EFFICIENT IN PLANTA GENETIC TRANSFORMATION THROUGH Agrobacterium-MEDIATED FLORAL-DIP TECHNIQUE IN INDIAN FLAX
SOURAV DUTTA *
Biotechnology Department, Techno India University, EM- 4/1, EM Block, Sector V, Salt Lake, Kolkata 700 091, West Bengal, India and ICAR-Central Research Institute for Jute and Allied Fibres, Barrackpore 700120, West Bengal, India.
PRANIT KUMAR MUKHERJEE
ICAR-Central Research Institute for Jute and Allied Fibres, Barrackpore 700120, West Bengal, India.
ABHIJIT MITRA
Department of Marine Science, University of Calcutta, 35 BC Road, Kolkata 700019, India.
SUFIA ZAMAN
Biotechnology Department, Techno India University, EM- 4/1, EM Block, Sector V, Salt Lake, Kolkata 700 091, West Bengal, India.
ASIT B. MANDAL
ICAR-Central Research Institute for Jute and Allied Fibres, Barrackpore 700120, West Bengal, India.
*Author to whom correspondence should be addressed.
Abstract
An efficient in planta genetic transformation technique through Agrobacterium-mediated floral-dipping involving an Indian flax (Linum usitatissimum L.) var. Tiara has been developed. The technique is very simple, reproducible and found to be efficient in development of transgenic lines. Floral buds of Tiara were infected with Agrobacterium strain LBA 4404 harbouring cry1A(b) in a binary vector pCAMBIA1301. Agrobacterium cells at an O.D.600 of 0.5 in infiltration medium (IM) containing 8.0% sucrose and 0.1% Tween 20 were used for floral-dip transformation. Transgenic status of the transformants were established by assessing transgene expression at transient level through GUS histochemical assay and PCR at T1 generation and stable integration of the intruded transgene at genomic level was confirmed through PCR and semi-quantitative RT-PCR analysis at T2 generation. Successful stable integration of cry1A(b) transgene was observed in 19 lines at T2 generation. Progenies of those lines deemed to impart tolerance to combat attack of serious lepidopteran insect pests in Indian flax. This technique prospects amply in augmenting the quality and productivity of Indian flax, especially at the interface of discernible global climate changes and increase of resilient insect pests damage to this crop of economic importance.
Keywords: Indian flax (Linum usitatissimum L.), Agrobacterium-mediated genetic transformation, Lepidopteran insect pest management, floral-dipping, Gus histochemical assay, semi-quantitative RT- PCR