Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.pdf
RESEARCH Open Access Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata Sushil Kumar Yadav1*, Sweety Katikala1, Varalaxmi Yellisetty1, Annapurna Kannepalle2, Jyothi Lakshmi Narayana1, Vanaja Maddi1, Maheswari Mandapaka1, Arun Kumar Shanker1*, Venkateswarlu Bandi1 and Kirti Pulugurtha Bharadwaja3 Abstract 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. Keywords: Agrobacterium mediated transformation, Annexin, Double cotyledonary node, Vigna radiata Background Grain legumes constitute an important dietary consti- tuent for humans and animals. They associate with ni- trogen 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 pro- tein 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 di- gestibility. In India, it is cultivated mainly under limited and erratic rainfall conditions and on marginal and sub- marginal lands where numerous biotic and abiotic stres- ses limit its productivity (Jaiwal and Gulati 1995; Jaiwal and Singh 2003). Conventional breeding for enhancing biotic and abiotic stress tolerance in crop plants has se- veral constraints and since the available genetic variabi- lity 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 regene- ration 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 * Correspondence: skyadav@crida.in; arunshank@gmail.com 1Central Research Institute for Dryland Agriculture, Santoshnagar, Hyderabad 500 059, India Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Yadav et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yadav et al. SpringerPlus 2012, 1:59 http://www.springerplus.com/content/1/1/59 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 transform- ation, 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 re- producible 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 environ- mental 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 Ara- bidopsis 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 Earlier, hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced trans- genic calli on B(5) basal medium supplemented with 5 × 10(−6) M Benzlaminopurine (BAP), 2.5 × 10(−6) M each of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and 1-Napthaleneacetic acid (NAA) and 50 mg l(−1) kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing beta-glucuronidase (gusA) and neomycin phospho- transferase II (nptII) marker genes at a frequency of 0.9% (Jaiwal et al. 2001). However, we in our study for the first time, transformed green shoots showing strong GUS activity regenerated directly from cotyle- donary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B(5) medium containing 6-benzylaminopurine (5 × 10(−7) M) and 100 mg l(−1) kanamycin. Molecular analysis of putative transformed plants revealed the integration and expression of trans- genes in T(0) plants and their seeds. Transformation efficiency observed was 4.2% (Table 1). Various parameters were optimized to establish a re- producible 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 regene- ration and targeted gene delivery (Chandra and Pental 2003). Transformation of cotyledonary nodes leading to the recovery of transgenic plants has been reported ear- lier in mungbean (Jaiwal et al. 2001). Optimizing kanamycin concentration to select transformants Prior to transformation, an effective concentration of antibiotic for the selection of transformed cells was determined by culturing DCN explants on Murashige and Skoog with B5 vitamins (MSB5) media containing various concentrations of kanamycin (0, 25, 50, 75, 100, 150 mg/l) (Figure 1). The effect of various concentrations of kanamycin on regeneration response is described below (Table 2). Kanamycin over a concentration of 100 mg/l was observed to be effective to select the transformants derived from DCN explants and caused complete necrosis of the untransformed explants within three weeks and thus was successfully used for selection of transformants. Hence kanamycin at a concentration of 100 mg/l was used for transformants. Earlier Phogat et al. (1999) selected transformed calli of Vigna radiata on 100 mg/l of kana- mycin concentration. Factors effecting genetic transformation Experiments were designed to work out the most opti- mal 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 kanamy- cin 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 trans- formation. Similar results were also reported by Jaiwal et al. (1998, 2001). Contrary to present report, a decline in Yadav et al. SpringerPlus 2012, 1:59 Page 2 of 8 http://www.springerplus.com/content/1/1/59 explants excised and cultured for efficient shoot regene- ration as described by Yadav et al. 2010a. Agrobacterium strain and gene construct The disarmed Agrobacterium strain LBA4404 was used for the genetic transformation of greengram. The gene construct contained a binary vector pCAMBIA 2301 which had β-glucuronidase (GUS) reporter (uidA) gene, a neomycin phosphotransferase (nptII) gene as plant se- lection marker driven by cauliflower mosaic virus (CaMV) 35S promoter. The uidA gene contains an in- tron in the coding region to ensure that the observed GUS activity occurred in the plant cell and not due to residual Agrobacterium cells. Annexin1bj gene cassette (−35S promoter-annexin-nos terminater-) of 1.5 kb in size was cloned in pCAMBIA 2301 vector at Pst I site (Figure 6). The recombined vector was transferred into E. coli (DH5α) by heat- shock method and finally trans- ferred into Agrobacterium tumefaciens strain LBA 4404 by freeze-thaw method. Plasmid DNA isolated from E. coli cell upon digestion with Pst I resulted in re- lease of 1.5 Kb annexin gene cassette. The transformed Agrobacterium strain LBA 4404 was used for genetic transformation of DCN explants of greengram. The cDNA of annexin1bj gene was 954 bp length frag- ments, it had Bam HI restriction site at 5' end and Sal I site at 3' end. Annexin cDNA also had two restriction sites for Hind III enzyme resulting in 3 fragments of 368, 439 and 147 bp lengths (Figure 7). Npt II and annexin1bj gene specific primers were designed which gave 645 and 941 bp product, respectively. Agrobacter- ium strain was grown over night at 28°C in YEB medium containing either 50 mg/l kanamycin. Bacterial cells were pelleted and resuspended in liquid shoot regene- ration medium for further use in standardization of va- rious transformation parameters. Kanamycin kill curve for selection of transformants Prior to transformation, an effective concentration of kanamycin was determined for selection of transfor- mants by culturing untransformed DCN explants on shoot bud induction medium containing various concen- trations 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 cul- tured 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 re- gion. 