Skip to main content
SpringerLink
Log in

Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker

  • Research Article
  • Published:
Physiology and Molecular Biology of Plants Aims and scope Submit manuscript

Abstract

Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l−1) and Paramomycin (70 mg l−1) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Anon, (2014). [Online] Available at: http://oryza.com/news/rice-news/usda-post-forecasts-india-my-2014-15-rice-production-104-million-tons [Accessed 1 Jul. 2014].

  • Baisakh N, Datta K, Rashid H, Oliva N, Datta SK (2000) Agrobacterium tumefaciens-mediated transformation of an elite indica rice maintainer line IR68899B with a reconstructed T-DNA carrying multiple genes. Rice Genet Newsl 17:122–125

    Google Scholar 

  • Battraw MJ, Hall TC (1990) Histochemical analysis of CaMV 35S promoter-β-glucuronidase gene expression in transgenic rice plants. Plant Mol Biol 15:527–538

    Article  CAS  PubMed  Google Scholar 

  • Bellini CP, Guerche A, Spielmann J, Goujaud C, Lesaint CM (1989) Genetic analysis of transgenic tobacco plants obtained by liposome-mediated transformation: absence of evidence for the mutagenic effect of inserted sequences in sixty characterized transformants. J Hered 80:361–367

    CAS  Google Scholar 

  • Caplan A, Dekeyser R, van Montagu M (1992) Selectable markers for rice transformation. Methods Enzymol 216:426–441

    Article  CAS  PubMed  Google Scholar 

  • Chan MT, Lee TM, Chang HH (1992) Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Plant Cell Physiol 33:577–583

    CAS  Google Scholar 

  • Chan MT, Chang HH, Ho SL, Tong WF, Yu SM (1993) Agrobacterium-mediated production of transgenic rice plants ex-pressing a chimeric α-amylase promoter/β-glucuronidase gene. Plant Mol Biol 22:491–506

    Article  CAS  PubMed  Google Scholar 

  • Chaudhury A, Maheshwari SC, Tyagi AK (1995) Transient expression of gus gene in intact seed embryos of indica rice after electroporation-mediated gene delivery. Plant Cell Rep 14:215–220

    Article  CAS  PubMed  Google Scholar 

  • Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci 71:3672–3676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chu CC, Wang CC, Sun CS, Msu C, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther cultures of rice through comparative experiments on nitrogen sources. Sci Sinica 18:659–668

    Google Scholar 

  • Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, Muthukrishnan S, Datta SK (1999) Over-expression of cloned rice thaumatin like protein (PR-5) in transgenic rice plants enhances environmental-friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 98:1138–1145

    Article  CAS  Google Scholar 

  • Datta K, Koukolíková-Nicola Z, Baisakh N, Oliva N, Datta SK (2000) Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor Appl Genet 100:832–839

    Article  CAS  Google Scholar 

  • Datta K, Tu JM, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, Datta SK (2001) Enhanced resistance to sheath blight by constitutive expression of infection related rice chitinase in transgenic elite indica rice cultivar. Plant Sci 160:405–414

    Article  CAS  PubMed  Google Scholar 

  • Dekeyser R, Claes B, Marichal M, Montagu MV, Caplan A (1989) Evaluation of selectable markers for rice transformation. Plant Physiol 90:217–223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini preparation: version II. Plant Mol Biol Report 1:19–21

    Article  CAS  Google Scholar 

  • Escandon A, Hahne G (1991) Genotype and composition of culture medium are factors important in the selection for transformed sunflower (Helianthus annus) callus. Physiol Plant 81:367–376

    Article  CAS  Google Scholar 

  • Eustice DC, Wilhelm JM (1984) Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis. Antimicrob Agents Chemother 26(1):53–60

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guerche P, Charbonnier M, Jouanin L, Tourneur C, Paszkowski J, Pelletier G (1987) Direct gene transfer by electroporation in Brassica napus. Plant Sci 52:111–116

