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Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

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Abstract

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2–3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.

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References

  • Atkinson RG, Gardner R (1991) Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants. Plant Cell Rep 10:208–212

    Article  CAS  PubMed  Google Scholar 

  • Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep 16:513–519

    Article  Google Scholar 

  • Bhomkar P, Upadhyay CP, Saxena M, Muthusamy A, Prakash NS, Pooggin M, Hohn T, Sarin NB (2008) Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by over expression of the glyoxalase 1 gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter. Mol Breed 22:169–181

    Article  CAS  Google Scholar 

  • Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang CH, Fonger TM, Degg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bruning JL, Kintz BL (1977) Computational handbook of statistics, vol 2, 2nd edn. Scott, Foresman and Co, Glenview

    Google Scholar 

  • Chaudhary D, Madanpotra S, Jaiwal R, Saini A, Kumar P, Jaiwal PK (2007) Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L.Walp) cultivar and transmission of transgene into progeny. Plant Sci 172:692–700

    Article  Google Scholar 

  • Dan Y (2008) Biological functions of antioxidants in plant transformation. In Vitro Cell Dev Biol Plant 44:146–161

    Article  Google Scholar 

  • Das DK, Bhomkar P, Shiva Prakash N, Bhalla Sarin N (2002a) Improved method of regeneration of blackgram (Vigna mungo L.) through liquid culture. In Vitro Cell Dev Biol Plant 38:456–459

    Article  Google Scholar 

  • Das PK, Roy M, Mandal N (2002b) In vitro organogenesis from shoot tip in blackgram. Indian J Genet Plant Breed 62:91–92

    Google Scholar 

  • Dayal S, Lavanya M, Devi P, Sharma KK (2003) An efficient protocol for shoot regeneration and genetic transformation of pigeonpea (Cajanus cajan L.) using leaf explants. Plant Cell Rep 21:1072–1079

    Article  CAS  PubMed  Google Scholar 

  • De Clercq J, Zambre M, Van Montagu M, Dillen W, Angenon G (2002) An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolius. A. Gray. Plant Cell Rep 21:333–340

    Article  Google Scholar 

  • Delic D, Stajkovic O, Kyzmanovic D, Rasulic N, Knezevic S, Milicic B (2009) The effects of rhizobial inoculation on growth and yield of Vigna mungo L. in Serbian soils. Biotechnol Anim Husb 25:1197–1202

    Google Scholar 

  • Dillen W, DeClercq J, Kapila J, Zhambre M, Montaque MV, Angenon G (1997) The effect of temperature on Agrobacterium-mediated gene transfer to plants. Plant J 12:1459–1463

    Article  CAS  Google Scholar 

  • Fillati JJ, Sellmer J, Mc Cown B, Haissing B, Comai L (1987) Agrobacterium-mediated transformation and regeneration of Populus. Mol Gen Genet 206:192–199

    Article  Google Scholar 

  • Franklin G, Ignacimuthu S (2000) Differential morphogenetic response of cotyledonary explants of Vigna mungo. Biol Plant 43:1–4

    Article  Google Scholar 

  • Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178:1498–1504

    PubMed Central  CAS  PubMed  Google Scholar 

  • Fullner KJ, Lara JC, Nester EW (1996) Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273:1107–1109

    Article  CAS  PubMed  Google Scholar 

  • Gamborg OL, Miler RA, Ojima K (1968) Nutrient requirement of suspension culture of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Gawali MP, Akhare AA, Gahukar SJ (2010) In vitro regeneration of pigeonpea from leaf with petiole explants. Asian Sci 5:34–38

    Google Scholar 

  • Godwin I, Todd G, Ford-Lloyd B, Newbury HJ (1991) The effect of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9:671–675

    Article  CAS  PubMed  Google Scholar 

  • Grant JE, Cooper PA, Gilpin BJ, Hoglund SJ, Reader JK, Pither Joyece MD, Timmerman-Vaughan GM (1998) Kanamycin is effective for selecting transformed peas. Plant Sci 139:159–164

    Article  CAS  Google Scholar 

  • Harisaranraj R, Saravana BS, Suresh K (2008) Callus Induction and plant regeneration of Vigna mungo (L.) Hepper via half seed explant. Ethnobot Leafl 12:577–585

    Google Scholar 

  • Hess D, Dressler K, Nimmrichter R (1990) Transformation experiments by pipetting Agrobacterium into the spikelets of wheat ( Triticum aestivum L.). Plant Sci 72:233–244

    Article  CAS  Google Scholar 

  • Ignacimuthu S, Franklin G (1999) Regeneration of plantlets from cotyledon and embryogenic axis explants of Vigna mungo L. Hepper. Plant Cell Tissue Organ Cult 55:75–78

    Article  Google Scholar 

  • Jaiwal PK, Kumari R, Ignacimuthu S, Potrykus I, Sautter C (2001) Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) a recalcitrant grain legume. Plant Sci 161:239–247