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 cefota- xime (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. Figure 6 Schematic representation of T-DNA region of pCAMBIA 2301 having annexin gene (6345bp). Figure 7 Restriction enzyme sites in annexin1bj gene cassetter (1.5Kb). Yadav et al. SpringerPlus 2012, 1:59 Page 6 of 8 http://www.springerplus.com/content/1/1/59 Transformation The conditions optimized for the best regeneration ear- lier were practiced to get the finest transformation re- sponse to develop transgenic greengram with annexin 1bj gene by Agrobacterium mediated approach. The transformation of three day old DCN explants was car- ried 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 con- trol on kanamycin free media. Selection of putative transgenics The transformants were selected on 100 mg/l kanamy- cin in shoot bud induction medium (MS B5 contain- ing 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 contain- ing 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 pri- mary hardening. GUS histochemical analysis Transient and stable histochemical GUS assay was car- ried 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 iso- lated by Cetyl Trimethyl Ammonium Bromide (CTAB) method and used for molecular characterization of puta- tive 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 Bio- systems 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 un- transformed 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 visua- lized with ethidium bromide. Southern Blot Hybridization Leaf genomic DNA was isolated by CTAB method from the putative T0 annexin greengram transgenics deve- loped by Agrobacterium mediated transformation. Inte- gration 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 solu- tion (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 auto- radiography 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 trans- gene was present. Abbreviations DCN: Double Cotyledonary Node; GUS: β-glucuronidase; PCR: Polymerase Chain Reaction; BAP: Benzlaminopurine; MSB5: Murashige and Skoog with B5 vitamins; CaMV: Cauliflower Mosaic Virus (CaMV); CTAB: Cetyl Trimethyl Ammonium Bromide. Competing interests The authors declare that they have no competing interests Authors’ contributions SKY conceived the study and has contributed to the experimental concept and design. KS, YV, NJL and MV contributed to the acquisition of data by carrying out different experiments. KA has contributed to Southern analysis. MM has made substantial contribution to experimental conception and design and in the initial draft preparation of the manuscript. AKS has helped in critically drafting and revising the manuscript for important intellectual content. BV has been involved in various suggestions while carrying out the study and critically going through the manuscript. PBK has helped in initial concept design and gene construct preparation. All authors read and approved the final manuscript. Acknowledgement 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. Yadav et al. SpringerPlus 2012, 1:59 Page 7 of 8 http://www.springerplus.com/content/1/1/59 Author details 1Central Research Institute for Dryland Agriculture, Santoshnagar, Hyderabad 500 059, India. 2Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110 012, India. 3Department of Life Sciences, University of Hyderabad, Hyderabad 500 046, India. Received: 11 September 2012 Accepted: 29 November 2012 Published: 10 December 2012 References Amutha S, Muruganantham M, Ganapathi A (2006) Thidiazuron induced high frequency axillary and adventitious shoot regeneration in Vigna radiata L. Wilczek. In Vitro Cell Dev Biol Plant 42:26–30 Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep 16:513–519 Bidney D, Seelonge C, Martich J, Burrs M, Sims L et al (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. 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J Plant Biochem Biotechnol 15:131–134 Yadav SK, Gopala Krishna M, Maheswari M, Vanaja M, Venkateswarlu B (2010a) High frequency induction of multiple shoots and plant regeneration from cotyledonary node explant of mung bean (Vigna radiata L Wilczek). J Plant Biochem Biotech 19:267–270 Yadav SK, Sreenu P, Maheswari M, Vanaja M, Venkateswarlu B (2010b) Efficient shoot regeneration from double cotyledonary node explants of green gram (Vigna radiata L Wilczek). Indian J Biotech 9:403–407 doi:10.1186/2193-1801-1-59 Cite this article as: Yadav et al.: Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata. SpringerPlus 2012 1:59. 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Title: Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata
Authors: Sushil Kumar Yadav, Sweety Katikala, Varalaxmi Yellisetty, Annapurna Kannepalle, Jyothi Lakshmi Narayana, Vanaja Maddi, Maheswari Mandapaka, Arun Kumar Shanker, Venkateswarlu Bandi, Kirti Pulugurtha Bharadwaja ​
Publisher: SpringerPlus
Date: 2012-12-10 00:00:00
Abstract: 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 1bj 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. ​