    Article  CAS  Google Scholar 

  • Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hyper virulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bac 168:1291–1301

    CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. Embo j 6:3901–3907

    CAS  PubMed  PubMed Central  Google Scholar 

  • KaviKishor PB, Sangam S, Naidu KP (1999) Sodium, potassium, sugar alcohol and proline mediated somatic embryogenesis and plant regeneration in recalcitrant rice callus. Plant Tissue Culture and Biotechnology: Emerging Trends, Proceedings of a symposium held in Hyderabad, India, pp 78–85

  • Khaleda L, Al-Forkan M (2006) Stimulatory effects of casein hydrolysate and proline in in vitro callus induction and plant regeneration from five Deepwater rice (Oryza sativa L.). Biotech 5(3):379–384

    Article  CAS  Google Scholar 

  • Khanna HK, Raina SK (1999) Agrobacterium-mediated transformation of indica rice cultivars using binary and super binary vectors. Aust J Plant Physiol 26:311–324

    Article  CAS  Google Scholar 

  • Lin YJ, Zhang Q (2005) Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell 23:540–547

    Article  CAS  Google Scholar 

  • Manimaran P, Kumar GR, Reddy MR, Jain S, Rao TB, Mangrauthia SK, Sundaram RM, Ravichandran S, Balachandran SM (2013) Infection of early and young callus tissues of indica rice BPT 5204 enhances regeneration and transformation efficiency. Rice Sci 20:415–426

    Article  Google Scholar 

  • Miki B, McHugh S (2004) Selection marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 170:193–232

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • 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 Cell Physiol 31:805–813

    CAS  Google Scholar 

  • Papademetriou MK (2000) Rice production in the Asia-Pacific region: issues and perspectives. Bridging Rice Yield Gap Asia-Pacific Region 220

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

    Article  CAS  PubMed  Google Scholar 

  • Praveen P, Dinesh TK (2012) Modern techniques and agronomic packages for hybrid rice cultivation in India. Adv Agri Bota 4(1):17–21

    Google Scholar 

  • Raineri DM, Bottino P, Gordon MP, Nester EW (1990) Agrobacterium mediated transformation of rice Oryza sativa L. Nat Biotechnol 8:33–38

    Article  CAS  Google Scholar 

  • Saharan V, Yadav RC, Yadav NR, Ram K (2004) Studies on improved agrobacterium mediated transformation in two indica rice (Oryza sativa L) varieties. Afr J Biotechnol 3:572–575

    CAS  Google Scholar 

  • Shin Y-M, Choe G, Shin B, Yi G, Yun PY, Yang K, Lee JS, Kwak SS, Kim KM (2007) Selection of nptII transgenic sweetpotato plants using G418 and paromomycin. J Plant Biol 50(2):206–212

    Article  CAS  Google Scholar 

  • Syaiful BP, Siti NAA, Maheran AA, Sariah M, Othman O (2009) Somatic embryogenesis from scutellar embryo of Oryza sativa L. var. MR 219. Pertanika J Tropl Agri Sci 32:185–194

    Google Scholar 

  • Tariq M, Ali G, Hadi F, Ahmad S, Ali N, Shah AA (2008) Callus induction and in vitro plant regeneration of rice (Oryza sativa L.) under various conditions. Pak J Biol Sci 11:255–259

    Article  CAS  PubMed  Google Scholar 

  • Tu J, Ona I, Zhang Q, Mew TW, Khush GS, Datta SK (1998) Transgenic rice variety ‘IR72’ with Xa21is resistant to bacterial blight. Theor Appl Genet 97:31–36

    Article  CAS  Google Scholar 

  • Tu J, Datta K, Oliva N, Zhang G, Xu C, Khush GS, Zhang Q, Datta Sk (2003) Site-independently integrated transgenes in the elite restorer rice line minghui 63 allow removal of a selectable marker from the gene of interest by self-segregation. Plant Biotechnol J 1:155–165

