Micropropagation from apical and nodal segments of Bituminaria bituminosa and the furanocoumarin content of propagated plants

An efficient micropropagation protocol has been developed for Bituminaria bituminosa, a species considered to be of pharmaceutical interest for its furanocoumarin (FC) content. Four accessions of B. bituminosa with different FC contents were used: variety albomarginata ("albo-tedera"), variety bituminosa ("bitu-Calnegre"), var. albomarginata × var. bituminosa ("albo-hybrid"), and var. bituminosa × var. albomarginata ("bitu-hybrid"). Different concentrations of sodium troclosene solution were evaluated for in vitro establishment of apical and nodal explants cultured on modified Murashige and Skoog (MS) medium. The effects of the type of agar and the addition of activated charcoal (AC) were also evaluated. The best treatment for surface sterilising the explants was 3.5 g l -1sodium troclosene. The lowest frequency of hyperhydrated shoots was achieved using 0.8% (w/v) American bacteriological agar. Apical explants resulted in greater shoot proliferation than nodal explants. The addition of AC increased the quality and development of regenerated shoots. The highest average numbers of shoots ≥ 5 mm per explant were obtained from apical explants of "albo-tedera" (7.8 shoots) and "albo-hybrid" (7.6 shoots) on a modified MS medium supplemented with plant growth regulators (T2 media) and for "bitu-Calnegre" (6.3 shoots) and "bitu-hybrid" (5.3 shoots) on a modified MS medium without plant growth regulators (T1 media). The regenerated shoots rooted efficiently on different media depending on the plant material. Plantlets were acclimatised and maintained in a greenhouse until they were transferred to the experimental field. The total FC contents in micropropagated field grown plants were found to be higher or equal to those in the non-micropropagated mother plants. The accession "bitu-Calnegre" was found to have higher, or equivalent FC levels to those in other species such as Psoralea corylifolia and Ruta graveolens, reported as sources of FC. All micropropagated plants maintained the characteristics of the non-micropropagated mother plants.