Narcissus and Daffodil

Production of galanthamine in vitro 221
subcultured and developed in a multiplication medium, also constituted good plant
material for further experiments on the production of galanthamine type alkaloids.
EFFECT OF GROWTH REGULATORS ON GROWTH AND
MORPHOLOGY OF EXPLANTS AND ALKALOID PRODUCTION
Although the growth of aggregates of both buds and shoots of Narcissus in liquid
medium has been succesfully achieved without adding any kind of phytohormones
(Bergoñón et al., 1992), the influence of two kinds of growth regulators has been
studied: cytokinins (BA and kinetin), which are responsible for both cell division
and growth of the aerial part of the shoots, and paclobutrazol, an inhibitor of the
synthesis of gibberellins, which has been previously tested with other bulbous
plants such as Gladiolus (Steinitz and Lilien-Kipnis, 1989; Steinitz et al., 1991; Ziv,
1992) and Nerine (Lilien-Kipnis et al., 1992). The main action of paclobutrazol is to
inhibit the growth of the aerial part of the explants, thus producing wider bulbs.
Effect of cytokinins
It has been observed that higher levels of growth regulators are needed in Narcissus
plants than in other monocots to induce adventitious shoots (Seabrook et al., 1976;
Seabrook, 1990) and bulblets (Keller, 1993), but their influence in liquid medium
cultures has not been previously studied.
Although the addition of cytokinins to the liquid medium is not very advisable,
especially at high concentrations, because they induce vitrification of the plant
tissues (Hussey, 1986), this experiment was carried out using seed-derived shootclumps
with a few (four or five) emergent leaves (Sellés, 1996). The clusters were
grown for two months in baby-food jars with MS liquid medium supplemented
with 6% sucrose and cytokinin (BA or kinetin) at concentrations of 0, 1, 3, 5 or
10 mg/l. Every two weeks, coinciding with the subculturing steps, the alkaloids
released to the liquid medium were measured. The alkaloids accumulated in the
tissues were determined at the end of the experiment. In the present chapter, we
have considered the ‘total production’ of alkaloids as the sum of the alkaloids
released to the liquid medium during the successive subculturing steps, and those
accumulated in the tissues at the end of the experiment.
Benzyladenine
In general, in the shoot-clumps growing in a liquid medium with cytokinins, the
aerial part was more developed than the bulb, exhibiting narrow, intensely green
leaves. Under these culture conditions the percentage of survival was around 90%,
with the exception of the clusters treated with 10 mg/l of BA, 80% of which became
vitrified and necrotic.
Accumulation of alkaloids in the liquid medium
The results concerning the levels of alkaloids released by the shoot-clumps to the
liquid medium during the two-month experiment are shown in Figure 7.5. The