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Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 105 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in Solanaceae species. [ABSTRACT FROM AUTHOR]

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L−1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L−1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L−1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L−1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa. [ABSTRACT FROM AUTHOR]

This study aims to examine the metabolic discrimination between in vitro grown adventitious roots and the standard medicinal parts of Atractylodes macrocephala. To achieve this goal, firstly, in vitro culture conditions of adventitious roots such as indole-3-butyric acid (IBA) concentrations, types of media, inorganic salt strength of culture medium, and elicitor types and concentrations were optimized. The optimal culture conditions for proliferation of adventitious roots was found to consist of Murashige and Skoog (MS) medium containing 5 mg L−1 IBA. Whole cell extracts from adventitious roots and the standard medicinal parts of A. macrocephala were subjected to Fourier transform infrared spectroscopy (FT-IR). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from FT-IR spectral data showed that adventitious roots and standard medicinal parts were clearly distinguished in the PCA and PLS-DA score plot. Furthermore, the overall metabolite pattern from adventitious roots was changed depending on the dose-dependent manner of chemicals. These results suggest that FT-IR spectroscopy can be applied as an alternative tool for the screening of higher metabolic root lines and for discriminating metabolic similarity between in vitro grown adventitious roots and the standard medicinal parts. In addition, the adventitious roots proliferation system established in this study can be directly applied as an alternative means for the commercial production of A. macrocephala. [ABSTRACT FROM AUTHOR]

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species. [ABSTRACT FROM AUTHOR]