In-vitro regeneration of Calendula maritima Guss. (Asteraceae), a threatened plant endemic to Western Sicily

Calendula maritima is a critically endangered endemic plant of Western Sicily. Besides habitat destruction, the hybridization with the contiguous congener species C. fulgida is a major threat to its conservation. For this reason, seed-based propagation and seed storage are not appropriate for conservation purposes. In the present paper we describe a rapid and prolific in vitro plant regeneration method by direct organogenesis from leaves of C. maritima. Leaf explants were cultured on solid Murashige and Skoog (MS) medium in the presence of several plant growth regulator combinations. The best shoot multiplication rate (2.5 shoots/explant) was obtained on the medium containing 4.4 µM 6-benzylaminopurine in combination with 10 µM ß-naphthoxyacetic acid. Regenerated shoots were successfully rooted on solid MS medium supplemented with several auxins and the best result was obtained with 1.0 µM indole-3-acetic acid (35% of plantlets rooted). Plantlets were thereafter established in the greenhouse (survival frequency 75%) and no phenotypic variations were observed between regenerants and the mother plants.