Cryopreservation of in vitro-grown shoot tips of Cleome rosea Vahl (Cleomaceae) using the V cryo-plate technique
The present study is the first account of in vitro carotenoid pigment induction in Cleome rosea, a Brazilian herbaceous species frequently found near the coast in ecosystems submitted to intense anthropic degradation. Micropropagated plants obtained from in vitro roots were used as source of internodal explants for callus cultures. The callus cultures were induced on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) in the light or in the dark. The use of different culture media (B5, Nitsch and White) supplemented with 2,4-D was also evaluated. Although callogenesis was obtained in all treatments, a high production of biomass was achieved from the cultures that were maintained in light. The highest biomass accumulation was reached by cultures established on MS medium supplemented with 0.2 mg L−1 2,4-D. The exposure of cultures to light was an essential factor for carotenoid production. Pigment induction was observed on calli maintained in all tested media, and the highest pigment yield was obtained by cultures established on MS medium with 0.2 mg L−1 2,4-D. Callus cultures were subjected to treatments with elicitors (chitosan, methyl jasmonate, and yeast extract) at different concentrations and exposure times. The highest pigment production was achieved on cultures treated with 60 mg L−1 methyl jasmonate (MeJa), which resulted in a six-fold increase in the carotenoid yield when compared to non-elicited cultures. A chromatographic analysis showed that the addition of MeJa induced β-carotene production. Elicited and non-elicited calli were used to establish cell suspension cultures. These cultures were evaluated during three subsequent subcultures, showing an increased production of carotenoids during the first subculture. This study showed that in vitro cultures of C. rosea, especially after elicitation, may become an efficient alternative source of carotenoids indicating the success of plant tissue culture techniques for secondary metabolite production.