Plant Cell, Tissue and Organ Culture

, Volume 81, Issue 1, pp 55–63 | Cite as

Development of cotton transgenics with antisense AV2 gene for resistance against cotton leaf curl virus (CLCuD) via Agrobacterium tumefaciens

  • Sanjaya
  • V. V. Satyavathi
  • V. Prasad
  • N. Kirthi
  • S. P. Maiya
  • H. S. Savithri
  • G. Lakshmi. Sita
Article

Abstract

Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l−1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T1 plants were obtained from T0 seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T1 lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance.

Keywords

cross protection gemini virus genetic transformation Gossypium hirsutum virus resistance 

Abbreviations

BA

6-benzylaminopurine

CLCuD

cotton leaf curl virus

GA3

gibberellic acid

IBA

indole-3-butyric acid

NAA

α-naphthalene acetic acid

nptII

gene encoding neomycin phosphotransferase

T-DNA

DNA region transferred to plant genome from the Agrobacterium tumefaciens Ti plasmid

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Asad, S, Haris, WAA, Bashir, A, Xafar, Y, Malik, KA, Malik, NN, Lichtenstein, CP 2003Transgenic tobacco expressing Gemini viral RNAs are resistant to the serious viral pathogen causing cotton leaf curl diseaseArch. Virol.14823412352Google Scholar
  2. Beachy, RN 1997Mechanisms and applications of pathogen-derived resistance in transgenic plantsCurt. Opin. Biotech.8215220Google Scholar
  3. Bendahmane, M, Gronenborn, B 1997Engineering resistance against tomato yellow leaf curl virus (TYLCU) using antisense RNAPlant Mol. Biol.33351357Google Scholar
  4. Cooper, B, Lapidot, M, Heiks, JA, Dodds, JA, Beachy, RN 1995A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibilityVirology206307313Google Scholar
  5. Firoozabady, E, De Boer, DL, Merl, DJ, Halk, EL, Amerson, LN, Raashka, KE, Murray, EE 1987Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plantsPlant Mol. Biol.10105116Google Scholar
  6. Gizant, J, Weintraub, H 1984Inhibition of thymidine kinase gene expression by antisense RNA; a molecular approach to genetic analysisCell3610071015Google Scholar
  7. Golemboski, DB, Lomonosoff, GB, Zaitlin, M 1990Plants transformed with tobacco mosaic virus non-structural gene sequence are resistant to the virusProc. Natl. Acad. Sci. USA8763116315Google Scholar
  8. Gonsalves, D, Chee, P, Provviden, R, Seem, R, Lsightom, J 1992Comparison of coat protein mediated and genetically derived resistance in cucumber mosaic virus under field conditions by vectorsBio/Technology1014661472Google Scholar
  9. Goodwin, J, Chapman, K, Swaney, S, Park, TD, Wernsman, EA, Doughetry, WG 1996Genetic and biochemical dissection of transgenic RNA-mediated virus resistancePlant Cell895105CrossRefPubMedGoogle Scholar
  10. Gould, J, Banister, S, Hasegawa, O, Fahima, M, Smith, R 1991Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissue for transformationPlant Cell Rep.101216Google Scholar
  11. Holsters, M, Waele, D, Depicker, , Messens, E, Van Montagu, M, Schell, J 1978Transfection and transformation of Agrobacterium tumefaciensMol. Gen. Genet.163181187Google Scholar
  12. Kaniewski, WK, Lawson, C, Sammons, B, Haley, L, Hart, J, Delanny, X, Turner, NE 1990Field resistance of transgenic russet Burbank potato to effects of infection by potato virus X and potato virus YBiotechnology6750754Google Scholar
  13. Lapidot, M, Gafny, R, Ding, B, Wolf, S, Lucas, WJ, Beachy, RN 1993A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spreadPlant J.4959970Google Scholar
  14. Lindbho, JA, Dougherty, WG 1992Pathogen derived resistance to potyvirus immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequenceMol. Plant-Micro. Inter.5144153Google Scholar
