Composition of the medium affected shoot survival and
growth. The proportion of shoots growing on MS2 and MS3
medium was 90 and 50%, respectively (Table 1). IBA
(0.05 mM) combined with 1.4 mM kinetin added to 1/2 MS
basal salts decreased the viability of shoots.
On medium MS1 and MS5 all explants turned brown and
died within 2 weeks.
As shoots grew better on 1/2 MS basal salts than on 1/4
MS basal salts, MS2 medium was used for further
subcultures. Within 6 months most of the shoots developed
dichotomies and the sporophylls leaves developed with
sporangia. After 8 months of culture the shoots reached the
height of approximately 10 cm (Fig. 3B and C).
After 3-month of culture some shoots stopped to elongate
and compact callus was visible in the apical part of the
shoots. Over the next 4 weeks the callus became round and
continued to grow surrounding 4–5 leaves below.
Nodular structures (Fig. 4) were developing on the callus
surface and during the next 2 weeks they developed into
somatic embryos (Fig. 5A). Somatic embryos separated
spontaneously from the callus and fell down on to the
medium. It was also possible to separate the embryos using
tweezers. The average number of embryos, which could be
separated from one callus was 8–10. Somatic embryos