Narcissus and Daffodil

µ g/ml
6.0
5.0
4.0
3.0
2.0
1.0
0.0
2 4
Time (weeks)
6
NFNGAL GAL HAEM TAZ
A
µ g/g DW
Production of galanthamine in vitro 219
At the end of the experiment, the major alkaloids in both tissues and liquid
medium were haemanthamine and N-formylnorgalanthamine, with a maximum
content of 1.38 and 0.75 mg/g DW, respectively (Figure 7.3B). The haemanthamine
type alkaloids were mainly accumulated in the tissue, whereas those of the galanthamine
type were mainly released to the liquid medium, especially in the last
subculture, coinciding with a remarkable necrosis of the root tissue.
In general, the kind of explant used influenced the type of alkaloid obtained
in vitro, so most of the subsequent experiments were carried out with shootclumps,
or with small bulblets, as galanthamine has been the main alkaloid
accumulated in these explants.
In vitro cultured plant cells grown in an undifferentiated state either produce
insignificant amounts of secondary metabolites or they lose their ability to produce
them when maintained long-term. This is the case, for instance, in Urginea indica
(Jha et al., 1991). This loss, however, can be restored when shoots or roots are
differentiated from disorganised tissue (Payne et al., 1991). The present results
show that embryogenic callus of N. confusus may accumulate nearly the same
amounts of alkaloids as shoot-clumps (Table 7.1). Since the globular structures
(embryogenic callus) which start alkaloid accumulation are rather simply organised,
1200
1000
800
600
400
200
0
NFNGAL GAL HAEM TAZ
Alkaloid
tissue medium
Figure 7.3 A: levels of alkaloids released by root clusters to the culture medium throughout
the experiment. B: accumulation of alkaloids in both tissue (root cluster) and
liquid medium at the end of the experiment (values represent means of three
replicates; thin bars represent SD). For abbreviations, see Figure 7.1.
Table 7.1 Alkaloid content (µg/g DW ± SD calculated from three replicates) at various development
stages of Narcissus confusus tissue cultures a
Tissue culture NFNGAL GAL HAEM TAZ
De-differentiated callus 0.08 ± 0.02a 0.03 ± 0.02a 0.06 ± 0.00a 0.02 ± 0.00a
Embryogenic callus 0.25 ± 0.17a 0.11 ± 0.05a 0.13 ± 0.08a 0.04 ± 0.00a
Shoot-clumps 0.10 ± 0.01a 0.14 ± 0.01a 0.08 ± 0.00a 0.03 ± 0.00a
Plantlets 0.73 ± 0.22b 1.43 ± 0.51b 1.02 ± 0.63b 0.68 ± 0.15b
Note
a means followed by the same letter are not significantly different from each other at p ≤ 0.05. Abbreviations:
DW, dry weight; FW, fresh weight; SD, standard deviation; NFNGAL, N-formylnorgalanthamine;
GAL, galanthamine; HAEM, haemanthamine; TAZ, tazettine.
B