    Article  CAS  PubMed  Google Scholar 

  • Twyman RM, Stoger E, Kohli A, Capell T, Christou P (2002) Selectable and screenable markers for rice transformation. In: Jackson JF, Linskens HF (eds) Molecular methods of plant analysis, volume 22 (testing for genetic manipulation in plants). Springer-Verlag, NY, pp. 1–18

    Chapter  Google Scholar 

  • Urushibara S, Tozawa Y, Kawagishi-Kobayashi M, Wakasa K (2001) Efficient transformation of suspension-cultured rice cells mediated by Agrobacterium tumefaciens. Breed Sci 51:33–38

    Article  CAS  Google Scholar 

  • US Food and Drug Administration (FDA) (1994) Secondary food additives permitted in food for human consumption: food additives permitted in feed and drinking water of animals; aminoglycoside 3-phosphotransferase II; final rule. Fed Regist 59:26700–26711

    Google Scholar 

  • Vardi A, Bleichman S, Aviv D (1990) Genetic transformation of citrus protoplasts and regeneration of transgenic plants. Plant Sci 69:199–206

    Article  CAS  Google Scholar 

  • Visarda KBRS, Sarma NP (2004) Transformation of indica rice through particle bombardment: factors influencing transient expression and selection. Biol Plant 48(1):25–31

    Article  Google Scholar 

  • Wani SH, Gosal SS (2010) An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding indica rice (Oryza sativa L.) variety PAU 201. New Biotechnol 28(4):418–422

    Article  Google Scholar 

  • Yang R, Zhou Y, Cao Y, Yin Z, Yang L, Li J (2013) The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-waxy gene. Breed Sci 63(2):147–153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Routine procedures for growing rice plants in culture solution. In: Cock J H, Gomez K A. Laboratory Manual for Physiological Studies of Rice. Los Banos, Philippines: IRRI: 61–66.

  • Zhang SP, Song WY, Chen LL, Ruan DL, Taylor N, Ronail P, Beachy R, Na dFauquet C (1998) Transgenic elite indica rice varieties, resistant to Xanthomonas oryzae pv. Orzae. Mol Breed 4: 551–558.

  • Zhao ZY, Gu W, Tishu C, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–233

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are thankful to Mr. Sanjay Gupta, Director JK Agri. Genetics Ltd., Hyderabad, India and all research scientists and staffs of organization. Authors, also acknowledge the partial funding support of Biotechnology Industry Research Assistance Council (BIRAC), New Delhi, India and Department of Biotechnology, Government of India (BIPP grant no. BT/BIPP/0320/07/10)

Author information

Author notes

  1. Debashis Rana

    Present address: Bayer CropScience-Seeds, Bayer (South East Asia) Pte Ltd, Singapore, Singapore

Authors and Affiliations

  1. Department of Biotechnology, Jawaharlal Nehru Technological University (JNTU), Hyderabad, Telangana, 500072, India

    M. Chakraborty & M. Laxmi Narasu

  2. Biotechnology Division, J.K Agri. Genetics Pvt. Ltd., Hyderabad, Telangana, 500016, India

    M. Chakraborty, P. Sairam Reddy, Gaurav Krishna & Debashis Rana

  3. Jacob School of Biotechnology & Bioengineering, Sam Higginbottom Institute of Agriculture, Technology & Sciences (Formerly Allahabad Agricultural Institute), Deemed University, Allahabad, 211007, Uttar Pradesh, India

    Gaurav Krishna

Authors
  1. M. Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Sairam Reddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Laxmi Narasu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gaurav Krishna
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Debashis Rana
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gaurav Krishna.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chakraborty, M., Sairam Reddy, P., Laxmi Narasu, M. et al. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker. Physiol Mol Biol Plants 22, 51–60 (2016). https://doi.org/10.1007/s12298-015-0334-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-015-0334-y

Keywords

Search

Navigation