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The propagation potential of the clones was determined through the initial propagation
coefficient (PC) and the subcultivation propagation coefficient when considering
the number of the shoots and bulblets obtained for a period of 6 months on
a single primary or secondary explant. The subcultivation was shown to stimulate
the new shoot formation and the subcultivation propagation coefficient was significantly
higher than the initial one. Clones with fast-growing bulblets were with
a high yearly PC and were subcultivated to form new shoots. Clones which bulblets
grow slower showed very low PC due to the slow formation of their shapeless shoots.
Most of the scientific data show a possible decrease
in the PC when the time is getting on. However, in our
experiments the second subcultivation was even more
successful than the first for several clones. Therefore we
concluded that the long-term cultivation and the continuous
subcultivation of the clones could ensure a high PC.
Of course we have to consider the clone-specific morphogenesis
and the very important period of subcultivation.
The content of Galanthamine, Lycorine and 4 additional alkaloids was
determined in the clones every 3 months for a period of 2 years by HPLC.
It was found that the clones keep their capacity of synthesizing alkaloids
in vitro and that they also keep the alkaloid profile of the donor plant.
Four clones were defined as Galanthamine-type and 2 of
them- as Lycorine-type clones. Most of the clones showed
a different content and proportion of Galanthamine and
Lycorine therefore they were defined as mixed-type clones.
The clones originating from the Danube L.aestivum populations
contained Homolycorine, Lycorine and Ungiminorine.
These results showed a genetically-dependant alkaloid synthesis and
gave us the opportunity to select highly alkaloid-productive clones.
Following the dynamic of the alkaloid content it was found that clones with a same
alkaloid profile could show also a similar dynamic of Galanthamine and Lycorine content.
Such a tendency was observed regarding the Galanthamine-type clones as well as regarding
the Lycorine-type clones and the clones originating from the Danube L.aestivum populations.
The selection of the clones was performed considering the following indices: morphology
of the obtained shoots and bulblets, stability of the propagation coefficient and
Galanthamine and Lycorine content. Five clones were selected as high Galanthamine-producing
clones and 1 clone was determined as perspective for Lycorine synthesis.
The results of ex vitro adaptation of the bulblets of clone 5.2 show that the diameter
has an important influence on the success rate. Small bulblets even rooted showed low % of
successful adaptation when planted in the soil in the autumn. In the most successful experiment
50% of the rooted bulblets with a diameter of 8-10mm survived when planted in the spring.
The Galanthamine content was determined in a sample of leaves of ex vitro adapted plants from clone 5.2.
The result (3.97 mg/g dry weight) was similar to the content of the alkaloid
in the respective cluster in situ, i.e. the plants kept their potential of synthesizing
alkaloids for the whole cycle of in vitro cultivation and even after their ex vitro adaptation.
Aiming at studying the capacity of biomass and alkaloid accumulation
we performed several experiments using solid and liquid media
with different composition. In the first experiment were used bulblets
with a same origin. They were cultivated for 2 months in liquid
medium using a stirrer-shaker. Different variations of the medium
(after Murashige and Skoog) were used concerning the type of the
auxin and the cytokinin, respectively with 30 g/l and 60 g/l sucrose.
The growth index was not dependant on the sucrose concentration.
The yield was higher in the variants with 60 g/l sucrose but
the Galanthamine content was low so the variants with 30 g/l sucrose were
preferable (moreover these variants were cheaper). Best results were
achieved using BAP and NAA as growth regulators and 30 g/l sucrose.
This medium led to the highest Galanthamine content and no
vitrification was observed as well so we chose this variant for the
next experiments. The Galanthamine content decreased in comparison
with the content of the bulblets grown in solid medium.
The next experiment was conducted using shoot-clumps from 6 clones with different
alkaloid profile which were cultivated in liquid medium for 2 months. All clones showed
a rapid growth and prolonged their leaves. The growth index varied a lot. The clones kept
the type of the main alkaloid and the Galanthamine content also decreased in comparison
with the content of the cultures grown in solid medium. This could be a result from the period
when the experiment was carried out and the established dynamic of the alkaloid content.
The rapid growth and a possible release of small quantities of the alkaloid
in the medium could be also a reason for the decreased Galanthamine content.