Establishment of first protocol of hypocotyl-based regeneration and callus transformation in waterhemp (Amaranthus tuberculatus)

Abstract

Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) is one of the most problematic weeds in the Midwestern agricultural systems in the USA. As a consequence of extensive herbicide selection pressure, many waterhemp populations across the USA evolved resistance to multiple herbicides, leading to limited options for chemical control. Genome editing tools, including CRISPR/Cas9, have great potential in weed science, especially when coupled with a gene drive system for a sustainable weed management. The application of modern biotechnological tools requires availability of an efficient regeneration and genetic transformation system. The objectives of this research were to develop an efficient protocol for successful regeneration of waterhemp via callus culture and optimize an Agrobacterium-mediated transformation method for waterhemp. Hypocotyl explants cultured on callus induction medium (CIM), C6 containing 1.0 mg L⁻¹ kinetin and 4.0 mg L⁻¹ 2,4-D for 2 wk followed by subculture into shoot induction medium (SIM), S8 containing 3.0 mg L⁻¹ BAP, 0.05 mg L⁻¹ 2,4-D, and 0.3 mg L⁻¹ GA₃ for 4 to 6 wk was successful in shoot regeneration. Leaf explants failed to respond to any treatment combinations tested for regeneration. Using the hypocotyl explant–based regeneration protocol optimized in this research, Agrobacterium-mediated transformation was performed using uidA and hpt genes as a proof of concept. Although transgenic plants were not regenerated, molecular characterization of the transformed calluses confirmed the presence of transgenic genes, for example uidA and hpt. This is the first report of the tissue-cultured-based regeneration and Agrobacterium-mediated transformation of hypocotyl derived calluses in waterhemp, which is a step forward in exploring the possibilities of genome editing research in weed science.