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Development of transgenic broccoli with cryIAa gene for resistance against diamondback moth (Plutella xylostella)

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Abstract

Transgenic broccoli (Brassica oleracea L. var. italica) cv. ‘Solan Green Head’ has been developed by using Agrobacterium tumefaciens strain harboring cryIAa gene for resistance against diamondback moth (Plutella xylostella). By combining the best treatments of 72 h pre-culturing and 48 h co-cultivation time period, a transformation frequency of 4.92 and 13.83% was obtained from cotyledon and hypocotyl explants, respectively. Supplementing the co-cultivation medium with acetosyringone in a concentration of 100 µM enhanced the transformation frequency to 17.92 and 32.11% in cotyledon and hypocotyl explants, respectively. The transgene (cryIAa) integration was confirmed by polymerase chain reaction using gene-specific primers and Southern blot analysis using digoxigenin nonradiolabelled DNA probe. Gene expression in the PCR-positive transgenic events had been confirmed by reverse transcriptase-PCR and quantitative real time-PCR. Insect bioassay proved the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae. To the best of our knowledge, this is the first report of optimization of a highly efficient transformation system and transgenic development in broccoli using cry1Aa gene for insect resistance.

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References

  • Awasthi M, Srivastava DK (2013) Pyramiding of Bt-genes (cry1Ab and cry1Aa) in cauliflower (Brassica oleracea L. var. botrytis) for insect resistance using an Agrobacterium-mediated gene transfer technique. In: Proceedings of National symposium on plant tissue culture and biotechnology for food and nutritional security, Abstract p. 72

  • Bhalla PL, Singh MB (2008) Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat Protoc 3:181–189

    Article  CAS  PubMed  Google Scholar 

  • Chen LFO, Hwang JY, Charng YY, Sun CW, Yang SF (2001) Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation. Mol Breed 7:243–257

    Article  CAS  Google Scholar 

  • Chen LO, Chin HL, Kelkarc SM, Chang YM, Shawa JF (2007) Transgenic broccoli (Brassica oleracea L. var. italica) with antisense chlorophyllase (BOCLH1) delays post harvest yellowing. Plant Sci 174(1):25–31

    Article  CAS  Google Scholar 

  • Costa MS, Miguel CL, Oliveira MM (2006) An improved selection strategy and the use of acetosyringone in shoot induction medium increase almond transformation efficiency by 100-fold. Plant Cell Tiss Org Cult 85:205–209

    Article  Google Scholar 

  • Deng-Xia Y, Lei C, Yu-Mei L, Mu Z, Yang-Yong Z, Zhi-Yuan F, Li-Mei Y (2011) Transformation of cabbage (Brassica oleracea L. var. capitata) with Btcry1Ba3 gene for control of diamondback moth. Agric Sci China 10(11):1693–1700

    Article  CAS  Google Scholar 

  • Dhiman K, Verma S, Srivastava DK (2015) Plant regeneration, genetic transformation and expression of gus gene in broccoli. Veg Sci 41(8):129–134

    Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissues. Focus 12:13–15

    Google Scholar 

  • Gambhir G (2014) Studies on Agrobacterium-mediated insect resistance gene transfer in cabbage (Brassica oleracea L. var. capitata) and molecular analysis of regenerated plantlets. Ph. D. Thesis, Dr YS Parmar University of Horticulture and Forestry, Nauni. Solan (H.P.), India

  • Gaur A (2015) Studies on Agrobacterium-mediated insect resistance gene [cry1A(a)] transfer in cauliflower (Brassica oleracea L. var. botrytis). Thesis PhD, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.), India

  • Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York

    Google Scholar 

  • Husaini AM (2010) Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants. Plant Cell Rep 29:97–110

    Article  CAS  PubMed  Google Scholar 

  • Keshavareddy G, Kumar ARV (2016) Bacillus thuringiensis. In: Omkar (ed) Ecofriendly pest management for food security. Academic Press, London, pp 443–477

    Chapter  Google Scholar 

  • Kumar P, Srivastava DK (2015a) Effect of potent cytokinin thidiazuron (TDZ) on in vitro morphogenic potential of broccoli (Brassica oleracea L. var. italica), an important vegetable crop. Indian J Plant Physiol 20(4):317–323

