Cover Image

Standardization of Agrobacterium mediated genetic transformation in Indica rice cv BPT-5204

Anuradha G., Mahendranath Gandikota, Krishnakanth Yadav T., Jagadish P., Raghurami Reddy M., Balachandran S.M.,, Siddiq E.A., Yamini K.N.


The BPT-5204 genotype from Indica rice cultivar (cv) is recalcitrant and showed low transformation frequency compare to japonica rice cv. Here we have optimized the efficient transformation protocol to minimize the time scale and enhance the transformation frequency by altering the key parameters like, acetosyringone (AS) concentration, optical density of bacterial culture and co-cultivation time. Highly proliferated 21 days old Scutellum derived embryogenic calli were infected with Agrobacterium tumefaciens harbouring pCAMBIA2300-Ds-En-Bar binary vector. T-DNA contains tetrameric CaMV35S enhancer elements along with bar gene which embedded between the transposable Ds element. At 0.5 OD600nm of Agrobacterium culture and co-cultivation for 2 days on MS co-cultivation medium containing 100 μM acetosyringone proved to be optimal and attained 6.2 % of transformation efficiency. The transformed calli and regenerated plantlets were proliferated on Murashige and Skoog (MS) medium containing phosphinothricin (PPT). Polymerase chain reaction (PCR) confirmed that intact T-DNA was successfully integrated in the rice genome. This protocol can be employed to develop transgenic rice plants with gain of functional mutagenesis.


Agrobacterium tumefaciens; BPT-5204; Embryogenic calli; Activation tagging

Full Text:



Aananthi N, C.R Anandakumar, R Ushakumari and P Shanthi. “Regeneration study of some indica rice cultivars followed by Agrobacterium-mediated transformation of highly regenerable cultivar Pusa Basmati.” Electron. J. Plant Breed 1(2010): 1249-1256.

Baskaran P and I Dasgupta. “Gene delivery using micro injection of Agrobacterium to embryonic shoot apical meristem of elite indica rice cultivars.” J. Plant Biochem Biotech 21 (2012): 268-274.

Cervera M, JA Pina, J Juarez, L Navarro and L Pena. “Agrobacterium mediated transformation of citrange factors affecting transformation and regeneration.” Plant Cell Reports 18 (1998): 271-278

Dellaporta SL, J Wood, JB Hicks. “A plant DNA mini-preparation Version II.” Plant MolBiol Rep 1 (1983):19-21

Fitch MMM, TCW Leong, Mc Cafferty, HRK, YJ Zhu, PH Moore, D Gonsalves, HS Aldwincle and HJ Atkinson “Improved transformation of Anthurium.” Horticultural Science 46 (2011): 358-364.

Gelvin SB. “Agrobacterium and plant genes involved in T-DNA transfer and integration.” Annu Rev Plant Physiol Plant MolBiol.51 (2000): 223-56

Gelvin SB. “Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool.” Microbiol. Mol. Biol. Rev. 67 (2003):16-23.

Hiei Y, T Komari. “Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed.” Nat Protocols. 3 (2008): 824-834.

Hao ZN, J Wang, LP Wang, RX Tao. “Influences of the disease resistance conferred by the individual transgenes, Pi-d2, Pi-d3 and Xa21, on the transgenic rice plants in yield and grain quality.” African J Biotech. 8 (2009):4845-4848

Jung KH, G An, PC Ronald. “Towards a better bowl of rice: assigning function to tens of thousands of rice genes.” Nat Rev Genet 9 (2008):91-101

Khush GS. “Origin, dispersal, cultivation and variation of rice.” Plant MolBiol 35 (1997):25-34

Krishnaiah K, NRG Varma. “Changing insect pest scenario in the rice ecosystem. A national perspective.” Directorate of Rice Research Rajendranagar, Hyderabad. (2012):2-8

Kavitha G, F Taghipour and F Huyop. “Investigation of factors in optimizing Agrobacterium-mediated gene transfer in Citrulluslanatus cv. Round Dragon.” Journal on Botanical Sciences. 10(3) (2010): 209-216

Kumlehn J, L Serazetdinora, G Hensel, D Becker and H Loerz. “Genetic transformation of barley (Hordeum vulgare L.) via infection of andro genetic pollen culture with Agrobacterum tumefaciens.” Plant Biotechnology 4 (2006): 251-258.

Manimaran P, G Ravikumar , M Raghurami Reddy, S Jain, TB Rao, SK Mangrauthia, RM Sundaram, S Ravichandran, SM Balachandran . “Infection of early and young callus tissues of indica rice BPT-5204 enhances regeneration and transformation efficiency.” Rice Sci 20 (2013):415-426

Manimaran P, S Venkata Reddy, M Moin, M Raghurami Reddy, P Yugandhar, SS Mohanraj, SM Balachandran, PB Kirti. “Activation-tagging in indicarice identifies a novel transcription factor subunit, NF-YC13 associated with salt tolerance.” Sci Rep 7 (2017):9341

Mourgues F, E Chevreau, C Lambert and A Bondt. “Efficient Agrobacterium mediated transformation and recovery of transgenic plants from pear (PyruscommunisL).” Plant Cell Reports.16 (1996): 245-249

Rance IM, W Tian, H Mathews, AD Kochko, RN Beachy, C Fauquet. “Partial desiccation of mature embryo-derived calli, a simple treatment that dramatically enhances the regeneration ability of indica rice.” Plant Cell Rep.13 (1994): 647-651.

Rashid H, S Yokoi, K Toriyama and K Hinata. “Transgenic plant production mediated by Agrobacterium in indica rice.” Plant Cell Rep.15 (1996):727-730.

Skamnioti P, SJ Gurr. “Against the grain: safeguarding rice from rice blast disease.” Trends Biotechnol 27(3) (2009): 141–150

Shimoda N, A Toyoda-Yamamoto, J Nagamine, S Usami, M Katayama, Y Sakagami and Y Machida. “Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides.” Proceedings of National Academic Sciences.87 (1990): 6648-6688

Tripathi R, H Bisht and R Singh. “Effect of Acetosyringone and Callus Age on Transformation for Scutel-lum-Derived Callus of Rice.” International Journal of Pharma and Bio Sciences. 4 (2010): 163-170

Upadhyaya NM, QH Zhu, RS Bhat. “Transposon insertional mutagenesis in rice. In: Pereira A (ed) Plant reverse genetics, methods and protocols, methods in molecular biology.” Springer Science+Business Media. LLC (2011): 147-177

Urushibara S, Y Tozawa, MK Kobayashi and K Wakasa. “Efficient transformation of suspension-cultured rice cells mediated by Agrobacterium tumefaciens.” Breeding Sci. 51 (2001):33-38

Zhao Q, J Jing, G Wang, JH Wang, YY Feng, HW Xing and CF Guan. “Optimization in Agrobacterium-mediated transformation of Anthurium andraeanum using GFP as a reporter.” Electronic Journal of Biotechnology.13 (2010):1-3

Zhang Y. and T Sompong. “Optimization of Certain Parameters for Transformation of indica Rice HomKra Dang Ngah Variety via Agrobacterium-Mediated Transformation.” Kasetsart J. (Nat. Sci.). 49 (2015): 676- 686.



  • There are currently no refbacks.

Copyright (c) 2018 Annals of Plant Sciences

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.