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A modified in planta method of Agrobacterium-mediated transformation enhances the transformation efficiency in safflower (Carthamus tinctorius L.)

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Abstract

Plant transformation has emerged as an important tool to integrate foreign genes in the plant genome to modify the plants for desired traits. Though many techniques of plant transformation are available; getting single copy transgenic events and cost associated remains a big challenge. Thus Agrobacterium-mediated transformation remains the method of choice due to multiple advantages. In the present work a tissue culture free protocol of Agrobacterium-mediated transformation was optimized in safflower, an oil seed crop recalcitrant to transformation. As a proof of concept we selected pCAMBIA2300 gene cassette containing Arabidopsis specific delta 15 desaturase (FAD3) downstream to truncated seed specific promoter beta-conglycinin and optimized tissue culture free protocol of Agrobacterium-mediated transformation using embryos as explants. Addition of silwet L-77, sonication treatment, vacuum infiltration in infection medium and use of paper wicks in co-cultivation period increased the transformation efficiency to 19.3%. Further, success in transformation was confirmed via product accumulation in 21 independent transgenic events wherein oil in transformed seeds showed significant accumulation of alpha-linolenic acid (ALA; 18:3; n3) which is generated from linoleic acid (LA; 18:2; n3) in a FAD3 catalyzed reaction. The present protocol can be utilized to produce transgenic safflower with different desired characters.

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Abbreviations

ALA:

Alpha linolenic acid

BAP:

6-Benzylaminopurine

βCG:

Beta- conglycinin

FAD:

Fatty acid desaturase

GLA:

Gamma linolenic acid

IBA:

Indole butyric acid

LA:

Linoleic acid

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Acknowledgements

Authors are thankful to Department of Biotechnology [DBT; Ref No. (BT/PR4077/AGR/2/832/2011)], India, for providing financial assistance. Arti Rani is thankful to the Department of Science and Technology (DST) for providing fellowship under women scientist [WOS (A); Ref no. SR/WOS-A/LS-580/2013 dated 1.8.2014]. Technical help provided by Dr. Karunakara Alageri Chandrashekhara for GC analysis of the samples and by Mrs Kamla for maintaining the plants in green house is highly acknowledged. Authors are thankful to Dr. Kshitish Acharya, Shodhaka Life Sciences Pvt. Ltd. (www.shodhaka.com), Bengaluru for statistical analysis of the data.

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Authors and Affiliations

  1. Vittal Mallya Scientific Research Foundation, #23, 5th Main J. C. Industrial Layout, Kanakapura main Road, Bangalore, 560062, India

    Arti Rani, Asha Panwar, Manjary Sathe & Anil Kush

Authors
  1. Arti Rani
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  2. Asha Panwar
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  3. Manjary Sathe
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  4. Anil Kush
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Contributions

Dr Arti Rani designed the experiments, performed few experiments and wrote the manuscript. Asha Panwar and Manjary Sathe optimized various parameters of Agrobacterium-mediated in planta transformation. Molecular work such as RT-PCR to screen transformed plants was done by Arti Rani and Asha Panwar. Oil extraction from the transformed seeds for GC analysis was done by Manjary Sathe. Dr Anil Kush did overall supervision of the project work.

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Correspondence to Anil Kush.

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The authors declare that they have no conflict of interest.

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Rani, A., Panwar, A., Sathe, M. et al. A modified in planta method of Agrobacterium-mediated transformation enhances the transformation efficiency in safflower (Carthamus tinctorius L.). J. Plant Biochem. Biotechnol. 27, 272–283 (2018). https://doi.org/10.1007/s13562-017-0437-3

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  • DOI: https://doi.org/10.1007/s13562-017-0437-3

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