Agrobacterium tumefaciens -mediated in planta transformation strategy for development of transgenics in cotton ( Gossypi
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Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker Karthik Kesiraju1,2 • Pragya Mishra1 • Akansha Bajpai1 • Manju Sharma2 Uma Rao3 • Rohini Sreevathsa1
•
Received: 6 June 2020 / Revised: 5 September 2020 / Accepted: 22 September 2020 Ó Prof. H.S. Srivastava Foundation for Science and Society 2020
Abstract Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8–6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.
& Rohini Sreevathsa [email protected] 1
ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi 110012, India
2
Amity Institute of Biotechnology, Amity University Haryana, Manesar, Gurugram 122413, India
3
ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi 110012, India
Keywords Transgenic cotton Genotype independent In planta transformation Genetic engineering Agrobacterium tumefaciens
Introduction Cotton is an important crop globally and planted in an area of 329.49 million hectares (Directorate of Cotton Development Government of India 2017). Despite the use of extra cultivable land, productivity of cotton is severely affected by several biotic and abiotic factors (Mohapatra and Saha 2019). Thus, cotton improvement by introducing genes for superior traits is the need of the hour. Transgenic technology has emerged as a very effective tool for crop improvement, offering the feasibility to selectively introduce one or more genes for mitigation of various stresses (Birch 1997). In vitro regeneration in cotton has been limited to nonindigenous coker cultivars and its closely related genotypes (Hussain et al. 2009; Zhu et al. 2011) and hence the major obstacle for genetic transformation of cotton. Efforts to improve regenerability in cotton (Aydin et al. 2004; Kumar and Tuli 2004; Wu et al. 2008) we
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