Micropropagation of Hohenbergia penduliflora (A. Rich.) Mez. for sustainable production of plant proteases

Hohenbergia penduliflora (A. Rich.) Mez. inhabits the protected ecological area of Cunagua Highland, Ciego de Ávila, Cuba. The availability of this plant for experimental purposes is exceedingly limited. Tissue cultured plants of this specie, would be useful for propagation purposes. Experiments were carried out to optimize the micropropagation process from the disinfection of fruits to ex vitro hardening of regenerated plantlets. The best results were obtained when seeds were disinfected with 2 % (v:v) sodium hypochlorite for 20 min and placed in vitro for 45 days for seed germination. Tissue cultured shoots (1 cm) with vertical wounds in the basal region (5 mm long) were placed in a medium containing Murashige and Skoog (MS) salts, 100 mg l myo-inositol, 0.1 mg l thiamine-HCl, 30 g l sucrose, 8.8 μM 6-benzyladenine (BA) and 1.6 μM naphthalene acetic acid (NAA). Shoots were proliferated for 45 days to obtain 8.21 new shoots per explant; they were subsequently divided and rooted on a medium containing 1.6 μM NAA for 30 days. For ex vitro hardening, plastic trays containing 82 cm of filter-cake-sugarcane ashes were used; 100 % survival rate was recorded. After 6 months of hardening, plants were established ex vitro and ready for protease extraction. Comparisons between protein contents, proteolytic activities and specific proteolytic activities of extracts from stems of macro- and micropropagated plants were acquired. Tissue cultured stems showed statistically lower figures which is why Ethrel was tested here to increase proteolytic activity in micropropagated plant stems. After Ethrel applications, protein contents, proteolytic activities and specific proteolytic activities of extracts from stems were the three main indicators recorded. However, other biochemical effects of Ethrel were also evaluated, such as, levels of chlorophyll pigments, malondialdehyde and other aldehydes; and superoxide dismutase, and guaiacol peroxidase activities. Rising concentrations of Ethrel (0, 1.5, 3.0, 4.5 and 6.0 mg l) decreased protein contents at 72 h but increased proteolytic and specific proteolytic activities of stem extracts. Ethrel was effective in increasing proteolytic activity in in vitro culture-derived plant stems, at a level higher than in field-grown plant stems. Moreover, Ethrel increased superoxide dismutase and guaiacol peroxidase-specific activities in leaves; and decreased chlorophyll pigments. Ethrel did not affect levels of malondialdehyde and other aldehydes. © 2013 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.