Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocol for Black Rice (Oryza sativa L. cv. Chakhao Poireiton)

Abstract

Aims: To develop and optimise an efficient in vitro regeneration and Agrobacterium-mediated transformation protocol for black rice (Oryza sativa L. cv. Chakhao poireiton), a nutritionally rich but low-yielding and stress-susceptible variety, thereby establishing a reproducible system for its genetic improvement. Study Design: The study used an experimental design with seven callus-induction treatments (combinations of VACA and 2,4-D) and four shoot-regeneration treatments (various kinetin and kinetin–BAP combinations). Each treatment was replicated three times, with multiple callus samples per replicate. Afterwards, we performed Agrobacterium-mediated genetic transformation using strain LBA4404 to evaluate gene delivery efficiency. Place and Duration of Study: This research was conducted at the Agricultural Biotechnology Laboratory, Faculty of Agricultural Science and Technology, Assam Agricultural University, Jorhat, India. Methodology: Mature black rice embryos were cultured on N6-based media with varying concentrations of 2,4-D (1.0 to 2.5 mg L⁻¹) and VACA (0 to 1.0 g L⁻¹) to assess callus induction. Regeneration potential was evaluated with different combinations of kinetin and BAP in N6RH50 medium. To determine the minimum lethal dose for selecting transformants, hygromycin sensitivity tests were performed at concentrations ranging from 0 to 60 mg L⁻¹. Transformation was conducted using Agrobacterium tumefaciens strain LBA4404 with the binary vector pCAMBIA1304, and successful T-DNA delivery was confirmed through GUS histochemical assays. Results: The combination of 1 g L⁻¹ VACA and 2 mg L⁻¹ 2,4-D yielded the highest callus induction frequency (60%) with friable, embryogenic calli. The maximum shoot regeneration rate (55%) was achieved with both 4 mg L⁻¹ and 5 mg L⁻¹ kinetin4 - –5   mg L⁻¹ kinetin in N6RH50 medium. Hygromycin at 30 mg L⁻¹ was found to be the minimum lethal concentration for selection. Transformed calli exhibited strong blue colouration in GUS assays, confirming successful gene delivery. Conclusion: The optimised regeneration and transformation system provides a reliable, reproducible platform for the genetic improvement of black rice, laying the foundation for future molecular breeding and functional genomics to enhance yield and stress tolerance.