Skip to main content
SpringerLink
Log in

Development of Agrobacterium-mediated in planta transformation protocol through coleoptile in rice

  • Research
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Genetic modification of rice is mainly carried out by Agrobacterium-mediated transformation of callus accompanied by tissue culture. It is time consuming, laborious and unapplicable for cultivars unable to induce callus. In this study, we have reported a novel gene transfer protocol that involves pulling out primary leaf from coleoptile and injection of Agrobacterium culture into the empty channel. Out of 25 plants survived after injection of Agrobacterium tumefaciens EHA105 culture harboring pCAMBIA1301-RD29A-AtDREB1A, 8 T0 plants revealed the expected size of around 811 bp corresponding to AtDREB1A gene and Southern blotting analysis on 18 T1 plants suggested introgression of AtDREB1A. 3 T2 lines (7–9, 12–3, 18–6) exhibited accumulation of free proline and soluble sugars, yet increase of chlorophyll content, but decrease of electrolyte leakage and methane dicarboxylic aldehyde under cold stress condition at the vegetative growth stage. Yield components investigation on T2 lines showed earlier heading date and no yield loss compared to wild type plants grown under normal condition. GUS expression analysis and integrated transgene detection in T0 and T1 plants followed by evaluation of cold stress tolerance in T2 lines suggest the advantage of this in planta transformation protocol to obtain transgenic rice.

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

Similar content being viewed by others

Abbreviations

AtDREB :

Arabidopsis thaliana dehydration responsive element binding

hpt :

Hygromycin phosphotransferase

GUS:

Beta-glucuronidase

References

  • Abhishek A, Kumari R, Karjagi CG, Kumar P, Kumar B, Dass S, Kumar RS, Ramteke PW (2016) Tissue culture independent Agrobacterium tumefaciens mediated In Planta transformation method for tropical maize (Zea mays L.). Proc Natl Acad Sci India Sect B Biol Sci 86(2):375–384

    Article  CAS  Google Scholar 

  • Ahmed T, Biswas S, Elias SM, Rahman MS, Tuteja N, Seraj ZI (2018) In Planta transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (Oryza sativa L.) cultivar. Vitro Cell Dev Biol Plant 54(2):154–165

    Article  CAS  Google Scholar 

  • Aminedi R, Dhatwalia D, Jain V, Bhattacharya R (2019) High efficiency in planta transformation of Indian mustard (Brassica juncea) based on spraying of floral buds. Plant Cell Tissue Organ Cult 138(2):229–237

    Article  Google Scholar 

  • Bahari Z, Sazegari S, Niazi A, Afsharifar A (2020) The application of an Agrobacterium-mediated in planta transformation system in a Catharanthus roseus medicinal plant. Czech J Genet Plant Breed 56(1):34–41

    Article  CAS  Google Scholar 

  • Barton MK (2010) Twenty years on: the inner workings of the shoot apical meristem, a developmental dynamo. Dev Biol 341:95–113

    Article  CAS  PubMed  Google Scholar 

  • Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, Datta SK (1998) Constitutive and tissue-specific differential expression of cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theor Appl Genet 97:20–30

    Article  CAS  Google Scholar 

  • Datta K, Baisakh N, Ganguly M, Krishnan S, Yamaguchi-Shinozaki K, Datta SK (2012) Overexpression of arabidopsis and rice stress genes’inducible transcription factor confers drought and salinity tolerance to rice. Plant Biotechnol J 10:579–586

    Article  CAS  PubMed  Google Scholar 

  • Ding Y, Li H, Zhang X, Xie Q, Gong Z, Yang S (2015a) OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis. Dev Cell 32:278–289

    Article  CAS  PubMed  Google Scholar 

  • Ding ZT, Li C, Shi H, Wang H, Wang W (2015b) Pattern of CsICE1 expression under cold or drought treatment and functional verification through analysis of transgenic Arabidopsis. Genet Mol Res 14:11259–11270

