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Agrobacterium-mediated transformation of indica rice cv. ADT 43

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Abstract

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T0 plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l−1 (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l−1 timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.

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Abbreviations

BA:

N6-Benzyladenine

LS:

Linsmaier and Skoog medium

MS:

Murashige and Skoog medium

NAA:

Naphthalene acetic acid

GUS:

(β—glucuronidase)

HPT:

(Hygromycin phosphotransferase)

2,4-D:

2,4-dichlorophenoxyacetic acid

References

  • Amina R, Patrice S (2011) Effects of exogenous application of polyamines on wheat anther cultures. Plant Cell Tiss Organ Cult 105:345–353

    Article  Google Scholar 

  • Amoah BK, Wu H, Sparks CA, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Bot 52:1135–1142

    Article  PubMed  CAS  Google Scholar 

  • Armstrong CL, Rout JR (2001) A novel Agrobacterium-mediated plant transformation method. Int Patent Publ WO01/09302 A2

  • Bais HP, George J, Ravishankar GA (1999) Influence of polyamines on growth of hairy root cultures of witloof chicory (Cichorium intybus L cv. Lucknow Local) and formation of coumarins. J Plant Growth Regul 18:33–37

    Article  PubMed  CAS  Google Scholar 

  • Barro F, Cannell ME, Lazzeri PA, Barcelo P (1998) The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants. Transgenic Res 97:684–695

    CAS  Google Scholar 

  • Birch RG (1997) Plant transformation: problems and strategies for practical application. Ann Rev Plant Physiol Plant Mol Biol 48:297–326

    Article  CAS  Google Scholar 

  • Carlos Henrique S, Carvalho Usha, Zahr B, Gunaratna Nilupa, Anderson Joseph, Halina H, Halina H, Kononowicz AK, Hodges TK, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum; factors that affect transformation efficiency. Gene Mol Biol 27:259–269

    Article  Google Scholar 

  • Cheng M, Fry JE, Pang S, Zhou H, Hironaka C, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    PubMed  CAS  Google Scholar 

  • Chilton MD, Currier T, Farrand S, Benddich A, Gordon M, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown call tumors. Proc Nat Acad USA 71:3672–3676

    Article  CAS  Google Scholar 

  • De Clercq J, Zambre M, Van Montagu M, Dillen W, Angenon G (2002) An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolious A. Gray Plant Cell Rep 21:333–340

    Article  Google Scholar 

  • Diah R, Takehiko H, Eiichi I, Hiroyuki A (2004) Agrobacterium-mediated transformation of javanica rice cv Rojolele. Biosci Biotechnol Biochem 68:1193–1200

    Article  Google Scholar 

  • Enriquez-Obregon GA, Prieto-Samsonov DL, De La Riva GA, Perez M, Selman Housein G, Vazquez-Padron RI (1999) Agrobacteirum–mediated japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tiss Organ Cult 59:159–168

    Article  CAS  Google Scholar 

  • Fits LVD, Deakin EA, Hoge JHC, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502

    Article  Google Scholar 

  • Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321–325

    Article  CAS  Google Scholar 

  • Guo-qing S, Aaron W, James FH (2011) Factors influencing Agrobacterium-mediated transformation of switchgrass cultivars. Plant Cell Tiss Organ Cult doi:10.1007/s11240-011-0056-y

  • Hauptmann RM, Vasil V, Ozias-Akin P, Tabaeizadeh Z, Roger G, Fraley RT, Horch RB, Vasil IR (1988) Evaluation of selectable marker for obtaining stable transformation in the Gramineae. Plant Physiol 86:602–606

    Article  PubMed  CAS  Google Scholar 

  • He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS, Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L var durum cv stewart) with improved efficiency. J Exp Bot 61:1567–1581

    Article  PubMed  CAS  Google Scholar 

  • 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 T-DNA. Plant J 6:271–282

    Article  PubMed  CAS  Google Scholar 

  • Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205–218

    Article  PubMed  CAS  Google Scholar 

  • Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Kaeppler HF, Pederson JF (1996) Media effects on phenotype of callus cultures initiated from photoperiod-insensitive, elite inbred sorghum lines. Maydica 41:83–89

