Abstract
The examinations of conditions for establishing a variety independent Agrobacterium-mediated transformation procedure for wheat are preferable since many of cultivars and breeding lines remain recalcitrant for biotechnological manipulation, mainly due to low efficiency of plant regeneration in vitro, which is highly genotype specific. This paper describes and discusses an improved protocol for enhanced and low-genotype dependent Agrobacterium-mediated transformation using a super-binary vector LBA4404/pTOK233 carrying reporter gus-intron gene and hygromycin (hpt) and kanamicyn (nptII) selectable marker genes. The protocol was optimized on highly responsive common wheat cv. Vesna. Transient expression monitored by the gus-intron on explants after 3, 6 and 25 days of co-cultivation, followed by GUS expression and hygromycin resistance in whole plants indicated the protocol including a co-cultivation of freshly isolated immature embryos in the presence of ascorbic acid, and acetosyringone added only in the bacteria-containing infection medium combined with a delayed and stepwise increasing hygromycin B selection procedure significantly enhanced the transformation efficiency in cv. Vesna that exceed 7% of treated explants from previously 0.41%. Explant pre-cultivation did not additionally improve transformation efficiency. The optimized protocol was successful in evoking satisfactory transformation efficiencies from 3.6% to 10.8% in 5 less-responsive wheat genotypes. All 57 T0 hygromycin-resistant and GUS-positive lines were phenotypically normal and fertile. Therefore, the conditions employed in this study may serve as a base to facilitate the transformation in other, particularly recalcitrant wheat cultivars.
Similar content being viewed by others
References
Ahsan N., Lee S-H., Lee D-G., Anisuzzaman M., Alam M.F., Yoon H-S., Choi M-S., Yang J-K. & Lee B-H. 2007. The effects of wounding type, preculture, infection method and cocultivation temperature on the Agrobacterium-mediated gene transfer in tomatoes. Ann. Appl. Biol. 151: 363–372.
Bhalla P.L. 2006. Genetic engineering of wheat—current challenges and opportunities. Trends Biotechnol. 24: 305–311
Cheng M., Fry J.E., Pang S., Zhou H., Hironaka C.M., Duncan D.R., Conner T.W. & Wan Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115: 971–980.
Cheng M., Lowe B.A., Spencer T.M., Ye X. & Armstrong C.L. 2004. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev. Biol. Plant 40: 31–45.
Chilton M.D., Currier T.C., Farrand S.K., Bendich A.J., Gordon M.P. & Nester E.W. 1974. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc. Natl. Acad. Sci. USA 71: 3672–3676.
Costa M.S., Miguel C. & Oliveira M.M. 2006. An improved selection strategy and the use of acetosyringone in shoot induction medium increase almond transformation efficiency by 100-fold. Plant Cell Tiss. Organ Cult. 85: 205–209.
Dan Y. 2008. Biological functions of antioxidants in plant transformation. In Vitro Cell Dev. Biol. Plant 44: 149–161.
Dutt M. & Grosser J.W. 2009. Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tiss. Organ Cult. 98: 331–340.
Enríquez-Obregón G.A., Vázquez-Padrón R.I., Prieto-Samsónov D.L., Pérez M. & Selman-Housein G. 1997. Genetic transformation of sugarcane by Agrobacterium tumefaciens using antioxidants compounds. Biotecnología Aplicada 14: 169–174.
Enríquez-Obregón G.A., Prieto-Samsónov D.L., de la Riva G.A., Pérez M., Selman-Housein G. & Vázquez-Padrón R.I. 1999. Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an anti-necrotic treatment. Plant Cell Tiss. Organ Cult. 59: 159–168.
Frame B.R., Shou H., Chikwamba R.K., Zhang Z., Xiang C., Fonger T.M., Pegg S.E.K., Li B., Nettleton D.S., Pei D. & Wang K. 2002. Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol. 129: 13–22.
Hiei Y., Ohta S., Komari T. & Kumashiro T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the TDNA. Plant J. 6: 271–282.
Hu T., Metz S., Chay C., Zhou H. P., Biest N., Chen G., Cheng M., Feng X., Radionenko M., Lu F. & Fry J. 2003. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep. 21: 1010–1019.
Iser M., Feeting S., Scheyhing F., Viertel K. & Hess D. 1999. Genotype-dependent stable genetic transformation in german spring wheat varieties selected for high regeneration potential. J. Plant Physiol. 154: 509–516.
Janakiraman V., Steinau M., McCoy S.B. & Trick H.N. 2002. Recent advances in wheat transformation. In Vitro Cell Dev. Biol. Plant 38: 404–414.