  15. Majeed, A, Husnain, T, Riazuddin, S 2000Transformation of virus resistant genotype of Gossypium hirsutum L. with pesticidal genePlant Biotechnol.17105110Google Scholar
  16. Mansoor, S, Bedford, I, Pinner, MS, Stanley, J, Markham, PG 1993A whitefly-transmitted Gemini virus associated cotton leaf curl disease in PakistanProc. Natl. Acad. Sci. USA9238793883Google Scholar
  17. Mayshenko, SI, Kodakova, OA, Taliansky, ME, Atabekov, JA 1989Plant virus transport function: complementation by helper viruses is non-specificJ. Gen. Virol.7027512757Google Scholar
  18. McCabe, DE, Martinell, BJ 1993Transformation of elite cotton cultivars via particle bombardment of meristemsBio/Technology11596598Google Scholar
  19. Murashige, T, Skoog, F 1962A revised medium for rapid growth and bioassay with tobacco tissue culturesPhysiol. Plant.15473497Google Scholar
  20. Narayanaswamy K, Savithri HS (2003) Genetically engineered resistance to plant virus. In: Singh RP, Jaiwal PK (eds) Plant Genetic Engineering: Applications and Limitations.Vol. 1. Plant Genetic Engineering; Vol. I. Applications and Limitations (pp. 61–85). Scitech Pub. LLC, USAGoogle Scholar
  21. Nateshan, HM, Muniyappa, V, Swanson, MM, Harrison, BD 1996Host range, vector relations and serological relationships of cotton leaf curl virus in South IndiaAnn. Appl. Biol.126233244Google Scholar
  22. Noris, E, Accotto, GP, Tavazza, R, Brunetti, A, Crespi, S, Tavazza, M 1996Resistance to tomatoyellow leaf curl Gemini virus in Nicotiana benthamiana plants transformed with a truncated viral C1 geneVirology224130138Google Scholar
  23. Paterson, AH, Brubaker, CL, Wendel, JF 1993A rapid method for extraction of cotton (Gossypiumspp.) genomic DNA suitable for RFLP or PCR analysisPlant Mol. Biol. Rep.11122127Google Scholar
  24. Perlak, FJ, Deaton, RW, Armstrong, TA, Fuchs, RL, Sims, SR, Greenplate, JT, Fischhoff, DA 1990Insect resistant cotton plantsBio/Technology8939943Google Scholar
  25. Rajasekharan, K, Grula, JW, Hudspeth, RL, Pofelis, S, Anderson, DM 1996Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthaseMol. Breed.2307319Google Scholar
  26. Rishi, N, Chauhan, MS 1994Appearance of leaf curl diseases of cotton in North IndiaJ. Cotton Res. Dev.8179180Google Scholar
  27. Sambrook, J, Fritsch, EF, Maniatis, T 1989Molecular Cloning: a Laboratory Manual2Cold Spring Harbor Laboratory PressCold Spring Harbor, New York1.31.101Google Scholar
  28. Satyavathi, VV, Prasad, V, Gita, Lakshmi, Lakshmi Sita, G 2002High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciensPlant Sci.162215223Google Scholar
  29. Stark, DM, Beachy, RN 1989Protection against potyvirus in transgenic plants; Evidence for broad-spectrum resistanceBio/Technology712571262Google Scholar
  30. Sunilkumar, G, Rathore, KS 2001Transgenic cotton; factors influencing Agrobacterium-mediated transformation and regenerationMol. Breed.83752Google Scholar
  31. Zapata, Z, Park, SH, El-Zikand, KM, Smith, RH 1999Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apexTheor. Appl. Genet.98252256Google Scholar
  32. Zhou, X, Liu, Y, Robinson, JD, Harrison, BD 1998Four DNA-A variants among Pakistan isolates of cotton leaf curl virus and their affinities to DNA-A and Gemini virus isolates from OkraJ. Gen. Virol.79915923Google Scholar
  33. Ziegler-Graff, V, Guillford, PJ, Baulcombe, DC 1991Tobacco rattle virus RNA-1 29k gene product potentiates viral movement and also affects symptom induction in tobaccoVirology182145155Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Sanjaya
    • 1
  • V. V. Satyavathi
    • 1
  • V. Prasad
    • 1
  • N. Kirthi
    • 2
  • S. P. Maiya
    • 2
  • H. S. Savithri
    • 2
  • G. Lakshmi. Sita
    • 1
  1. 1.Department of Microbiology and Cell BiologyIndian Institute of ScienceBangaloreIndia
  2. 2.Department of BiochemistryIndian Institute of ScienceBangaloreIndia

Personalised recommendations