Agrobacterium‐mediated transformation via establishment of stable tissue culture system inZoysia matrella(L.) Merrill ‘Wakaba’

Abstract

Zoysia matrella (L.) Merrill is a perennial C4 warm-season turfgrass grown for landscapes, golf courses, sports fields and recreation parks. To create a new breeding method by genome editing, an efficient genetic transformation system is essential. In this study, we report the efficient protocol of Agrobacterium-mediated transformation through the establishment of a stable tissue culture system for Z. matrella 'Wakaba'.The embryogenic callus was induced from shoot apices of nodal segments incubated in Murashige and Skoog (MS) medium containing 2 mg/l 2,4-dichlorophnoxyacetic acid (2,4-D), 0.1 mg/l 6-benzylaminopurine (BAP) and 5 μM CuSO 4 (MS-DBC).Repeated subculture of compact high-quality callus in MS-DBC medium produced a highly regenerative callus with dense pre-embryogenic clusters, and it was used as a transformation target. Agrobacterium strain EHA105 harboring pANIC8B vector containing the β-glucuronidase gene (GUS) and hygromycin B phosphotransferase gene was used. Agrobacterium-infected calli were cocultured for 5 days with 100 μM acetosyringone and then subjected to selection pressure of 50 mg/l hygromycin. This optimized protocol yielded transformation efficiencies of up to 6.6%. Southern blot analysis verified one to three copies of the GUS gene in different independent transgenic plants. All transgenic plants were morphologically normal, and the GUS expressions were stable. Our optimized in vitro and transgenic system will facilitate the new breeding technology of genome editing in zoysiagrass.

CRISPR/Cas9-mediated knockout of NYC1 gene enhances chlorophyll retention and reduces tillering in Zoysia matrella (L.) Merrill

“…1 a). An all-in-one plasmid vector carrying both the gRNA and the Cas9 expression cassette was introduced into the embryogenic callus through Agrobacterium -mediated transformation system (Ng et al 2023 ) in Z. matrella ‘Wakaba’ (Fig. 1 b).…”

“…Embryogenic callus was induced from the stolon node and yellowish and compact callus with dense poly-embryogenic clusters on the surface were subcultured every 2 weeks. These highly regenerative embryogenic calli were cultured on MS medium supplemented with 2 mg/L 2,4-D, 0.1 mg/L BAP, and 5 μM CuSO 4 (MS-DBC medium) at 27 °C in the dark, according to our established procedure (Ng et al 2023 ).…”

“…Agrobacterium -mediated transformation of Z. matrella ‘Wakaba’ was conducted as described in our previous report (Ng et al 2023 ), with slight modifications. Calli were co-cultivated with the A. tumefaciens for 5 days at 25 °C in the dark.…”

CRISPR/Cas9-mediated knockout of NYC1 gene for the development of stay-green variety in Zoysia matrella (L.) Merrill

“…We also generated a high-quality draft genome sequence of Z. japonica, and draft genome sequences of Z. matrella and Z. paci ca, with 'Wakaba' serving as the representative material for Z. matrella (Tanaka et al 2016a). Additionally, by leveraging the experience of our research group in establishing tissue culture systems for various warm-season grasses (Muguerza et al 2022), we have established an e cient transformation and tissue culture system using this cultivar (Ng et al 2022).…”

“…Agrobacterium -mediated transformation of Z. matrella 'Wakaba' Agrobacterium-mediated transformation of Z. matrella 'Wakaba' was conducted as described in our previous report (Ng et al 2022), with slight modi cations. Calli were co-cultivated with the A. tumefaciens for 5 days at 25°C in the dark.…”