 70% alcohol for 2 min and the surface sterilized with 0.2% aqueous solution of HgCl2 (w/v) for 5 min. The seeds were subsequently washed several times with sterile distill water and cultured on MSB5 medium. The best conditions included three-day old DCN explants pre-cultured for 2 days and injured with fine needle at the axillary meristematic region. Over night grown cultures of Agrobacterium (0.8A, 1000 μl) were added to the flask containing infection medium and swirled well. Injured explants were added to the infection medium and swirled for 15 min. Infected explants were kept on co-cultivation medium containing 50, 100 and 200 μM acetosyringone for 2, 3 and 4 days. After co-cultivation explants were washed with cefotaxime (250 mg/l) and cultured on shoot bud induction medium containing 100 mg/l kanamycin as selection agent. Explants were sub-cultured onto fresh medium every 15 days.
The conditions optimized for the best regeneration earlier were practiced to get the finest transformation response to develop transgenic greengram with annexin 1bj gene by Agrobacterium mediated approach. The transformation of three day old DCN explants was carried out by using LBA 4404 strain of Agrobacterium tumefaciens harbouring pCAMBIA 2301 binary vector containing annexin 1bj gene under the control of CaMV 35S promoter. Experiments were repeated on a regular interval to generate more of independent events for selecting the promising transgenic plants of green gram. Untransformed explants were kept as regeneration control on kanamycin free media.
Selection of putative transgenics
The transformants were selected on 100 mg/l kanamycin in shoot bud induction medium (MS B5 containing BAP and NAA) for first 30 days of culture. Subsequently, the kanamycin concentration was reduced to 50 mg/l for next cycle of 30 days in shoot elongation and proliferation medium (MS B5 containing reduced levels of BAP and NAA). The regenerated shoots were rooted on ½ MS B5 medium and were taken to maturity in a transgenic glass house after primary hardening.
GUS histochemical analysis
Transient and stable histochemical GUS assay was carried out in different tissues essentially as described by Jefferson (1987).
PCR Analysis of putative transformants
The leaf genomic DNA from T0 and T1 plants was isolated by Cetyl Trimethyl Ammonium Bromide (CTAB) method and used for molecular characterization of putative transgenics by PCR using nptII and annexin gene specific primers. T0 plants were analyzed by PCR using npt II/annexin gene specific primers while T1 plants were analyzed using annexin gene specific primers only. The sequence of oligonucleotide for npt II primers was, Forward: 5’ - AAT ATC ACG GGT AGC CAA CG – 3’; Reverse: 5’ - GCT TGG GTG GAG AGG CTA TT - 3’ and annexin gene specific primers was, Forward: 5’- ATG GCG ACT CTT AAG GTT TCT T –3’; Reverse: 5’ - TCA CCG AGA AGT GCG ATG AG– 3’. PCR was carried out with 60 ng of purified genomic DNA, and Dream Taq polymerase (Genetix) in a Applied Biosystems thermal cycler with previously standardized run conditions which included initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 30 s and final extension at 72°C for 5 min. Plants confirming positive with PCR were taken to maturity and their seed was collected. The T0 seed so collected was sown in pots to raise T1 plants. The leaf genomic DNA from the T1 plants was analyzed by PCR using annexin gene specific primers. The genomic DNA from the untransformed control plants and pCAMBIA 2301 annexin were used as negative and positive controls, respectively. The amplified products were separated by electrophoresis on a 0.8% agarose gel and visualized with ethidium bromide.
Southern Blot Hybridization
Leaf genomic DNA was isolated by CTAB method from the putative T0annexin greengram transgenics developed by Agrobacterium mediated transformation. Integration of foreign gene in the host genome was determined by Southern analysis as per procedure described by Sambrook et al. 1989. Genomic DNA was digested with Pst I restriction enzyme which releases 1.5 kb gene cassette containing 954 bp annexin gene. The restricted DNA was blotted onto a Hybond N + membrane. Probe DNA was prepared from the PCR amplified product (941 bp) of annexin gene. The probe was made hot as per standard procedure with 32P. The Hybond N + membrane was incubated with pre-hybridization solution for 4 hrs and hybridization solution (containing hot annexin 941 bp probe DNA) for 20 hrs.
The Hybond N + membrane was washed and dried. Then the Hybond N + membrane was exposed to autoradiography film for two weeks. DNA isolated from an untransformed control plant was also tested for the presence of annexin gene in order to determine if transgene was present.
Double Cotyledonary Node
Polymerase Chain Reaction
Murashige and Skoog with B5 vitamins
Cauliflower Mosaic Virus (CaMV)
Cetyl Trimethyl Ammonium Bromide.
Authors are grateful to Department of Biotechnology, New Delhi for providing financial support to part of this work and Centre for Application of Molecular Biology to International Agriculture (CAMBIA), Australia for plasmid 2301.
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