Abstract
Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a destructive fungal disease of banana. Transferring antifungal genes into banana provides a feasible way to control fungal disease, but transformation frequencies for banana are often cultivar and cell line dependent. We investigated an efficient liquid medium selection protocol for an Agrobacterium-mediated transformation system for Furenzhi (Musa spp. AA group). Embryogenic cell suspensions (ECS) of Furenzhi were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring a plasmid containing the endochitinase gene chit42 from Trichoderma harzianum. After co-cultivation, GUS-positive ECS were selected in liquid medium with antibiotics, and compared to semi-solid medium selection. In total, 186 transgenic plantlets were obtained using M2S liquid medium (based on Murashige and Skoog salts) with hygromycin, whereas no transgenic lines were obtained in parallel experiments with semi-solid selection medium. Integration of the transgene was confirmed by PCR, and Southern blots which showed a single copy of the transgene had integrated into the banana genome in three plant lines. Expression of the transgene in regenerated plants was confirmed by ß-glucuronidase histochemical assays and real-time PCR. Both in vitro and ex vivo disease assays showed that the majority of transgenic lines (three of seven) expressing chit42 showed a higher level of resistance to Fusarium wilt (Foc race 4), whereas non-transgenic control plants were susceptible. These results imply a relationship between Foc4 disease resistance and the transcription levels of the transgene in the transgenic clones. This study may offer a promising approach to breed bananas resistant to the fungal disease Fusarium wilt.
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References
Arinaitwe G, Remy S, Strosse H, Swennen R, Sági L (2004) Agrobacterium and particle bombardment-mediated transformation of a wide range of banana cultivars. In: Jain SM, Swennen R (eds) Banana improvement, cellular, molecular biology and induced mutations. Science Publishers Inc., Enfield, pp 351–358
Atkinson HJ, Grimwood S, Johnston K, Green J (2004) Prototype demonstration of transgenic resistance to the nematode Radophylus similes conferred on banana by cystatin. Trans Res 13:135–142
Becker DK, Dugdale B, Smith MK, Harding RM, Dale JL (2000) Genetic transformation of Cavendish banana (Musa spp. AAA group) cv. ‘Grand Nain’ via microprojectile bombardment. Plant Cell Rep 19:229–234
Bolar JP, Norelli JL, Harman GE, Brown SK, Aldwinckle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10:533–543
Bretagne-Sagnard B, Chupeau Y (1996) Selection of transgenic flax plants facilitated by spectinomycin. Transgenic Res 5:131–137
Chakrabarti A, Ganapathi TR, Mukherjee PK, Bapat VA (2003) MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana. Planta 216:587–596
Chhikara S, Chaudhury D, Dhankher OP, Jaiwal PK (2012) Combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic Brassica juncea provides protection against Alternaria brassicae. Plant Cell Tiss Organ Cult 108:83–89
Chong-Pérez B, Reyes M, Rojas L, Ocaña B, Pérez B, Kosky RG, Angenon G (2012) Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of spermidine on transformation efficiency. Plant Cell Tiss Organ Cult 111:79–90
Côte FX, Domergue R, Monmarson S, Schwendiman J, Teisson C, Escalant JV (1996) Embryogenic cell suspensions from the male flower of Musa AAA cv. Grand Nain. Physiol Plant 97:285–290
Das DK, Rahman A (2012) Expression of a rice chitinase gene enhances antifungal response in transgenic litchi (cv. Bedana). Plant Cell Tiss Organ Cult 109:315–325
Esposito S, Colucci MG, Frusciante L, Filippone E, Lorito M, Bressan RA (2000) Antifungal transgenes expression in Petunia hybrida. Acta Hortic 508:157–161
Ganapathi TR, Higgs NS, Balint-Kurti PJ, Arntzen CJ, May GD, Eck JM (2001) Agrobacterium-mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB). Plant Cell Rep 20:157–162
Gentile A, Deng ZN, La Malfa S, Distefano G, Domina F, Vitale A, Polizzi G, Lorito M, Tribulato E (2007) Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breed 126:146–151
Ghag SB, Shekhawat US, Ganapathi TR (2012) Petunia floral defensins with unique prodomains as novel candidates for development of Fusarium wilt resistance in transgenic banana plants. PLoS ONE 7:e39557. doi:10.1371/journal.pone.0039557
Ghosh A, Ganapathi TR, Nath P, Bapat VA (2009) Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA). Plant Cell Tiss Organ Cult 97:131–139
Girhepuje PV, Shinde GB (2011) Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to Fusarium oxysporum f. sp. lycopersici. Plant Cell Tissue Org Cult 105:243–251
Hu CH, Wei YR, Liu K, Yi GJ, Huang BZ, Huang YH (2010) Cloning of chitinase gene and its genetic transformation of wild banana (Musa itinerans Cheesm.), Fenzi Zhiwu Yuzhong. Mol Plant Breed 8:719–724, in Chinese with English Abstract
Huang X, Huang XL, Xiao W, Zhao JT, Dai XM, Chen YF, Li XJ (2007) Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminate cv. Mas (AA) via a liquid co-cultivation system. Plant Cell Rep 26:1755–1762