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Kaneyashi J, Kobayashi S, Nakamura Y, Shigemoto N, Doi Y (1994) A simple and efficient gene transfer system of trifoliate orange. Plant Cell Rep 13:541–545

    Google Scholar 

  • Kapila J, DeRycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108

    Article  CAS  Google Scholar 

  • Karamany EL (2006) Double purpose (forage and seed) of mungbean production 1-effect of plant density and forage cutting date on forage and seed yields of mungbean (Vigna radiata (L.) Wilczeck). Res J Agric Biol Sci 2:162–165

    Google Scholar 

  • Karthikeyan AS, Sharma KS, Veluthambi K (1996) Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper. Plant Cell Rep 15:328–331

    Article  CAS  PubMed  Google Scholar 

  • Ko TS, Korban SS (2004) Enhancing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledon of soybean (Glycine max (L.) Merrill). In Vitro Cell Dev Biol Plant 40:552–558

    Article  CAS  Google Scholar 

  • Krishanamurthy KV, Suhasini K, Sagare AP, Meixner M, de Kathen PT, Schieder O (2000) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) embryo-axes. Plant Cell Rep 19:235–240

    Article  Google Scholar 

  • Langridge P, Brettschneider R, Lazzeri P, Lorz H (1992) Transformation of cereals via Agrobacteriumand the pollen pathway: a critical assessment. Plant J 2:631–638

    Article  CAS  Google Scholar 

  • Lin JJ, Garcia-Assad N, Kuo J (1994) Effect of Agrobacterium cell concentration on the transformation efficiency of tobacco and Arabidopsis thaliana. Focus 16:72–77

    Google Scholar 

  • Lukoki L, Marechal R, Otoul E (1980) Les ancetres sauvages des haricots cultives: Vigna radiata (L.) Wilczek et V. mungo (L.) Hepper. Bull Jard Bot Nat Belg 50:385–391

    Article  Google Scholar 

  • Mahalakshmi SL, Leela T, Manoj Kumar S (2006) Enhanced genetic efficiency of mungbean by use of primary leaf explants. Curr Sci 91:93–98

    CAS  Google Scholar 

  • Mony SA, Haque MS, Alam MM, Hasanuzzaman M, Nahar K (2010) Regeneration of blackgram (Vigna mungo L.) on changes of hormonal condition. Not Bot Hort Agrobot Cluj 38:140–145

    Google Scholar 

  • Mujib A (2005) In vitro regeneration of Sandal ( Santalum album L.) from leaves. Turk J Bot 29:63–67

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Muruganantham M, Ganapathi A, Amutha S, Vengadesan G, Selvaraj N (2005) Shoot regeneration from immature cotyledonary nodes in black gram (Vigna mungo (L.) Hepper). Indian J Biotechnol 4:551–555

    Google Scholar 

  • Muruganantham M, Amutha S, Selvaraj N, Vengadesan G, Ganapathi A (2007) Efficient Agrobacterium-mediated transformation of Vigna mungo using immature cotyledonary node explants and phosphinothricin as the selection agent. In Vitro Cell Dev Biol Plant 43:550–557

    Article  CAS  Google Scholar 

  • Muthukumar B, Marriyamma M, Gananam A (1995) Regeneration of plants from primary leaves of cowpea. Plant Cell Tiss Org Cult 44:153–155

    Article  Google Scholar 

  • Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA (2001) The role of thiol compounds increasing Agrobacterium-mediated transformation of soybean cotyledonary node cells. Plant Cell Rep 20:731–737

    Article  CAS  Google Scholar 

  • Olhoft PM, Flagel LE, Denovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary node method. Planta 216:723–735

    CAS  PubMed  Google Scholar 

  • Popelka C, Gollasch S, Moore A, Molvig L, Higgins TJV (2006) Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny. Plant Cell Rep 25:304–312

    Article  CAS  PubMed  Google Scholar 

  • Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. In: Gelvin SB, Schilperoot RA (eds) Plant molecular biology mannual. Kluwer Acad. Publ, Dordrecht, pp 1–11

    Google Scholar 

  • Sahoo L, Jaiwal PK (2008) Asiatic beans. In: Kole C, Hall TC (eds) A compendium of transgenic crop plants. Blackwell Publ, Oxford, pp 115–132

    Google Scholar 

  • Saini R, Jaiwal PK (2002) Age, position in mother seedling, orientation and polarity of the epicotyl segments of blackgram (Vigna mungo L. Hepper) determines its morphogenic response. Plant Sci 163:101–109

    Article  CAS  Google Scholar 

  • Saini R, Jaiwal PK (2005) Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures. Plant Cell Rep 24:164–171

    Article  CAS  PubMed  Google Scholar 

  • Saini R, Jaiwal PK (2007) Agrobacterium tumefaciens—mediated transformation of blackgram: an assessment of factors influencing the efficiency of uidA gene transfer. Biol Plant 51:69–74

    Article  CAS  Google Scholar 

  • Saini R, Makani S, Jaiwal PK (2003) Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens. Plant Cell Rep 21:851–859

