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 to 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 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 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. Key message Methodology for regeneration and transformation of Physalis pruinosa is a key component for the genetic improvement of this underutilized fruit crop for future agricultural production. Keywords AGL1 • Green fluorescent protein • Groundcherry • Kanamycin • Solanaceae Abbreviations GFP Green fluorescent protein GUS Beta-glucuronidase MS Murashige and Skoog nptII Neomycin phosphotranferase II

In vitro regeneration and Agrobacterium-mediated genetic transformation of Dragon’s Head plant (Lallemantia iberica)

“…In addition to nptII selectable marker, which is frequently used to select transformed plants, pXK2FS7 vector contains the GFP reporter gene. One advantage of GFP is that its expression analysis does not involve a destructive assay and visualization of GFP expression is performed with fluorescent microscopy allowing tracking progress throughout different stages 51 . In this study the putative transformant plants were initially screened using GFP signals.…”

A mutant allele of the flowering promoting factor 1 gene at the tomato BRACHYTIC locus reduces plant height with high quality fruit

“…Targeted mutagenesis of BRACHYTIC ( Solyc01g066980 ) using the CRISPR‐Cas9 system for processing tomato cultivar M82 was performed as previously described (Brooks et al, 2014 ; Swartwood & Van Eck, 2019 ; Van Eck et al, 2019 ). Briefly, the binary vectors were constructed through Golden Gate cloning as described by Werner et al ( 2012 ), and introduced into tomato by A. tumefaciens ‐mediated transformation as described by Swartwood and Van Eck ( 2019 ) and Van Eck et al ( 2019 ). 1st generation transgenic plants were transplanted in soil and genotyped to validate potential Cas9‐gRNA‐introduced mutations by Sanger sequencing of PCR products (Xu et al, 2015 ; Table S7).…”

Establishing Physalis as a new Solanaceae model system enables genetic reevaluation of the inflated calyx syndrome

“…Outstanding questions on ICS and our broad interest in Solanaceae biology and agriculture led us several years ago to begin establishing Physalis as a new model system. We developed efficient Agrobacterium-mediated transformation and CRISPR-Cas9 genome editing in the diploid groundcherry species P. grisea, and demonstrated the utility of these tools by mutating orthologs of tomato domestication genes in groundcherry to improve productivity traits (Lemmon et al, 2018;Swartwood & Van Eck, 2019) . More recently, P. grisea was critical in revealing pleiotropic functions of an ancient homeobox gene, and in dissecting evolution of redundancy between duplicated signaling peptide genes controlling stem cell proliferation in the Solanaceae (Hendelman et al, 2021;Kwon et al, 2022) .…”