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Two experiments were conducted using culture medium supplemented with precursors of
Galanthamine and Lycorine (phenylalanine, tyrosine and casein hydrolysate) in order to increase
the alkaloid content. The amino acids phenylalanine and tyrosine were added to the culture medium as
the only nitrogen source and this led to an increase in the Galanthamine content of the Galanthamine-type
clones and of the mixed-type clones. The precursor addition led to an increase also in the Lycorine content
of the Lycorine-type clones. Clones with longer leaves contained a much higher amount of Galanthamine.
The influence of the precursors was studied also by cultivation of the Galanthamine-type
clone 5.2 and the Lycorine-type clone 9.6 in a liquid medium for 3 months.
The growth index in the precursor-containing media was lower as compared to the control medium.
The Galanthamine content of clone 5.2 and the Lycorine content of clone 9.6 were increased
in comparison with the control variants. We studied the influence of a half volume of
the medium on the growth and the alkaloid content and found out an increase both in
the biomass and alkaloid accumulation in clone 5.2. In clone 9.6 such effect was not
observed but in all variants of the medium the growth index of this clone was very high.
Three experiments were conducted to study the effect of Jasmonic acid elicitation of L. aestivum shoot cultures.
Two Galanthamine-type clones (5.2 and 7.80) were compared.
The growth index of clone 7.80 was three times higher than those
of clone 5.2 but the yield was the same. The elicitor had different effect on the biosynthetic activity of both clones.
This could be due to the negative correlation between the Galanthamine
content dynamics of the two clones grown on solid medium.
Clone 5.2 showed an increased biosynthetic activity and clone 7.80 was in a period of decreased biosynthetic activity.
Probably in a period of increased biosynthetic activity the elicitor has no the expected effect. About a quarter of
the theoretical yield of Galanthamine was released in the medium. Small quantity of the alkaloid was determined in the medium
supplemented with amino acids in which clone 5.2 was cultivated. No alkaloid was found in the respective medium of clone 7.80.
Galanthamine release in the liquid medium of both clones was gradually from the 3rd to the 30th day of cultivation.
Shoot clumps of clone 5.2 were cultivated for the purposes of the second experiment.
The theoretical yield of Galanthamine in the medium supplemented with elicitor was higher than those
of the control medium. Small quantity of the alkaloid was determined in all three media.
Galanthamine release in the liquid medium was gradually until the 30th day of cultivation.
The aim of the third experiment was to study the dynamics of the Galanthamine content in the biomass and in the medium
in parallel. Galanthamine content in the liquid medium was low and increased gradually but no relation was found
to the alkaloid accumulation in the biomass. The influence of both factors "elicitor" and "time" on the Galanthamine
content in clone 5.2 was studied. The dispersion analysis of two factors showed that the factor "elicitor" had not
an influence but the factor "time" influenced the alkaloid content. Clone 5.2 was again in a period of increased
biosynthetic activity by the time of the experiment. All three experiments for elicitation were carried out without using
a stirrer-shaker. It was established that the growth index is the same and it is not necessary to shake the culture medium.
Thus the negative effect on the shoots was avoided. Moreover the expenses in such cultivation were lower.
Conclusions
1. In vitro obtained clones of Leucojum aestivum could be successful
subcultivated for a long-term period of at least 4 years.
2. The new shoots were formed through a direct organogenesis
between the bulb scales which proved their genetic
identity to the mother plants. Their development was speed up by addition of Abscisic acid to the culture medium.
3. The clones showed their own regeneration potential which could be stable long time.
4. The clones kept their alkaloid profile for the period of the in vitro cultivation.
The Galanthamine content was higher in the leaves and it was commensurable to
the alkaloid content in the respective clusters from the ex situ collection as
well as to the average Galanthamine content in the commercially used populations.
5. All the studied clones showed their own specific dynamics of the biosynthesis of the main alkaloids
which was different from the seasonal dynamics of the plants grown in situ and cultivated ex situ.
6. The ex vitro adaptation was more successful when the bulbs were over
8 mm in diameter and when they were planted in the soil in the spring.
7. The alkaloid biosynthesis in vitro could be stimulated through the
supplementation of the culture medium with Galanthamine precursors or elicitor.
8. Cultivation of shoot clumps in liquid medium could ensure
a very high growth index with or without using a stirrer-shaker.
The theoretical Galanthamine yield depended on the clone
features and on the dynamics of the alkaloid biosynthesis.
9. The result of sterilization of the donor bulbs was no
related to their origin and was more successful after
a chilling treatment of the plant material. This could ensure
successful in vitro cultivation of valuable genotypes.
Dissertation contributions:
1. Leucojum aestivum in vitro clones were obtained and
propagated through a subcultivation. Plants of known origin
and alkaloid content were collected from several Bulgarian
populations which consisted of different chemotypes.
2. It was proven that by long-term in vitro cultivation the clones kept
their ability to synthesize alkaloids as well as their alkaloid profile.
3. Direct organogenesis was histologically proven and it was observed
that the new shoots developed from the base of the bulb scales.
4. The sterilization protocol of the donor bulbs was improved which is important
for the success of the in vitro micropropagation of valuable plant material.
5. Five Leucojum aestivum clones were selected as high Galanthamine-producing
clones and 1 clone was determined as perspective for Lycorine synthesis.
These clones could be used for reintroduction and for creation of
plantations as well as for in vitro biosynthesis of Galanthamine.
6. Successful ex vitro adaptation of in vitro obtained bulblets from clone 5.2 was achieved.
Six papers were published including data from the dissertation.
One paper was published in the Journal In vitro Plant which has an impact factor.
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