Narcissus and Daffodil

Production of galanthamine in vitro 235
EFFECT OF CULTURE VESSEL SIZE ON GROWTH
AND ALKALOID PRODUCTION
After observing that the best treatment for the growth of the explants in liquid
medium was that of 90 g/l of sucrose, an experiment to produce alkaloids during a
longer period of time (two months) was designed. Unlike the previous experiments,
this was performed with small plantlets obtained from a strain of seedderived
organogenic callus, and two sizes of culture flasks (250 and 500 ml) were
used to check the possible influence of this factor on the growth and morphology
of explants and alkaloid production (Sellés, 1996). The selected plantlets showed a
well formed bulb (ca. 0.9–1.3 cm), with emerging leaves (2–3 cm in length) and
small shoots at the base of the bulb. They were cultured in a liquid MS medium
supplemented with 90 g/l of sucrose. The 250 ml and 500 ml erlenmeyer flasks
contained 50 ml and 100 ml of liquid medium, respectively. The samples were
analysed every two weeks, coinciding with subculturing, and the morphology of
the plantlets and the accumulation of alkaloids were determined.
Effect on growth and morphology of the explants
In general, plantlets grown in both sizes of vessels showed well formed bulbs, with
leaves up to 8–10 cm in length. The number of shoots developed by the end of the
experiment normally coincided with the number of small buds of the initial
explant, and they showed good development with apical dominance of the central
shoot. Some vitrification and necrosis were observed during the experiment in
about 7% of the explants. These problems were more notable in the 500 ml flasks,
probably because the explants were practically submerged in the liquid culture in
these vessels which prejudiced foliar development and promoted the growth of
bulbs.
Effect on alkaloid production
In the first sample, the excretion of alkaloids to the culture medium was not determined,
only accumulation in the tissue.
250 ml flasks experiment
Figure 7.15 shows the results of the alkaloids produced by the plantlets cultured in
the 250 ml flasks. In general, no significant fluctuations in the alkaloid production
were observed during liquid culture, although the highest levels were observed in
the first subculturing step. The main alkaloids were found to be galanthamine and
haemanthamine, which reached total (tissue and liquid culture) levels of 1.54 and
1.51 mg/g DW, respectively. The highest accumulation of N-formylnorgalanthamine
took place four weeks after starting the experiment, reaching 1.01 mg/g DW. The
total levels of tazettine were found to be more or less constant throughout the
experiment, but they were always lower than those accumulated by the explants
before starting the assay.