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RETRACTED ARTICLE: High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants

This article was retracted on 02 August 2013

Abstract

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD600 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T0 transgenic plants were generated, representing 3.6% transformation frequency. T0 transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T1 progeny. PCR, RT-PCR, and Southern hybridization analysis of T0 and T1 transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T0 and T1 transgenic chickpea plants was achieved maximum up to 116 ng mg−1 of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

Bt:

Bacillus thuringiensis

CIM:

Callus induction medium

Cry:

Crystal protein

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

MEA:

Mature embryonic axes

MS:

Murashige and Skoog medium

nptII:

Neomycin phosphotransferase

PGR:

Plant growth regulators

uidA :

β-Glucouronidase

References

  • Agarwal S, Singh R, Sanyal I, Amla DV (2008) Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants. Transgenic Res 17:881–896

    PubMed  Article  CAS  Google Scholar 

  • Ahmed K, Khalique F, Malik BA (1998) Modified artificial diet for mass rearing of Chickpea Pod borer, Helicoverpa armigera (H.). Pak J Biol Sci 1:183–187

    Article  Google Scholar 

  • Anwar F, Sharmila P, Saradhi PP (2010) No more recalcitrant: chickpea regeneration and genetic transformation. Afr J Biotechnol 9:782–797

    CAS  Google Scholar 

  • Araújo SS, Duque ASRLA, Santos DMMF, Fevereiro MPS (2004) An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong. Plant Cell Tissue Organ Cult 78:123–131

    Article  Google Scholar 

  • Babaoglu M, Davey MR, Power JB (2000) Genetic engineering of grain legumes: key transformation events. AgBiotechNet 2:1–8

    Google Scholar 

  • Bakhsh A, Rao AQ, Shahid AA, Husnain T, Riazuddin S (2009) Insect resistance and risk assessment studies in advance lines of Bt cotton harboring Cry1Ac and Cry2A genes. Am Eur J Agric Environ Sci 6(1):1–11

    CAS  Google Scholar 

  • Barna KS, Wakhlu AK (1993) Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Rep 12:521–524

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    PubMed  Article  CAS  Google Scholar 

  • Coram TE, Mantri NL, Ford R, Pang ECK (2007) Functional genomics in chickpea: an emerging frontier for molecular-assisted breeding. Funct Plant Biol 34:861–873

    Article  CAS  Google Scholar 

  • Dang W, Wei Z-M (2007) An optimized Agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes. Plant Sci 173:381–389

    Article  CAS  Google Scholar 

  • Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, Datta SK (1998) Constitutive and tissue-specific differential expression of the cry1Ab gene in transgenic rice plants conferring resistance to rice insect pests. Theor Appl Genet 97:20–30

    Article  CAS  Google Scholar 

  • Droste A, Pasquali G, Bodanese-Zanettini MH (2000) Integrated bombardment and Agrobacterium transformation system: an alternative method for soybean transformation. Plant Mol Biol Rep 18:51–59

    Article  CAS  Google Scholar 

  • Fontana GS, Santini L, Caretto S, Frugis G, Mariotti D (1993) Genetic transformation in the grain legume Cicer arietinum L. (chickpea). Plant Cell Rep 12:194–198

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:150–158

    Article  Google Scholar 

  • Glaser JA, Matten SR (2003) Sustainability of insect resistance management strategies for transgenic Bt corn. Biotechnol Adv 22:45–69

    PubMed  Article  CAS  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    PubMed  Article  CAS  Google Scholar 

  • Indurker S, Misra HS, Eapen S (2007) Genetic transformation of chickpea (Cicer arietinum L.) with insecticidal crystal protein gene using particle gun bombardment. Plant Cell Rep 26:755–763

    PubMed  Article  CAS  Google Scholar 

  • Jayanand B, Sudarsanam G, Sharma KK (2003) An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro germinated seedlings. In Vitro Cell Dev Biol 39:171–179

    Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Kar S, Johnson TM, Nayak P, Sen SK (1996) Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.). Plant Cell Rep 16:32–37

    Article  CAS  Google Scholar 

  • Kar S, Basu D, Das S, Ramakrishanan NA, Mukherjee P, Nayak P et al (1997) Expression of cry1Ac gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod borer (Heliothis armigera) larvae. Transgenic Res 6:177–185

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Kumar VD, Kirti PB, Sachan JKS, Chopra VL (1994) Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Cell Rep 13:468–472

    Article  CAS  Google Scholar 

  • Leelavathi S, Sunnichan VG, Kumria (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22(7):465–470

  • Mandaokar AD, Goyal RK, Shukla A, Bisaria S, Bhalla R, Reddy VS, Chaurasia A, Sharma RP, Altosaar I, Kumar PA (2000) Transgenic tomato plants resistant to fruit borer (Helicoverpa armigera Hubner). Crop Prot 19:307–312

    Article  CAS  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 

  • Nhut DT, Hanh NTM, Tuan PQ, Nquyet LTM, Tram NTH, Chinh NC, Nguyen NH, Vinh DN (2006) Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum. Sci Hortic 110:93–97

    Article  Google Scholar 

  • Pandey V, Misra P, Chaturvedi P, Mishra MK, Trivedi PK, Tuli R (2010) Agrobacterium tumefaciens mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Plant Cell Rep 29:133–141

