Milk thistle (Silybum marianum (L.) Gaertn.), a valuable medicinal plant with economic importance, serves as a source of both therapeutic compounds and vegetable oil for human use. Genome editing techniques have been widely applied to improve key agronomic traits in various species. Therefore, this study aims to develop an efficient method for seed sterilization and protoplast isolation in milk thistle. For seed sterilization, seed treated with hydrogen peroxide for either 4 hours or 7 hours with ethanol and detergent showed more sufficient to sterilize seed of milk thistle for in vitro growth than ethanol, sodium hypochlorite, and chlorine gas. For protoplast isolation in milk thistle, high-yielding, viable protoplasts were successfully isolated from the leaves of 21-day-old plants using a 2 hours enzymatic treatment containing 2% Viscozyme^® L, 1% Celluclast^® 1.5 L, and 1% Pectinex^® Ultra SP-L. Additionally, transient expression of green fluorescent protein was observed following polyethylene glycol-mediated transfection. Therefore, the seed sterilization and protoplast isolation methods developed in this study can facilitate in vitro culture, gene function analysis, and genome editing aimed at improving agronomic traits in milk thistle.

Adzuki bean (Vigna angularis [Willd.] Ohwi & Ohashi) has recently become widely cultivated in Asia. Generally, it is sensitive to flooding stress. Wild relatives of adzuki bean have shown higher tolerances to abiotic stressors than cultivars, but their responses to flooding are not well understood. In this study, the waterlogging tolerance levels of 202 accessions from two cultivated Vigna species including V. angularis var. angularis (cultivated adzuki bean), V. umbellata (rice bean), and two wild ones consisting of V. angularis var. nipponensis (wild adzuki bean), and V. nakashimae, were evaluated at the early vegetative stage using a foliar damage score. Additionally, the responses of 119 accessions from V. angularis var. nipponesis bean and V. nakashimae to submergence at the germination stage were evaluated using germination-related traits. Among the tested species, cultivated adzuki bean showed the lowest tolerance to waterlogging, while V. nakashimae showed the greatest. At the germination stage, seeds of V. nakashimae were more tolerant of submergence than seeds of V. angularis var. nipponesis, exhibiting significantly higher normal seedling indexes. There was no significant correlation between the early vegetative-stage index and either germination-stage index for both V. angularis var. nipponensis and V. nakashimae, suggesting that the genetic bases regulating flooding stress responses at the two growth stages differ. However, we identified four V. nakashimae accessions that exhibited tolerant or very tolerant responses to flooding at both stages, suggesting their potential for use in breeding flood-tolerant adzuki bean varieties.