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Khanna H, Becker D, Kleidon DJ (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
Kiggundu A, Pillay M, Viljoen A, Gold C, Tushemereirwe W, Kunert K (2003) Enhancing banana weevil (Cosmopolites sordidus) resistance by plant genetic modification: a prospective. Afr J Biotechnol 2:369–563
Kovács G, Sági L, Jacon G, Arinaitwe G, Busogoro JP, Thiry SE, Swennen R, Remy S (2013) Expression of a rice chitinase gene in transgenic banana (‘Gros Michel’, AAA genome group) confers resistance to black leaf streak disease. Transgenic Res 22:117–130
Lorito M, Woo SL, Fernadez IG, Colucci G, Harman GE, Pintor-Toro JA, Fillipone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, Scala F (1998) Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860–7865
Marroquin CG, Paduscheck C, Escalant JV, Teisson C (1993) Somatic embryogenesis and plant regeneration through cell suspensions in Musa acuminata. In Vitro Cell Dev Biol–Plant 29:43–46
May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzen CJ (1995) Generation of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation. Biotechnol 13:486–492
Mora AA, Earle ED (2001) Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene. Mol Breed 8:1–9
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nookaraju A, Agrawal DC (2012) Enhanced tolerance of transgenic grapevines expressing chitinase and β-1, 3-glucanase genes to downy mildew. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-012-0166-1
Novak FJ, Afza R, Van Duren M, Perea-Dallos M, Conger BV, Xiaolang T (1989) Somatic embryogenesis and plant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (Musa spp.). Bio/Technol 7:147–158
Pei XW, Chen SK, Wen RM, Ye S, Huang JQ, Zhang YQ, Wang BS, Wang ZX, Jia SR (2005) Creation of transgenic bananas expressing human lysozyme gene for panama wilt resistance. J Integr Plant Biol 47:971–977
Sági L, Remy S, Panis B, Swennen R, Volckaert G (1994) Transient gene expression in electroporated banana (Musa spp.; cv. Bluggoe, ABB group) protoplasts isolated from regenerable embryogenic cell suspensions. Plant Cell Rep 13:262–266
Sági L, Panis B, Remy S, Schoofs H, De Smet K, Swennen R, Cammus B (1995) Genetic transformation of banana (Musa spp.) via particle bombardment. Bio/Technol 13:481–485
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204
Sreeramanan S, Maziah M, Abdullah MP, Sariah M, Xavier R, Nor’Aini MF (2005) Physical and biological parameters affecting transient GUS and GFP expression in banana via particle bombardment. Asia Pacific J Biol Biotechnol 13:35–57
Tripathi L, Tripathi JN, Jd'A H (2005) Agrobacterium-mediated transformation of plantain (Musa spp.) cultivar Agbagba. Afr J Biotechnol 4:1378–1383
Tripathi JN, Muwonge A, Tripathi L (2012) Efficient regeneration and transformation of plantain cv. “Gonja manjaya” (Musa spp. AAB) using embryogenic cell suspensions. In Vitro Cell Dev Biol–Plant 48:216–224
Vishnevetsky J, White TL Jr, Palmateer AJ, Flaishman M, Cohen Y, Elad Y, Velcheva M, Hanania U, Sahar N, Dgani O, Perl A (2011) Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain. Transgenic Res 20:61–72
Wei YR, Huang XL, Li J, Huang X, Li Z, Li XJ (2005a) Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminate cv. Mas (AA). Chinese J Biotechnol 21:58–65
Wei YR, Huang XL, Huang X, Jia L, Xiao W, Li XJ (2005b) The induction of multiple buds and somatic embryogenesis of Musa AAB Silk-Guoshanxiang. Acta Hort 32:414–419
Wei YR, Huang XL, Huang BZ, Yang H, Qiu JS, Xu LB (2006) Establishment of embryogenic cell suspensions and regeneration of Musa itinerans from immature seeds. J Fruit Sci 23:41–45
Wong WC, Jalil M, Abdullah MO, Othman RY, Khalid N (2005) Comparison of β-glucuronidase expression and anatomical localization in bombarded immature embryos of banana cultivar Mas via biolistic transformation. Asia Pacific J Mol Biol Biotechnol 13:15–22
Wu YL, Yi GJ, Peng XX (2010) Rapid screening of Musa species for resistance to Fusarium wilt in an in vitro bioassay. Eur J Plant Pathol 128:409–415
Yang L, Hu CH, Li N, Zhang JY, Yan JW, Deng ZN (2011) Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease. Plant Mol Bio 75:11–23
Yao JL, Cohen D, Atkinson R, Richardson K (1995) Morris B regeneration of transgenic plants from the commercial apple cultivar ‘Royal Gala’. Plant Cell Rep 14:407–412
Yip MK, Lee SW, Su KC, Lin YH, Chen TY, Feng TY (2011) An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt. Plant Biotechnol Rep 5:245–254
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This research was supported by the National Natural Science Fund of China (31000903) and Guangdong Provincial Natural Science Fund (10451064001006326). The authors thank Prof. Deng Ziniu for his critical review of this paper.
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Hu, CH., Wei, YR., Huang, YH. et al. An efficient protocol for the production of chit42 transgenic Furenzhi banana (Musa spp. AA group) resistant to Fusarium oxysporum . In Vitro Cell.Dev.Biol.-Plant 49, 584–592 (2013). https://doi.org/10.1007/s11627-013-9525-9
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DOI: https://doi.org/10.1007/s11627-013-9525-9