    PubMed  Article  CAS  Google Scholar 

  • Pathak MR, Hamzah RY (2008) An effective method of sonicated assisted Agrobacterium-mediated transformation of chickpea. Plant Cell Tissue Organ Cult 93:65–67

    Article  Google Scholar 

  • Polowick PL, Baliski DS, Mahon JD (2004) Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.); gene integration, expression and inheritance. Plant Cell Rep 23:485–491

    PubMed  Article  CAS  Google Scholar 

  • Ramesh S, Nagadhara D, Pasalu IC, Padma Kumari A, Sarma NP, Reddy VD, Rao KV (2004) Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice. J Biotechnol 111:131–141

    PubMed  Article  CAS  Google Scholar 

  • Sagare AP, Suhasini K, Krishnamurthy KV (1993) Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Cell Rep 12:652–655

    Article  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sanyal I, Singh AK, Kaushik M, Amla DV (2005) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci 168:1135–1146

    Article  CAS  Google Scholar 

  • Sardana R, Dukiandjiev S, Giband M, Cheng X, Cowan K, Sauder C, Altosaar I (1996) Construction and rapid testing of synthetic and modified toxin gene sequences CryIA (b & c) by expression in maize endosperm culture. Plant Cell Rep 15:677–681

    Article  CAS  Google Scholar 

  • Sarmah BK, Moore A, TateW MorvigL, Morton RL, Rees RP et al (2004) Transgenic chickpea seeds expressing high levels of a bean α-amylase inhibitor. Mol Breed 14:73–82

    Article  CAS  Google Scholar 

  • Senthil G, Williamson B, Dinkin RD, Ramsay G (2004) An efficient transformation system for chickpea. Plant Cell Rep 23(5):297–303

    PubMed  Article  CAS  Google Scholar 

  • Sharma KK, Mathur PB, Thorpe TA (2005) Genetic transformation technology: status and problems. In Vitro Cell Dev Biol 4:102–112

    Google Scholar 

  • Singh PK, Kumar M, Chaturvedi CP, Yadav D, Tuli R (2004) Development of a hybrid δ-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura. Transgenic Res 13:397–410

    PubMed  Article  CAS  Google Scholar 

  • Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899

    PubMed  Article  CAS  Google Scholar 

  • Stewart CN Jr, Adang MJ, All JN, Boerma HR, Cardineau G, Tucker D, Parrott WA (1996) Genetic transformation, recovery and characterization of fertile soybean transgenic for a synthetic B. thuringiensis cry1Ac gene. Plant Physiol 112:121–129

    PubMed  Article  CAS  Google Scholar 

  • Suhasini K, Sagare AP, Krishnamurthy KV (1994) Direct somatic embryogenesis from mature embryo axes in chickpea (Cicer arietinum L.). Plant Sci 102:189–194

    Article  CAS  Google Scholar 

  • Suzuki S, Supaibulwatana K, Mii M, Nakano M (2001) Production of transgenic plants of Liliaceous ornamental plant Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated transformation of embryogenic calli. Plant Sci 161:89–97

    Article  CAS  Google Scholar 

  • Tewari-Singh N, Sen J, Kiesecker J, Reddy VS, Jacobsen HJ, Guha-Mukherjee S (2004) Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea. Plant Cell Rep 22:576–583

    PubMed  Article  CAS  Google Scholar 

  • Trick HN, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.] embryogenic suspension culture tissue. Plant Cell Rep 17:482–488

    Article  CAS  Google Scholar 

  • Wiebke B, Ferreira F, Pasquali G, Bodanese-Zanettini MH, Droste A (2006) Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation. Bragantia 65:543–551

    Article  CAS  Google Scholar 

  • Wu S-J, Wang H-H, Li F-F, Chen T-Z, Zhang J, Jiang Y-J, Ding Y, Guo W, Zhang T-Z (2008) Enhanced Agrobacterium-mediated transformation of embryogenic calli of upland cotton via efficient selection and timely subculture of somatic embryos. Plant Mol Biol Rep 26:174–185

    Article  CAS  Google Scholar 

  • Yu TA, Yeh SD, Yang JS (2001) Effects of carbenicillin and cefotaxime on callus growth and somatic embryogenesis from adventitious roots of papaya. Bot Bull Acad Sin Nan 42:281–286

    CAS  Google Scholar 

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Acknowledgments

We are thankful to Council of Scientific and Industrial Research, New Delhi for providing funds and research fellowships. We thankfully acknowledge Prof. I. Altosaar, Department of Biochemistry, University of Ottawa, Ottawa, Canada for providing synthetic modified Bt-cry1Ac gene. This work was carried out under the CSIR Network Project NWP0003 and OLP0031.

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Correspondence to Meenakshi Mehrotra.

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This article has been retracted on the demand of the authors because of error in one figure panel. Consequently, the data is not unambiguous. All of the authors have agreed with the retraction notice and sincerely regret the inconvenience that this retraction causes to PCR and its readership.

The retraction note to this article can be found online at http://dx.doi.org/10.1007/s00299-013-1485-3.

Communicated by P. Kumar.

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Mehrotra, M., Sanyal, I. & Amla, D.V. RETRACTED ARTICLE: High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants. Plant Cell Rep 30, 1603–1616 (2011). https://doi.org/10.1007/s00299-011-1071-5

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Keywords

  • Chickpea
  • Somatic embryogenesis
  • Mature embryonic axes
  • Agrobacterium-mediated transformation
  • Bacillus thuringiensis
  • Insect-resistant transgenic plants