Narcissus and Daffodil

218 C. Codina
easily lost on transfer to a regeneration medium, as happens, for instance, with
Allium cepa (Havel and Novák, 1988).
Regeneration of the meristematic callus (strains A and B) was induced by growing
it in several MS culture media supplemented with 3% of sucrose and with and
without 0.5 or 1 mg/l of the cytokinins benzyladenine (BA) and kinetin. Under
these conditions, the globular callus became green and developed aggregates of
young shoots, whereas numerous roots were formed in the most friable callus,
especially in presence of cytokinins. The alkaloid content in the organogenic
tissues was then determined in two types of aggregates, shoot and root clusters.
Alkaloids in shoot clusters
These organogenic tissues were constituted by aggregates of eight to ten young
shoots per explant, although they also showed meristematic buds which were far
less abundant in the callus grown with kinetin. The alkaloid content of these clusters
was similar, in global terms, to that of the meristematic callus, but the alkaloid
profile was not the same (Figure 7.2). One can observe that, in that callus with a
higher degree of differentiation, the main alkaloid was galanthamine.
Alkaloids in root clusters
After seeing that the clusters derived from the B strain treated with 0.5 mg/l of
kinetin grew well in solid medium, they were transferred to a liquid medium without
growth regulators and supplemented with 6% of sucrose. They were maintained
for 6 weeks, and alkaloid levels were determined every two weeks (Figure 7.3A).
The alkaloids were progressively released into the liquid medium throughout the
experiment. In general, the removal of alkaloids was higher in the first subculture,
with the exception of the alkaloids haemanthamine and galanthamine, which
reached a maximum percentage in the second and third subcultures, respectively.
N-formylnorgalanthamine was the alkaloid released to the medium in the highest
proportion, which was also observed in other assays carried out in liquid medium.
µg/g DW
200
160
120
80
40
0
NFNGAL GAL HAEM TAZ
Alkaloid
A strain B strain
A
µg/g DW
200
160
120
80
40
0
NFNGAL GAL HAEM TAZ
Alkaloid
A strain B strain
Figure 7.2 Alkaloid content in shoot clusters grown in MS solid medium supplemented
with 1 mg/l of BA (A) or kinetin (B) (values represent means of three replicates).
For abbreviations, see Figure 7.1.
B