Efficient and rapid system of plant regeneration via protoplast cultures of Fagopyrum esculentum Moench

Abstract

In the present study, a high yield of isolated protoplasts from the agronomically important crop Fagopyrum esculentum was obtained by applying a mixture of cellulase, pectolyase, and driselase. We demonstrated that the yield of morphogenic callus-derived protoplasts was 1 × 106 protoplasts per g of fresh tissue. For hypocotyls used as the protoplast source, the number of released cells was twice lower. The protoplasts, embedded in an agarose matrix and cultured in a modified Kao and Michayluk media supplemented with phytosulfokine, re-enter the cell cycle and start to develop, forming microcalli. The plating efficiency was about 20% in the case of hypocotyl- and morphogenic callus-derived protoplasts. For plant regeneration, the medium was supplemented with different combinations of cytokinin. Somatic embryogenesis and organogenesis occur during the cultivation of the protoplast-derived tissues, depending on the applied protoplast source. For the first time, an effective protoplast-to-plant system for F. esculentum has been developed.

Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum

“…Notably, an optimized protocol for protoplast regeneration in F. tataricum was developed by our group (Zaranek et al, 2023b). We have also recently published an efficient and rapid system of plant regeneration via protoplast cultures of F. esculentum, which opens up the possibility of utilizing the ribonucleoprotein complex in these species, while avoiding the issue of mosaicism (Zaranek et al, 2023a). For F. esculentum, an alternative approach of transformation is also available.…”

“…The plants were grown under field conditions from May to September. The morphogenic calli lines were derived from immature zygotic embryos in the dark at 26 ± 1°C using an RX medium as described previously (Betekhtin et al, 2017;Betekhtin et al, 2019;Zaranek et al, 2023a). The RX medium consisted of B5 mineral salts supplemented with 2.0 mg/L thiamine-HCl, 1.0 mg/L pyridoxine-HCl, 1.0 mg/L nicotinic acid, 2000 mg/L casein hydrolysate, 2.0 mg/L 2.4-D, 0.5 mg/L indolylacetic acid, 0.5 mg/L naphthylacetic acid, 0.2 mg/L kinetin, 2.5% sucrose and 0.8% agar (Gamborg et al, 1968).…”