High-yielding repetitive somatic embryogenesis in cultures of Narcissus L. ‘Carlton’

An innovatory protocol for large-scale production of narcissus ‘Carlton’ somatic embryos was developed based on the repetitive somatic embryogenesis (RSE). RSE was established as a stepwise process beginning with primary somatic embryogenesis (PSE) on ovary explants followed by secondary somatic embryogenesis (SSE) and continuously repeating cycles of SSE. A highly embryogenic lines of callus were sourced from primary embryogenic tissue, callus and embryos, which were continuously exposed to Amino-3,5,6-trichloropicolinic acid (Picloram) or 2,4-Dichlorophenoxyacetic acid (2,4-D) (both 25 µM) and 6-Benzyladenine (BA) (5 µM). Thus obtained calluses were multiplicated in six-week repetitive cycles and the efficiency of multiplication was 2.1- to 2.3-fold. Using RSE protocol, on regeneration medium containing 5 µM BA and 0.5 µM α-Naphthaleneacetic acid (NAA) it was possible to receive more than 20 embryos per 100 mg of callus. PSE yielded only 3.3–11 embryos. RSE resulted in the production of creamcoloured soft calluses which retained high efficiency of multiplication and differentiation of somatic embryos for over two years.