Assessment of biolistic and Agrobacterium -mediated genetic transformation methods in Cenchrus ciliaris
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ORIGINAL ARTICLE
Assessment of biolistic and Agrobacterium‑mediated genetic transformation methods in Cenchrus ciliaris Sundari Devi Laishram1 · Shipra Goyal1 · Shashi1 · Vishvas M. Kulkarni2 · Suresh Kumar3 · Vishnu Bhat1 Received: 25 March 2020 / Accepted: 10 August 2020 © Archana Sharma Foundation of Calcutta 2020
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 Agrobacteriummediated transformation requires further optimization of transformation protocols. Keywords Buffelgrass · Embryogenic calli · Agrobacterium tumefaciens · Biolistic · Genetic transformation · GUS expression · Shoot development Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid BAP 6-Benzylaminopurine GUS β-Glucuronidase psi Pound per square inch kPa Kilo pascal Corresponding Editor: Manoj Prasad. * Vishnu Bhat [email protected] 1
Plant Developmental Biology, Department of Botany, University of Delhi, New Delhi 110007, India
2
Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India
3
Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India
Introduction Cenchrus ciliaris L. (syn. Pennisetum ciliare), commonly known as buffelgrass, belongs to family Poaceae and reproduces predominantly
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