Maesa perlarius is a medicinal plant that produces maesabalides, which possess selective and strong anti-leishmania activity. In this study, M. perlarius plants were regenerated from leaf-derived calli. Shoots were induced in Murashige and Skoog medium in the presence of thidiazuron in combination with α-naphthalene acetic acid. In contrast to seed-derived plants, callus-derived regenerants were tetraploid showing typical characteristics of higher ploidy phenotypes. We assessed the impact of indirect plant regeneration and associated increase in ploidy on the production of saponin by means of LC–MS analysis. Tetraploid M. perlarius produce a saponin profile, which was not significantly different from seed grown plants. Based on this study, we concluded that saponin production in M. perlarius is not qualitatively changed by a genome-doubling event.

A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.

Saponins are secondary metabolites that are widely distributed in the plant kingdom and are often the active components in medicinal herbs. Hence, saponins have a potential for the pharmaceutical industry as antibacterial, virucidal, anti-inflammatory, and anti-leishmanial drugs. However, their commercial application is often hindered because of practical problems, such as low and variable yields and limited availability of natural resources. In vitro cultures provide an alternative to avoid problems associated with field production; they offer a system in which plants are clonally propagated and yield is not affected by environmental changes. Additionally, treatment of in vitro cultures with elicitors such as methyl jasmonate may increase the production of saponins up to six times. In vitro cultures are amenable to metabolic engineering by targeting specific genes to enhance saponin production or drive production towards one specific class of saponins. Hitherto, this approach is not yet fully explored because only a limited number of saponin biosynthesis genes are identified. In this paper, we review recent studies on in vitro cultures of saponin-producing plants. The effect of elicitation on saponin production and saponin biosynthesis genes is discussed. Finally, recent research efforts on metabolic engineering of saponins will also be presented.

Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects.

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell culture. Our results suggest that molecular mechanisms behind CFTD and NM may be partly related.

Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding α-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed α-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells.