Sporadic development of shoots
occurred in a period of 2–3 wk when embryos were transferred
to medium containing 2.22 μM BA+2.68 μM NAA (2%;
Fig. 1I–K), 4.54 μM TDZ+0.53 μM NAA+200 mg/L
glutamine (3%; Fig. 1L), or 44.39 μM BA+0.53 μM
NAA+200 mg/L glutamine (3%; Fig. 1M, N).
Re-harvesting of embryos from primary explants. The primary
explants, after 60 d, became almost brownish-black in
color. After removal of embryos from the explants, these
brownish explants were re-cultured back in fresh medium
(MS medium with 4.14 μM picloram and 0.22 μM BA).
Interestingly, these explants produced new embryos though
the number of embryos formed was less (50 embryos per
explant) compared to the primary culture (347 embryos per
explant) in medium containing with 4.14 μM picloram and
0.22 μM BA. This shows that these primary explants did not
senesce after 60 d of incubation in embryo induction medium
even though the explants were highly discolored.
Initiation and maintenance of suspension cultures. When
transferred to liquid (MS) agitated cultures, primary embryos
together with embryogenic callus resulted in the formation of
a fine cell suspension within 7–10 d. During subculturing,
large cell clumps and embryos were removed manually and the
cell suspension was allowed to settle for 2 min