Advertisement

Standardization of Regeneration, Agrobacterium-Mediated Transformation, and Introduction of Nucleocapsid Gene of Watermelon Bud Necrosis Virus in Watermelon

  • Rakesh Kumar
  • A. Swapana Geetanjali
  • M. Krishnareddy
  • P. K. Jaiwal
  • Bikash MandalEmail author
Research Article
  • 12 Downloads

Abstract

Seven types of explants (proximal cotyledon, distal cotyledon, distal cotyledon leaves, proximal cotyledon leaves, distal hypocotyls, proximal hypocotyls, and basal petioles) of watermelon (Citrullus lanatus) cv. Sugar Baby were tested for their regeneration capacity on MS medium supplemented with different concentrations and combinations of hormones (BAP 0–3 mg/l alone and IAA 0–0.5 mg/l and BAP 0–3 mg/l together). The results showed that the proximal petiole cultured in MS + BAP (3 mg/l) showed the highest percentage of callus induction (28%) and shoot production (24%). The proximal cotyledons cultured in MS + BAP (2 mg/l) + IAA (0.1 mg/l) showed the highest regeneration frequency (76%). Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pBI121 containing the GUS gene (ß-glucuronidase) and kanamycin-resistance gene, nptII, was used to transform petiole explants of watermelon cv. Sugar Baby. Various conditions such as the agrobacterial concentration of OD600 0.6, infection time of 20 min, co-cultivation duration of 2 days and selection at 100 mg/l kanamycin were found as important parameters for the successful Agrobacterium-mediated transformation of watermelon. Further, a transgene construct using the nucleocapsid protein (NP) gene from watermelon bud necrosis virus (genus Tospovirus family Peribunyaviridae) was developed in pBI121 and used to transform watermelon. The successful transformation of petiole explant of watermelon with the GUS gene as well as the NP gene was confirmed by molecular assays, which showed a transformation efficiency of 14.2% and 0.375% with the GUS and NP gene, respectively.

Keywords

Citrullus lanatus Sugar Baby Watermelon bud necrosis virus Regeneration GUS assay Genetic transformation 

Notes

Acknowledgements

The financial support from the Department of Biotechnology, Govt. of India (BT/PR7866/AGR/02/379/2006) and Indian Agricultural Research Institute, New Delhi is thankfully acknowledged.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

Supplementary material

40011_2019_1130_MOESM1_ESM.doc (126 kb)
Supplementary material 1 (DOC 126 kb)