    Article  CAS  Google Scholar 

  • Kumar P, Srivastava DK (2015b) High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop. Physiol Mol Biol Plants 21(2):279–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar P, Srivastava DK (2016a) Biotechnological advancement in genetic improvement of broccoli (Brassica oleracea L. var. italica), an important vegetable crop: a review. Biotech Lett 38(7):1049–1063

    Article  CAS  Google Scholar 

  • Kumar P, Srivastava DK (2016b) Biotechnological application in in vitro plant regeneration studies of Broccoli (Brassica oleracea l. var. italica), an important vegetable crop: a review. Biotech Lett 38(4):561–571

    Article  CAS  Google Scholar 

  • Kumar P, Gambhir G, Gaur A, Srivastava DK (2015) Molecular analysis of genetic stability in in vitro regenerated plants of broccoli (Brassica oleracea L. var. italica). Curr Sci 109(8):1470–1475

    CAS  Google Scholar 

  • Min BW, Cho YN, Song MJ, Noh TK, Kim BK, Chae WK, Park YS, Choi YD, Harn CH (2007) Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker. Plant Cell Rep 26:337–344

    Article  CAS  PubMed  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 

  • Parmar N, Singh KH, Sharma D, Singh L, Kumar P, Nanjundan J, Khan YJ, Chauhan DK, Thakur AK (2017) Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review. 3 Biotech 7:239. https://doi.org/10.1007/s13205-017-0870-y

    Article  PubMed  PubMed Central  Google Scholar 

  • Rafat A, Aziz MA, Rhashid AA, Abdullah SNA, Kamaladini H, Sirchi MHT, Javadi MB (2010) Optimization of Agrobacterium tumefaciens-mediated transformation and shoot regeneration after co-cultivation of cabbage (Brassica oleracea subsp. capitata) cv. KY cross with At hsp101 gene. Sci Hortic 124:1–8

    Article  CAS  Google Scholar 

  • Ravanfar SA, Aziz MA (2014) Shoot tip regeneration and optimization of Agrobacterium tumefaciens-mediated transformation of Broccoli (Brassica oleracea var. italica) cv. Green Marvel. Plant Biotech Rep. https://doi.org/10.1007/s11816-014-0340-5

    Article  Google Scholar 

  • Ravanfar SA, Aziz MA, Rashid AA, Shahida S (2014) In vitro adventitious shoot regeneration from cotyledon explant of Brassica oleracea subsp. italica and Brassica oleracea subsp. capitata using TDZ and NAA. Pakistan J Bot 46(1):329–335

    CAS  Google Scholar 

  • Song GQ, Walworth AE, Hancock JF (2012) Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”. In Vitro Cell Dev Biol Plant 48:383–389

    Article  CAS  Google Scholar 

  • Verma H, Kumar P, Gambhir G, Srivastava DK (2014) Agrobacterium-mediated transformation of broccoli. Crop improv 41(2):139–147

    Google Scholar 

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Acknowledgements

Authors are highly thankful to the Project Director, National Research Centre on Plant Biotechnology, New Delhi, for providing Agrobacterium strain and for allowing to do molecular analysis work. The senior author (PK) thankfully acknowledges Department of Science and Technology, New Delhi, for providing INSPIRE fellowship during doctoral studies.

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Authors and Affiliations

  1. Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, 173 230, India

    Pankaj Kumar, Ayesh Gaur, Krishan C. Sharma & Dinesh K. Srivastava

  2. ICAR-Indian Agricultural Research Institute, New Delhi, 110 012, India

    Geetika Gambhir

  3. ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur, Rajasthan, 321 303, India

    Ajay K. Thakur

Authors
  1. Pankaj Kumar
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  2. Geetika Gambhir
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  3. Ayesh Gaur
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  4. Krishan C. Sharma
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  5. Ajay K. Thakur
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  6. Dinesh K. Srivastava
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Correspondence to Pankaj Kumar.

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Kumar, P., Gambhir, G., Gaur, A. et al. Development of transgenic broccoli with cryIAa gene for resistance against diamondback moth (Plutella xylostella). 3 Biotech 8, 299 (2018). https://doi.org/10.1007/s13205-018-1316-x

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