    Article  CAS  PubMed  Google Scholar 

  • Duan XQ, Zheng LR, Sun JH, Liu WB, Wang WQ, An HL (2019) Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos. Physiol Mol Biol Plants 25(2):549–560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet ED, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encoded transcription activators that function in drought-, high salt- and cold-responsive gene expression. Plant J 33:751–763

    Article  CAS  PubMed  Google Scholar 

  • Geda CK, Repalli SK, Dash GK, Swain P, Rao GJN (2019) Enhancement of drought tolerance in rice through introgression of arabidopsis DREB1A through transgenic approach. J Rice Res. https://doi.org/10.4172/2375-4338.1000208

    Article  Google Scholar 

  • Guo WF, Wang KY, Wang N, Li J, Li GQ, Liu DH (2018) Rapid and convenient transformation of cotton (Gossypium hirsutum L.) using in planta shoot apex via glyphosate selection. J Integr Agric 17(10):2196–2203

    Article  CAS  Google Scholar 

  • Hussain Z, Ali S, Hayat Z, Zia MA, Iqbal A, Ali GM (2014) Agrobacterium mediated transformation of DREB1A gene for improved drought tolerance in rice cultivars (Oryza sativa L.). Aust J Crop Sci 8(7):1114–1123

    Google Scholar 

  • Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of rice DREB1⁄ CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kalbande BB, Patil AS (2016) Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum). J Genet Eng Biotechnol 14(1):9–18

    Article  PubMed  PubMed Central  Google Scholar 

  • Karkute SG, Krishna R, Ansari WA, Singh B, Singh PM, Singh M, Singh AK (2019) Heterologous expression of the AtDREB1A gene in tomato confers tolerance to chilling stress. Biol Plant 63:268–277

    Article  CAS  Google Scholar 

  • Keshamma E, Rohini S, Rao KS, Madhusudhan B, Udaya Kumar M (2008) Molecular biology and physiology tissue culture-independent In Planta transformation strategy: an Agrobacterium tumefaciens-mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.). J Cotton Sci 12:264–272

    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  CAS  PubMed  Google Scholar 

  • Kumar R, Mamrutha HM, Kaur A, Venkatesh K, Sharma D, Singh GP (2019) Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos. Mol Biol Rep 46(2):1845–1853

    Article  CAS  PubMed  Google Scholar 

  • Latha GM, Raman KV, Lima JM, Pattanayak D, Singh AK, Chinnusamy V, Bansal KC, Rao KRSS, Mohapatra T (2019) Genetic engineering of indica rice with AtDREB1A gene for enhanced abiotic stress tolerance. Plant Cell Tissue Organ Cult 136:173–188

    Article  CAS  Google Scholar 

  • Maher MF, Nasti RA, Vollbrecht M, Straker CG, Clark MD, Voytas DF (2019) Plant gene editing through de novo induction of meristems. Nat Biotechnol. https://doi.org/10.1038/s41587-019-0337-2

    Article  PubMed  PubMed Central  Google Scholar 

  • Man L, Xiang D, Wang L, Zhang W, Wang XD, Qi GC (2017) Stress-responsive gene RsICE1 from Raphanus sativus increases cold tolerance in rice. Protoplasma 254:945–956

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muthurajan R, Ramanathan V, Bansilal SA, Madhuri PS, Shankarrao CN, Rahman H, Kambale R, Nallathambi J, Tamilselvan S, Madasamy P (2021) Controlled over-expression of AtDREB1A enhances tolerance against drought and salinity in rice. Agronomy 11(1):159

    Article  CAS  Google Scholar 

  • Paul S, Gayen D, Datta SK, Datta K (2015) Dissecting root proteome of transgenic rice cultivars unravels metabolic alterations and accumulation of novel stress responsive proteins under drought stress. Plant Sci 234:133–143