    Google Scholar 

  • Karthikeyan A, Karutha Pandian S, Ramesh M (2009) High frequency plant regeneration from embryogenic callus of a popular indica rice (Oryza sativa L.). Physiol Mol Biol Plants 15:371–375

    Article  Google Scholar 

  • Katiyar SK, Chandel G, Singh P, Pratibha R (1999) Genetic variation and effect of 2, 4- D in in vitro plant regeneration in indica rice cultivars. Oryza 36:254–256

    Google Scholar 

  • Kewei Z, Juan W, Xiaorui H, Aifang Y, Juren Z (2010) Agrobacterium-mediated transformation of shoot apices of Kentucky bluegrass (Poa pratensis L.) and production of transgenic plants carrying a betA gene. Plant Cell Tiss Organ Cult 102:135–143

    Article  Google Scholar 

  • Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436

    PubMed  CAS  Google Scholar 

  • Khush GS (2005) What it will take to feed 5.0 billion rice consumers in 2030. Plant Mol Biol 59:1–6

    Article  PubMed  CAS  Google Scholar 

  • Khush GS, Virk PS (2000) Rice Breeding: achievement and future strategies. Crop Improv 27:115–144

    Google Scholar 

  • Kumar KK, Maruthasalam S, Loganathan M, Sudhakar D, Balasubramanian P (2005) An improved Agrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars. Plant Mol Biol Rep 23:67–73

    Article  CAS  Google Scholar 

  • Lin YJ, Jhang Q (2005) Optimising tissue culture conditions for high efficiency trasformation of indica rice. Plant CelI Rep 23:540–547

    Article  CAS  Google Scholar 

  • Ling HQ, Kirseleit D, Ganal MW (1998) Effect of ticarcillin/potassium clavulanate on callus growth and shoot regeneration in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill). Plant Cell Rep 17:843–847

    Article  CAS  Google Scholar 

  • Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127

    Article  CAS  Google Scholar 

  • Mikami T, Kinoshita T (1988) Genotypic effect on the callus formation from different explants of rice (Oryza sativa L.). Plant Cell Tiss Org Cult 12:311–314

    Article  Google Scholar 

  • Mohammad HK, Hamid R, Zahoor Swati A, Zubeda C (2007) Agrobacterium-mediated transformation to build resistance against bacterial blight in rice. Pak J Bot 39:1285–1292

    Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of higher molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

    Article  PubMed  CAS  Google Scholar 

  • Nauerby B, Billing K, Wyndaele R (1997) Influence of the antibiotic timentin on plant regeneration compared to carbenicillin and cefotaxime in concentration suitable for elimination of Agrobacterium tumefaciens. Plant Sci 123:169–177

    Article  CAS  Google Scholar 

  • Ozawa K (2009) Eatablishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Mol Biol 176:522–527

    CAS  Google Scholar 

  • Park SH, Pinson SMR, Smith RH (1996) T-DNA integration into genomic DNA of rice following Agrobacterium tumefaciens inoculation of isolated shoot apices. Plant Mol Biol 32:1135–1148

    Article  PubMed  CAS  Google Scholar 

  • Rajam MV, Weinstein LH, Galston AW (1985) Prevention of a plant disease by specific inhibition of fungal polyamine biosynthesis. Proc Natl Acad Sci 82:6874–6878

    Article  PubMed  CAS  Google Scholar 

  • Ramana RMV, Behera KS, Baisakh N, Datta SK, Rao GJN (2009) Transgenic indica rice cultivar ‘Swarna’ expressing a potato chymotrypsin inhibitor pin2 gene show enhanced levels of resistance to yellow stem borer. Plant Cell Tiss Organ Cult 99:277–285

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Saharan V, Yadav RC, Yadav NR, Chapagain BP (2004) High frequency plant regeneration from desiccated calli of indica rice. Afr J Biotechnol 3:256–575

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor, New York

    Google Scholar 

  • Selvaraj KN, Ramasamy C (2006) Drought, Agricultural risk and rural income: case of water limiting rice production environment, Tamil Nadu. Econo Politic Wkl 41:2739–2746

