Improved Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merr.] following optimization of culture conditions and mechanical techniques
- 21 Downloads
In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD600) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L−1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L−1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.
KeywordsAgrobacterium tumefaciens Half-seed explants Soybean transformation Regeneration
The authors are very grateful to Dr. Andy Ganapathi (Vice Chancellor, Bharathiar University, Coimbatore, India) for his valuable guidance in improving soybean transformation.
AS conceived and designed the experiments. AH and VK performed the experiments and compiled and analyzed the data. AK, MJ, and AH generated the pictures. AH, VK, and AS prepared the manuscript. SP and MSMJ helped in manuscript revision. All authors read and approved the final manuscript.
This work was supported by National Agriculture Science Fund (NASF) program by the Indian Council of Agricultural Research (ICAR), India.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
- Amoah BK, Wu H, Sparks C, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Biol 52:1135–1142Google Scholar
- An X, Wang B, Liu L, Jiang H, Chen J, Ye S, Chen L, Guo P, Huang X, Peng D (2014) Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmerianivea (L.) gaud]. Mol Biol Rep 41:3257–3269PubMedCrossRefPubMedCentralGoogle Scholar
- Ananthakrishnan G, Xia X, Amutha S, Singer S, Muruganantham M, Yablonsky S, Fischer E, Gaba V (2007) Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro. Plant Cell Rep 26:267–276PubMedCrossRefPubMedCentralGoogle Scholar
- Bechtold N, Pelletier G (1995) In-planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–326Google Scholar
- Bowen BA (1993) Markers for gene transfer. In: Kung S, Wu R (eds) Transgenic Plants: Engineering and Utilization, Academic Press, New York pp 89–123Google Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
- Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. John Wiley and Sons, New YorkGoogle Scholar
- Goodman RN, Novacky AJ (1994) The hypersensitive reaction in plants to pathogens. A resistant phenomenon. APS PRESS, St. Paul, MinnesotaGoogle Scholar
- Gupta S, Gupta S, Bhat V, Gupta MG (2006) Somatic embryogenesis and Agrobacterium-mediated genetic transformation in Indian accessions of Lucerne (Medicago sativa L.). Ind J Biotechnol 5:269–275Google Scholar
- Hada A, Krishnan V, Punjabi M, Basak N, Pandey V, Jeevaraj T, Marathe A, Gupta AK, Jolly M, Kumar A, Dahuja A, Manickavasagam M, Ganapathi A, Sachdev A (2016) Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335. Plant Biotechnol 33:341–350CrossRefGoogle Scholar
- Ismael KA, Antar EN (2014) Establishment of high-efficiency Agrobacterium-mediated transformation conditions of soybean callus. Ind J Biotechnol 13:459–463Google Scholar
- Joao L, Brown A (1993) Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58 CIRIF- R PGSFRI161 in the presence of acetosyringone. Plant Cell Rep 12:422–425Google Scholar
- Kumar B, Talukdar A, Verma K, Girmilla V, Bala I, Lal SK, Pal Singh K, Sapra RL (2014) Screening of soybean [Glycine max (L.) Merr.] genotypes for yellow mosaic virus (YMV) disease resistance and their molecular characterization using RGA and SSRs markers. Aust J Crop Sci 8:27–34Google Scholar
- Kumari S, Krishnan V, Dahuja A, Vinutha T, Jolly M, Sachdev A (2016) A rapid method for optimization of Agrobacterium-mediated transformation of Indian soybean genotypes. Indian J Biochem Biophys 53:218–226Google Scholar
- Kuta DD, Tripathi L (2005) Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. Afr J Biotechnol 4:752–757Google Scholar
- Leelavathi S, Sunnichan SG, Kumria R, Vijaykanth GP, Bhatnagar RK, Reddy VS (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470PubMedCrossRefGoogle Scholar
- Öz MT, Eyidoğan F, Yücel M, Öktem HA (2009) Optimized selection and regeneration conditions for Agrobacterium-mediated transformation of chickpea cotyledonary nodes. Pak J Bot 41:2043–2054Google Scholar
- Raj SK, Singh R, Pandey SK, Singh BP (2005) Agrobacterium-mediated tomato transformation and regeneration of transgenic lines expressing tomato leaf curl virus coat protein gene for resistance against TLCV infection. Curr Sci India 88:1674–1679Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Townsend JA, Thomas LA (1993) An improved method of Agrobacterium-mediated transformation of cultured soybean cells. US Patent WO 94:02620Google Scholar
- Tyagi H, Rajsubramaniam S, Dasgupta I (2007) Regeneration and Agrobacterium-mediated transformation of a popular indica rice variety, ADT39. Curr Sci India 93:678–673Google Scholar
- Wang G, Huang M (2002) Tissue culture and plant regeneration of Cerasus campanulata. J Nanjing Univ 26:73–76Google Scholar
- Weir B, Wang X, Upadhyaya N, Elliot A, Brettell R (2001) Agrobacterium tumefaciens transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aust J Plant Physiol 28:807–818Google Scholar
- Xinping YI, Deyue YU (2006) Transformation of multiple soybean cultivars by infecting cotyledonary-node with Agrobacterium tumefaciens. Afr J Biotechnol 5:1989–1993Google Scholar
- Zhong H, Que Q (2009) Method for transforming soybean (Glycine max). US Patent Number 20090023212Google Scholar
- Zia M, Zarrin RF, Rehman RU, Chaudhary FM (2010) Agrobacterium-mediated transformation of soybean (Glycine max L.): some conditions standardization. Pak J Bot 42:2269–2279Google Scholar