    Article  CAS  PubMed  Google Scholar 

  • Phillips RL, Kaeppler SM, Olhof P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl Acad Sci USA 91:5222–5226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ravikumar G, Manimaran P, Voleti SR, Subrahmanyam D, Sundaram RM, Bansal KC, Viraktamath BC, Balachandran SM (2014) Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice. Transgenic Res 23:421–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Razzaq A, Hafiz IA, Mahmood I, Hussalin A (2011) Development of in planta transformation protocol for wheat. Afr J Biotech 10(5):740–750

    CAS  Google Scholar 

  • Ren YJ, Zhao J (2008) Optimization of electroporation parameters for immature embryos of indica Rice (Oryza sativa). Rice Sci 15(1):43–50

    Article  Google Scholar 

  • Shan W, Kuang JF, Lu WJ, Chen JY (2014) Banana fruit NAC transcription factor MaNAC1 is a direct target of MaICE1 and involved in cold stress through interacting with MaCBF1. Plant Cell Environ 37:2116–2127

    Article  CAS  PubMed  Google Scholar 

  • Shiboleth Y, Tzfira T (2012) Agrobacterium-mediated plant genetic transformation. In: Altman A, Smith RH (eds) Introduction to basic procedures in plant biotechnology. Institute of Plant Sciences and Genetics in Agriculture Hebrew University of Jerusalem, Rehovot, Israel. https://doi.org/10.1016/B978-0-12-381466-1.00007-9

  • Wang QY, Guan YC, Wu YR, Chen HL, Chen F, Chu CC (2008) Overexpression of a rice OsDREB1F gene increase salt, drought and low temperature tolerance in both Arabidopsis and rice. Plant Mol Biol 67:589–602

    Article  CAS  PubMed  Google Scholar 

  • Zuraida AR, Rahiniza K, Nurul Hafiza MR, Roowi S, Zamri Z, Subramaniam S (2010) Factors affecting delivery and transient expression of gusA gene in Malaysian indica rice MR 219 callus via biolistic gun system. Afr J Biotech 9(51):8810–8818

    CAS  Google Scholar 

  • Zuraida AR, Zulkifli R, Seman A, Basirun N, Julkifle AL, Zainal Z, Subramaniam S (2011) Preliminary investigations of Agrobacterium-mediated transformation in indica rice MR219 embryogenic callus using gus A gene. Afr J Biotech 10(40):7805–7813

    Article  Google Scholar 

Download references

Acknowledgements

We thank all members who helped with vector construction, microscope sample preparation and photographing, plant regeneration and field works. This research was made possible by financial support of the Scientific and Technical Commission, D P R Korea.

Author information

Authors and Affiliations

  1. Faculty of Life Science, Kim Il Sung University, Pyongyang, Democratic People’s Republic of Korea

    Un-Hyang Ho, Sam-Rang Song, Kang Kim & Tong-Su Ho

  2. The Sci-Tech Complex, Pyongyang, Democratic People’s Republic of Korea

    Yong-Il Choe

  3. Pyongyang Floriculture Institute, Pyongyang, Democratic People’s Republic of Korea

    Myong-Hyok Pak

  4. Branch of Biotechnology, The State Academy of Science, Pyongyang, Democratic People’s Republic of Korea

    Mi-Hyang Kim

Authors
  1. Un-Hyang Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sam-Rang Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong-Il Choe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Myong-Hyok Pak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mi-Hyang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tong-Su Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

UH and YC conceptualized and designed the study, UH and SS conducted the experiments, and MP and MK constructed the vectors, KK, TH and YC analysed the data; UH and SS collected data and wrote the first draft; and all authors contributed substantially to revisions.

Corresponding author

Correspondence to Un-Hyang Ho.

Ethics declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The author declare that they have no conflict of interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 89 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ho, UH., Song, SR., Choe, YI. et al. Development of Agrobacterium-mediated in planta transformation protocol through coleoptile in rice. Transgenic Res 32, 339–349 (2023). https://doi.org/10.1007/s11248-023-00355-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-023-00355-9

Keywords

Search

Navigation