Abstract
One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana.
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Abbreviations
- ECS:
-
Embryogenic cell suspension
- RNAi:
-
RNA Interference
- GI:
-
Geographical indication
- SH:
-
Schenk and Hildebrandt
- pEB:
-
Plasmid Elaya Balan
- MS:
-
Murashige and Skoog
- RT-PCR:
-
Reverse transcription PCR
- BBTD:
-
Banana bunchy top disease
- THB:
-
Transgenic hill banana
- NTHB:
-
Non transformed hill banana
- NRCB:
-
National Research Center for Banana
- gDNA:
-
Genomic DNA
- dpt:
-
Days post transmission
- cDNA:
-
Complementary deoxyribonucleic acid
References
Beetham P, Hafner GJ, Harding RM, Dale JL et al (1997) Two mRNAs are transcribed from Banana bunchy top virus DNA-1. J Gen Virol 78:229–236
Broth W, Perez E, Cheah K, Chen Y, Xie WS, Gaskill D, Khalil S, Hu JS et al (2009) Banana bunchy top virus-resistant transgenic banana plants. Global perspectives on Asian Challenges (September 14–18) Guang Zhou, China. pp 84–85
Burns TM, Harding RM, Dale JL et al (1995) The genome organisation of banana bunchy top virus: analysis of six ssDNA components. J Gen Virol 76(192):1471–1482
Chellappan P, Masona M, Vanitharani R, Taylor N, Fauquet C et al (2004) Broad spectrum resistance to ssDNA viruses associated with transgene induced gene silencing in Cassava. Plant Mol Biol 56:601–611
Cote FX, Domergue R, Monmarson S, Schwendiman J, Teisson C, Escalant JV (1996) Embryogenic cell suspensions from the male flower of Musa AAA cv. Grand naine. Physiol Plant 97:285–290
Elayabalan S, Kalaiponmani K, Kumar KK, Angappan K, Balasubramanian P et al (2008) Genetic Engineering of Hill banana ecotypes against BBTV. In: International conference on Banana, Banana and plantain in Africa: Harnessing international partnerships to increase research impact (5–9th October) Mombasa, Kenya, pp 165–166
Engler D, Gutterson N, Nisbet GS et al (2000) Method of genetically transforming banana plants. United States Patent Application, Publication No. 6:133,035
Ganapathi TR, Higgs NS, Kurti PJB, Arntzen CJ, May MD et al (2001) Agrobacterium–mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB). Plant Cell Rep 20:157–162
Hafner GJ, Harding RM, Dale JL et al (1997) A DNA primer associated with banana bunchy top virus. J Gen Virol 78:479–486
Harding RM, Burns TM, Dale JL et al (1991) Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA. J Gen Virol 72:225–230
Hu JS, Wang M, Sether D, Xie W, Leonhardt KW et al (1996) Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa). Ann Appl Biol 128:55–64
Khanna H, Becker D, Kleidon J, Dale JL et al (2004) Centrifugation assisted Agrobacterium tumefaciens-mediated transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB). Mol Breed 14:239–252
May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzen CJ et al (1995) Generation of transgenic banana (Musa acuminata) plants via Agrobacterium- mediated transformation. Biotechnol J 13:486–492
Njoroge AM, Geijskes RJ, Harding RM, James AP, Tsao TT, Becker DK, Dale JL et al (2009) Towards transgenic resistance to banana bunchy top virus (BBTV) by expression of defective viral reps. Global perspectives on Asian challenges (September 14–18) Guang Zhou, China, p 164
Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T et al (2003) RNAi targeting of DNA virus in plants. Nat Biotechnology 21:131–132
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM et al (2000) Gene expression—total silencing by intron-spliced hairpin RNAs. Nat 407:319–320
Tenllado F, Llave C, Diaz-Ruiz JR et al (2004) RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res 102:85–96
Vanitharani R, Chellappan P, Fauquet CM et al (2003) Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci 100:9632–9636
Wanitchakorn R, Harding RM, Dale JL et al (1997) Banana bunchy top virus DNA-3 encodes the viral coat protein. Arch Virol 142:1673–1680
Wanitchakorn R, Hafner GJ, Harding RM, Dale JL et al (2000) Functional analysis of proteins encoded by Banana bunchy top virus DNA A4 to A6. J Virol 81:299–300
Acknowledgments
This work was financially supported by the Department of Biotechnology, Government of India. Mr. R. Pavalarajan, Planter and the Tamil Nadu hill banana growers association helped in Virupakshi inflorescence collection from lower Pulney hills.
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Elayabalan, S., Kalaiponmani, K., Subramaniam, S. et al. Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. World J Microbiol Biotechnol 29, 589–596 (2013). https://doi.org/10.1007/s11274-012-1214-z
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DOI: https://doi.org/10.1007/s11274-012-1214-z