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Santarem ER, Trick HN, Essig JS, Fines JJ (1998) Sonication assisted Agrobacterium-mediated transformation of soybean immature cotyledons: Optimization of transient expression. Plant Cell Rep 17:752–759

    Article  CAS  Google Scholar 

  • Sen J, Guha-Mukherjee S (1998) In vitro induction of multiple shoots and plant regeneration in Vigna. In Vitro Cell Dev Biol Plant 34:276–280

    Article  CAS  Google Scholar 

  • Sharma KK, Anjaiah V (2000) An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. Plant Sci 159:7–19

    Article  CAS  PubMed  Google Scholar 

  • Solleti SK, Bakshi S, Sahoo L (2008) Additional virulence genes in conjunction with efficient selection scheme and compatible culture regime enhance recovery of stable transgenic plants of cowpea via Agrobacterium tumefaciens- mediated transformation. J Biotechnol 135:97–104

    Article  CAS  PubMed  Google Scholar 

  • Song JY, Sivanesan I, Jeong RB (2012) Use of petal explants for successful transformation of Dendranthema × grandiflorum Kitamura ‘Orlando’ mediated by Agrobacterium tumefaciens. Afr J Biotechnol 11:9141–9148

    CAS  Google Scholar 

  • Sonia SR, Singh RP, Jaiwal PK (2007) Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris α-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker. Plant Cell Rep 26:187–198

    Article  CAS  PubMed  Google Scholar 

  • Srinivasan T, Verma VK, Kirti PB (2004) Efficient shoot regeneration in pigeon pea, Cajanus cajan (L.) Millsp. using seedling petioles. Curr Sci 86:30–32

    Google Scholar 

  • Srivastava P, Pandey A (2011) Standardization of callus induction and plant regeneration form leaf explants of black gram (Vigna mungo var. silvestris). Int J Inn Biol Chem Sci 1:1–6

    CAS  Google Scholar 

  • Stachel SE, Nester EW, Zambryski P (1986) A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci U S A 83:379–383

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Svabova L, Griga M (2008) The effect of cocultivation treatments on transformation efficiency in pea (Pisum sativum L.). Plant Cell Tissue Organ Cult 95:293–304

    Article  CAS  Google Scholar 

  • Varalaxmi V, Vijayalakshmi A, Yadav SK, Venkateswarlu B, Maheswari M (2007) Efficient plant regeneration from cotyledons of black gram (Vigna mungo (L.) Hepper). Indian J Biotechnol 6:414–417

    CAS  Google Scholar 

  • Villiers SD, Emongor Q, Njeri R, Gwata E, Hoisington D, Njagi I, Silim S, Sharma K (2008) Evaluation of the shoot regeneration response in tissue culture of pigeonpea (Cajanus cajan [L.] Millsp.) varieties adapted to eastern and southern Africa. Afr J Biotechnol 7:587–590

    Google Scholar 

  • Wright MS, Ward DV, Hinchee MA, Carnes MG, Kaufman RJ (1987) Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue. Plant Cell Rep 6:83–89

    CAS  PubMed  Google Scholar 

  • Yamada T, Teraishi M, Hattori K, Ishimoto M (2001) Transformation of azuki bean by Agrobacterium tumefaciens. Plant Cell Tiss Org Cult 64:47–54

    Article  CAS  Google Scholar 

  • Zeng P, Vadnais DA, Zhang Z, Polacco JC (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean (Glycine max. (L.) Merill). Plant Cell Rep 22:478–482

    Article  CAS  PubMed  Google Scholar 

  • Zeven AC, de Wet JMJ (1982) Dictionary of cultivated plants and their regions of diversity excluding most ornamentals forest trees and lower plants, 2nd edn. Pudoc, Wageningen

    Google Scholar 

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Acknowledgments

Authors are thankful to the Center for Application of Molecular Biology to International Agriculture (CAMBIA) for plasmid pCAMBIA2301 and Dr. P. A. Kumar, NRC on Plant Biotechnology, IARI, New Delhi for providing laboratory facilities for the Southern blot hybridization. PKJ is grateful to Department of Biotechnology, New Delhi for financial support to his laboratory for improvement of grain legumes. MS is thankful to Council of Science and Industrial Research, Department of Biotechnology and Department of Science and Technology, New Delhi for research fellowships.

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  1. Centre for Biotechnology, M. D. University, Rohtak, 124001, India

    Manish Sainger, Darshna Chaudhary, Savita Dahiya & Pawan K. Jaiwal

  2. Department of Zoology, M. D. University, Rohtak, 124001, India

    Ranjana Jaiwal

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  1. Manish Sainger
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  2. Darshna Chaudhary
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Correspondence to Pawan K. Jaiwal.

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Sainger, M., Chaudhary, D., Dahiya, S. et al. Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper). Physiol Mol Biol Plants 21, 505–517 (2015). https://doi.org/10.1007/s12298-015-0315-1

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