Jefferson R.A., Kava T.A. & Bevan M.W. 1987. GUS fusion: β-glucuronidase, a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.
Jones H.D., Doherty A. & Wu H. 2005. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1: 5.
Li J., Ye X., An B., Du L. & Xu H. 2012. Genetic transformation of wheat: current status and future prospects. Plant Biotechnol. Rep. 6: 183–193.
Mitić N., Nikolić R. & Nešković M. 1999. Somatic embryogenesis and plant regeneration from immature embryos of five wheat cultivars and their reciprocal hybrids. Arch. Biol. Sci. (Belgrade) 51: 99–104.
Mitić N., Nikolić R., Ninković S., Miljuš-Djukić J. & Nešković M. 2004. Agrobacterium-mediated transformation and plant regeneration of Triticum aestivum L. Biol. Plant. 48: 179–184.
Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
Murray M.G. & Thompson W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321–4325.
Nandakumar R., Chen L. & Rogers S.M.D. 2007. A stable and reproducible transformation system for the wetland monocot Juncus accuminatus (bulrush) mediated by Agrobacterium tumefaciens. In Vitro Cell Dev. Biol. Plant 43: 187–194.
Nehra N.S., Chibbar R.N., Leung N., Caswell K., Mallard C., Steinhauer L., Baga M. & Kartha K.K. 1994. Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with 2 distinct gene constructs. Plant J. 5: 285–297.
Ohta S., Mita S., Hattori T. & Nakamura K. 1990. Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Physiol. 31: 805–813.
Olhoft P.M., Flagel L.E., Donovan C.M. & Somers D.A. 2003. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216: 723–735.
Papadakis A.K., Siminis C.I. & Roubelakis-Angelakis K.A. 2001. Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts. Plant Physiol. 126: 434–444.
Parrott D.L., Anderson A.J. & Carman J.G. 2002. Agrobacterium induces plant cell death in wheat (Triticum aestivum L.). Physiol. Mol. Plant Pathol. 60: 59–69.
Pastori G.M., Wilkinson M.D., Steele S.H., Sparks C.A., Jones H.D. & Parry M.A.J. 2001. Age-dependent transformation frequency in elite wheat varieties. J. Exp. Bot. 52: 857–863.
Patnaik D., Vishnudasan D. & Khurna P. 2006. Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum. Curr. Sci. 91: 307–317.
Santra M., Ankrah N., Santra D.K. & Kidwell K.K. 2012. An improved wheat microspore culture technique for the production of doubled haploid plants. Crop Sci. 52: 2314–2320.
Shrawat A.K. & Lörz H. 2006. Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers. Plant Biotechnol. 4: 575–603.
Tao L.L., Yin G.X., Du L.P., Shi Z.Y., She M.Y., Xu H.J. & Ye X.G. 2011. Improvement of plant regeneration from immature embryos of wheat infected by Agrobacterium tumefaciens. Agric. Sci. China 10: 317–326.
Vasil V., Castillo A.M., Fromm M.E. & Vasil I.K. 1992. Herbicide resistant fertile transgenic wheat plants obtained by micro-projectile bombardment of regenerable embryogenic callus. Nat. Biotechnol. 10: 667–674.
Weeks J.T., Anderson O.D. & Blechl A.E. 1993. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.) Plant Physiol. 102: 1077–1084.
Weir B., Gu X., Wang M., Upadhyaya N., Elliott A.R. & Brettell R.I.S. 2001. Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aus. J. Plant Physiol. 28: 807–818.
Wu H., Sparks C., Amoah B. & Jones H.D. 2003. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep. 21: 659–668.
Xu X., Xie G., He L., Zhang J., Xu X., Qian R., Liang G. & Liu J-H. 2013. Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callusderived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco. Plant Cell Tiss. Organ Cult. 114: 161–169.
Yao Q., Cong L., He G., Chang J., Li K. & Yang G. 2007. Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency. Mol. Biol. Rep. 34: 61–67.
Yuan Z.C., Edlind M.P., Liu P., Saenkham P., Banta L.M., Wise A.A., Ronzone E., Binns A.N., Kerr K. & Nester E.W. 2007. The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium. Proc. Natl. Acad. Sci. USA 104: 11790–11795.
Zhang L., Rybczynski J., Langerberg W., Mitra A. & French R. 2000. An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration. Plant Cell Rep. 19: 241–250.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mitić, N., Vinterhalter, B., Ninković, S. et al. The procedure providing enhanced Agrobacterium-mediated transformation of wheat. Biologia 69, 1668–1677 (2014). https://doi.org/10.2478/s11756-014-0477-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.2478/s11756-014-0477-2