References

  1. 1.
    Holkar SK, Mandal B, Reddy MK, Jain RK (2019) Watermelon bud necrosis orthotospovirus—an emerging constraint in Indian subcontinents: an overview. Crop Prot 117:52–62CrossRefGoogle Scholar
  2. 2.
    Kumar R, Mandal B, Geetanjali AS, Jain RK, Jaiwal PK (2010) Genome organisation and sequence comparison suggest intraspecies incongruence in M RNA of watermelon bud necrosis virus. Arch Virol 155:1361–1365CrossRefGoogle Scholar
  3. 3.
    Tricoli DM, Carney KJ, Russell PF, McMaster JR, Groff DW, Hadden KC, Himmel PF, Hubbard JP, Boeshore ML, Quemada HD (1995) Field evaluation of transgenic squash containing single or multiple coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini mosaic virus. Biotechnology 13:1458–1465Google Scholar
  4. 4.
    Park SM, Lee JS, Jenal S, Jeon SL, Shin YS, Her NH, Lee JH, Lee M, Ryu KH, Yang SG, Harn CH (2005) Transgenic watermelon rootstock resistant to CGMMV (Cucumber green mottle mosaic virus) infection. Plant Cell Rep 24:350–356CrossRefGoogle Scholar
  5. 5.
    Yu TA, Chiang CH, Wu HW, Li CMM, Yang CF, Chen JH, Chen YW, Yeh SD (2011) Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W. Plant Cell Rep 30(3):359–371CrossRefGoogle Scholar
  6. 6.
    Liu L, Gu Q, Ijaz R, Zhang J, Ye Z (2016) Generation of transgenic watermelon resistance to Cucumber mosaic virus facilitated by an effective Agrobacterium-mediated transformation method. Sci Hortic 205:32–38CrossRefGoogle Scholar
  7. 7.
    Hung YC, Chiang CH, Li CM et al (2011) Transgenic watermelon lines expressing the nucleocapsid gene of watermelon silver mottle virus and the role of thiamine in reducing hyperhudricity in regenerated shoots. Plant Cell Tiss Organ Cult 106(1):21–29CrossRefGoogle Scholar
  8. 8.
    Gaba V, Zelcer A, Gal-On A (2004) Cucurbit biotechnology—the importance of virus resistance. In Vitro Cell Dev Biol Plant 40:346–358CrossRefGoogle Scholar
  9. 9.
    Chaturvedi R, Bhatnagar SP (2001) High-frequency shoot regeneration from cotyledon explants of watermelon cv. sugar baby. In Vitro Cell Dev Biol Plant 37:255–258CrossRefGoogle Scholar
  10. 10.
    Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci 77:7347–7351CrossRefGoogle Scholar
  11. 11.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:156–163CrossRefGoogle Scholar
  12. 12.
    Hoagland and Arnon (1950) The water-culture method for growing plants without soil. University of California, College of Agriculture, Agricultural Experiment Station, BerkeleyGoogle Scholar
  13. 13.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J6:3901–3907CrossRefGoogle Scholar
  14. 14.
    Rogers SO, Bendich AJ (1988) Extraction DNA from plant tissues. In: Gelvin SB, Schilperoot RA (eds) Plant molecular biology manual. Kluwer Academic Publishers, Dordrecht, p A6:1–10Google Scholar
  15. 15.
    Yesim Yalcin-Mendi N (2003) Effect of cotyledon age and explant location on regeneration of Cucumis sativus. Biotechnol Biotechnol Equip 17(1):38–43CrossRefGoogle Scholar
  16. 16.
    Srivastava DR, Andrianov VM, Pi’ruzian ES (1989) Tissue culture and plant regeneration of watermelon (Citrullus vulgsris Schrad. Cv. Melitopolski). Plant Cell Rep 8:300–302CrossRefGoogle Scholar
  17. 17.
    Compton ME, Gray DJ, Elmstrom GW (1993) A simple protocol for micropropagating diploid and tetraploid watermelon using shoot-tip explants. Plant Cell Tissue Organ Cult 33:211–217CrossRefGoogle Scholar
  18. 18.
    Choi PS, Soh WY, Kim YS, Yoo OJ, Liu J (1994) Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens. Plant Cell Rep 13:344–348CrossRefGoogle Scholar
  19. 19.
    Suratman F, Huyop F, Wagiran A, Rahmat Z, Ghazali H, Parvee GKA (2010) Cotyledon with hypocotyl segment as an explant for the production of transgenic Citrullus vulgaris Schrad (watermelon) mediated by Agrobacterium tumefaciens. Biotechnology 9(2):106–118CrossRefGoogle Scholar
  20. 20.
    Dong JZ, Jia SR (1991) High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad.). Plant Cell Rep 9:559–562CrossRefGoogle Scholar
  21. 21.
    Li J, Li XM, Qin YG, Tang Y, Wang L, Ma C, Li HX (2011) Optimized system for plant regeneration of watermelon (Citrullus lanatus Thumb.). Afr J Biotechnol 10(48):9760–9765Google Scholar
  22. 22.
    Mohiuddin AKM, Chowdhury MKU, Abdullah Zaliha C, Napis S (1997) Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tissue Organ Cult 51:75–78CrossRefGoogle Scholar
  23. 23.
    Li J, Yi Tang, Qin Yaoguo, Xiaomei Li, Huanxiu Li (2012) Agrobacterium-mediated transformation of watermelon (Citrullus lanatus). Afr J Biotechnol 11(24):6450–6456Google Scholar
  24. 24.
    Hu W, Phillips GC (2001) A combination of overgrowth-control antibiotics improves Agrobacterium tumefaciens-mediated transformation efficiency for cultivated tomato (L. esculentum). In Vitro Cell Dev Biol Plant 37:12–18CrossRefGoogle Scholar
  25. 25.
    Qin YH, Teixeira DA, Silva JA, Bi JH, Hu GB, Zhang SL (2011) Response of in vitro strawberry to antibiotics. Plant Growth Regul 65:183–193CrossRefGoogle Scholar
  26. 26.
    Murray SL, Vuuren RJ, Berger DK, Van Vuuren RJ (1998) Tomato transformation is influenced by acetosyringone and Agrobacterium tumefaciens cell density. J South Afr Soc Hortic Sci 8:60–64Google Scholar
  27. 27.
    Drori E, Altman A (2001) Transformation of tomato with the beta A gene to confer osmotic stress tolerance (glycine–betaine production). Isr J Plant Sci 49:152–153Google Scholar
  28. 28.
    Cho PS (2008) Agrobacterium-mediated transformation in Citrullus lanatus. Biol Plant 52:365–369CrossRefGoogle Scholar
  29. 29.
    Dabauza M, Bordas M, Salvador A, Roig LA, Moreno V (1997) Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis L. Schrad. Plant Cell Rep 16:888–892CrossRefGoogle Scholar
  30. 30.
    Ibrahim IA, Nower AA, Badr El-Den AM, Abd-Elaziem TM (2009) High efficiency plant regeneration and transformation of watermelon (Citrulus lanatus cv. Giza1). Res J Agric Biol Sci 5:689–697Google Scholar
  31. 31.
    Bezirganoglu I, Hwang SY, Shaw JF, Fang TJ (2014) Efficient production of transgenic melon via Agrobacterium-mediated transformation. Genet Mol Res 13:3218–3227CrossRefGoogle Scholar

Copyright information

© The National Academy of Sciences, India 2019

Authors and Affiliations

  1. 1.Division of Plant Pathology, Advanced Centre for Plant VirologyIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.Division of Plant PathologyIndian Institute of Horticultural ResearchBangaloreIndia
  3. 3.Maharishi Dayanand UniversityRohtakIndia
  4. 4.JK Agri Genetic LtdHyderabadIndia
  5. 5.SRM UniversityChennaiIndia

Personalised recommendations