In vitro micropropagation of endangered Rhododendron ponticum L. subsp. baeticum (Boissier & Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Anderson’s modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months)

Novel Electrospun Scaffolds for the Molecular Analysis of Chondrocytes Under Dynamic Compression

Mechanical training of engineered tissue constructs is believed necessary to improve regeneration of cartilaginous grafts. Nevertheless, molecular mechanisms underlying mechanical activation are not clear. This is partly due to unavailability of appropriate scaffolds allowing exposure of cells to dynamic compressive strains (DCS) in vitro while permitting subsequent molecular analyses. We demonstrate that three-dimensional macroporous electrospun poly(ɛ-caprolactone) scaffolds can be fabricated that are suitable for the functional and molecular analysis of dynamically loaded chondrocytes. These scaffolds encourage chondrocytic proliferation promoting expression of collagen type II, aggrecan, and Sox9 while retaining mechanical strength after prolonged dynamic compression. Further, they exhibit superior infiltration of exogenous agents into the cells and permit easy retrieval of cellular components postcompression to allow exploration of molecular mechanisms of DCS. Using these scaffolds, we observed that chondrocytes responded to DCS in a magnitude-dependent manner exhibiting antiinflammatory and proanabolic responses at low physiological magnitudes. Proinflammatory responses and decreased cellular viability were observed at hyperphysiological magnitudes. These scaffolds provide a means of unraveling the mechanotransduction-induced transcriptional and posttranslational activities involved in cartilage regeneration and repair