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Assessment of biolistic and Agrobacterium-mediated genetic transformation methods in Cenchrus ciliaris

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Abstract

Cenchrus ciliaris, commonly known as buffelgrass, is an apomictic perennial range grass usually grown in arid/semi-arid regions. Because of the difficulties faced in conventional breeding of this polymorphic polyploid grass, the development of an efficient protocol for genetic transformation is warranted. Such a protocol would enable functional genomic studies needed to elucidate the mechanism of apomixis in this species. The embryogenic calli for genetic transformation experiments were obtained by in vitro culture of the immature inflorescences of buffelgrass cv. IGFRI-3108. Four developmental stages of immature inflorescences were compared for callus induction, and the most mature stage produced most callus. Embryogenic calli were bombarded at three distances, 6 cm (L1), 9 cm (L2), or 12 cm (L3) with a marker gene uid A present in pCAMBIA1301, under the same vacuum (85 kPa) and at constant pressure (900 psi). The Agrobacterium-mediated genetic transformation was also performed using the same construct. Transient and stable expression as well as PCR amplification of the GUS gene was used for comparative analysis as well as for validation of the transformants. Transient GUS expression was present in a significantly higher percentage of bombarded calli (56.33%) than of Agrobacterium treated calli (11.17%), but the number of GUS positive cells per callus was similar. Among the three different bombardment distances, transient GUS expression was highest at L2, but stable GUS expression was highest at L1. Shoot development from the bombarded calli could be accomplished, which failed from the Agrobacterium-mediated transformed calli. Thus, the results indicate that C. ciliaris cv. IGFRI-3108 can be successfully transformed through Biolistic particle bombardment, while Agrobacterium-mediated transformation requires further optimization of transformation protocols.

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Abbreviations

2,4-D:

2,4-dichlorophenoxy acetic acid

BAP:

6-Benzylaminopurine

GUS:

β-Glucuronidase

psi:

Pound per square inch

kPa:

Kilo pascal

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Acknowledgements

Laishram Sundari Devi would like to thank University Grants Commission (UGC), India for providing financial assistance in the form of J.R.F and S.R.F. The authors express sincere gratitude to the Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy (DAE), Mumbai (37(1)/14/10/2016-BRNS/37024) for sponsoring this research work by providing financial assistance. We are also grateful to the University of Delhi for Research and Development grant at the initial stage of research work. We would like to thank Director, National Institute of Genome Research (NIPGR), New Delhi and Prof. Rajesh Tandon, Department of Botany, University of Delhi, for providing access to the CIF and laboratory facilities respectively, to perform particle bombardment experiments.

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Authors and Affiliations

  1. Plant Developmental Biology, Department of Botany, University of Delhi, New Delhi, 110007, India

    Sundari Devi Laishram, Shipra Goyal,  Shashi & Vishnu Bhat

  2. Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, 400085, India

    Vishvas M. Kulkarni

  3. Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India

    Suresh Kumar

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  1. Sundari Devi Laishram
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Correspondence to Vishnu Bhat.

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Laishram, S.D., Goyal, S., Shashi et al. Assessment of biolistic and Agrobacterium-mediated genetic transformation methods in Cenchrus ciliaris. Nucleus 63, 303–312 (2020). https://doi.org/10.1007/s13237-020-00332-1

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  • DOI: https://doi.org/10.1007/s13237-020-00332-1

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