Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

Abstract

Physalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production of P. pruinosa fruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration and Agrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing of P. pruinosa. Cotyledon and hypocotyl explants from 7 - 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected with Agrobacterium tumefaciens strain AGL1 containing the pJL33 binary vector that has the green fluorescent protein (GFP) reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on rooting of regenerated shoots on selective medium, GFP visualization, and PCR analysis for the presence of the nptII gene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement of P. pruinosa.