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.

Tiller development is an important agronomic trait in plant architecture and grain yield. Many plant hormones regulate axillary meristem formation, including bud outgrowth for shoot branching. However, the molecular mechanism underlying the brassinosteroid (BR) in tiller development is not yet well known. Therefore, in this study, we identified and characterized two novel T-DNA insertion mutants, osgras19 and osgras32, which showed the typical BR-deficient phenotype, such as fewer tiller numbers, dark-green leaves, and semi-dwarf phenotypes. Double knockout mutants, osgras19 osgras32, were then generated by crossing, and they showed similar phenotypic traits of each single mutant. Both OsGRAS19 and OsGRAS32 encoded the GRAS family proteins and were localized in the nucleus. We also confirmed that OsGRAS19 and OsGRAS32 did not directly interact with each other; however, OsGRAS19 interacted with MOC1 and SMALL ORGAN SIZE1 (SMOS1), an auxin-regulated APETALA2-type transcription factor, in yeast. Thus, we proposed OsGRAS19 as a component of the complex on the auxin-BR signaling pathway and plays role in the tiller development in rice.

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Chloroplasts are essential plant organelles that play important roles in photosynthesis and are involved in many fundamental metabolic pathways. Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development during chloroplast differentiation. In this study, we isolated and characterized T-DNA-tagged rice mutant (prpl13), which exhibited albino seedling lethality. PRPL13 is a nuclear gene encoding the 50S ribosomal protein L13, which is localized in chloroplasts. Transmission electronic microscopy analysis showed abnormal plastid development in the thylakoids of the chloroplasts of the prpl13 mutant seedlings. Chlorophyll and carotenoid contents were also significantly reduced in the leaves of the mutants. Quantitative reverse transcription-PCR analysis revealed that the prpl13 mutations altered the expression levels of genes involved in photosynthesis and chloroplast development. Thus, our data indicate that nuclear-encoded PRPL13 plays an important role in chloroplast development in rice.

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