    Google Scholar 

  • Sridevi G, Dhandapani M, Veluthambi K (2005) Agrobacterium-mediated transformation of white ponni a non-basmati variety of indica rice (Oryza sativa L.). Curr Sci 88:128–132

    CAS  Google Scholar 

  • Supaporn H, Kanyaratt S, Masahiro M, Ikuo N (2011) Genetic manipulation of Japonica rice using the OsBADH1 gene from indica rice to improve salinity tolerance. Plant Cell Tiss Organ Cult 104:79–89

    Article  Google Scholar 

  • Tang Wei, Luo Hongsong, Ronald Newton J (2004) Effects of antibiotics on the elimination of Agrobacterium tumefaciens from loblolly pine (Pinus taeda) zygotic embryo explants and on transgenic plant regeneration. Plant Cell Tiss Org Cult 70:71–81

    Article  Google Scholar 

  • Terada R, Asao H, Iida A (2004) A large–scale Agrobacterium-mediated transformation procedure with a strong positive–negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Rep 22:653–659

    Article  PubMed  CAS  Google Scholar 

  • Thodsaporn P, Sumontip B, Piyada T, Manit K (2003) Transformation of indica rice (Oryza sativa. L) cv. RD6 mediated by Agrobacterium tumefaciens. Songklanakarin J Sci Technol 26:1–13

    Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium-mediated barley transformation. Plant J 11:1369–1376

    Article  CAS  Google Scholar 

  • Tyagi H, Rajasubramanium S, Dasgupta I (2007) Regeneration and Agrobacterium-mediated transformation of popular indica rice variety ADT39. Curr Sci 93:678–683

    CAS  Google Scholar 

  • Vinod K, Campbell LAM, Keerti S (2011) Rapid recovery- and characterization of transformants following Agrobacterium-mediated T-DNA transfer to sorghum. Plant Cell Tiss Organ Cult 104:137–146

    Article  Google Scholar 

  • Visarada KBRS, Sailaja M, Sarma NP (2002) Effect of callus induction media on morphology of embryogenic calli in rice genotypes. Biol Planta 45:495–502

    Article  Google Scholar 

  • Wernicke W, Milkovitz L (1987) Effect of auxin on mitotic cell cycle in cultured leaf segments at different stages of development in wheat. Plant Physiol 69:16–22

    Article  CAS  Google Scholar 

  • Winans SC (1990) Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant released phenolic compounds, phosphate starvation and acidic growth media. J Bacteriol 172:2433–2438

    PubMed  CAS  Google Scholar 

  • Wu H, Sparks CA, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668

    PubMed  CAS  Google Scholar 

  • Zaidi MA, Narayanan M, Sardana R, Taga I, Postel S, Johns R, McNulty M, Mottiar Y, Mao J, Loit E, Altosaar I (2006) Optimizing tissue culture media for efficient transformation of different indica rice genotypes. Agronomy Res 4:563–575

    Google Scholar 

  • Zhao ZY, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798

    Article  PubMed  CAS  Google Scholar 

  • Zheng SJ, Khrustaleva L, Henken B, Jacobsen E, Kik C, Krens FA (2001) Agrobacterium-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots. Mol Breed 7:101–115

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a research grant from University Grants Commission, Govt. of India, New Delhi (F.No.34—251/2008). A. Karthikeyan is grateful to University Grants Commission for the award of UGC Research Fellowship in Sciences for Meritorious Students (F.4-3/2007(BSR)/11-61/2008). The authors gratefully acknowledge the use of the Bioinformatics Infrastructure facility, Alagappa University funded by the Department of Biotechnology, Ministry of Science and technology, Government of India (BT/BI/04/055/2001).

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  1. Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu, 630 003, India

    A. Karthikeyan, J. Shilpha, S. Karutha Pandian & M. Ramesh

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  1. A. Karthikeyan
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  2. J. Shilpha
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  3. S. Karutha Pandian
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  4. M. Ramesh
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Correspondence to M. Ramesh.

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Karthikeyan, A., Shilpha, J., Karutha Pandian, S. et al. Agrobacterium-mediated transformation of indica rice cv. ADT 43. Plant Cell Tiss Organ Cult 109, 153–165 (2012). https://doi.org/10.1007/s11240-011-0083-8

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