ABSTRACT Seeds of Pancratium maritimum collected from the natural populations along Bulgaria

B Pandova

M Stanilova

S Yanev

Y Bogdanova

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BIOSYNTHESIS OF LYCORINE BY IN VITRO CULTURES OF PANCRATIUM 
MARITIMUM L. (AMARYLLIDACEAE)  
Y. Bogdanova1, B. Pandova2, S. Yanev2, M. Stanilova1
1Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria 
2Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria 
Correspondence to: Marina Stanilova 
E-mail: maris@bio.bas.bg 
ABSTRACT 
Seeds of Pancratium maritimum collected from the natural populations along Bulgarian Black Sea coast were in vitro 
germinated in 2005 and cultures were maintained under equal conditions for three years (MS based medium with BAP and 
NAA; 23±2°C and 16/8 light/dark photoperiod). Directly regenerated bulblets with diameter of about 6-8 mm were cultured in 
parallel for two months on agar solidified and in liquid media containing 3 mg/L BAP and 0.1 mg/L NAA. Alkaloid 
determination by HPLC proved biosynthesis of lycorine which antiviral and antitumor properties are well known. Lycorine 
accumulation was up to 2.90 and 1.53 mg/g DW in the cultures grown on agar and in liquid medium, respectively, which was 
highly promising as first trials to produce this alkaloid in vitro. Additionally, bulblets were regenerated from callus cultures 
and comparison was done concerning the morphology of directly and indirectly obtained plantlets. Bulblet growth varied in 
accordance with the sucrose quantity in the medium as well.  
 
Keywords: alkaloids, long-term cultures, Sea Daffodil 
Introduction 
The alkaloid lycorine has been isolated from many 
Amaryllidaceae plant species belonging to various genera: 
Lycoris, Pancratium, Leucojum, Urginea, Narcissus, 
Galanthus, Amaryllis, Crinum, Hymenocallis, Nerine, 
Sternbergia, Zephyranthes, Eustephia, Haemanthus and 
Vallota (1). It is usually accompanying galanthamine and 
some other isoquinoline alkaloids. In P. maritimum plants 
originated form the Black Sea coast of Bulgaria there were 
detected 16 alkaloids some of them with pharmacological 
activities such as lycorine and galanthamine (2). Lycorine and 
galanthamine were also determined in the flowers of this species 
(3). 
Lycorine has been studied for its inhibitory activities 
against human immunodeficiency virus (HIV-1), severe acute 
respiratory syndrome-associated corona virus (SARS-CoV), 
poliovirus, coxsackie virus, measles virus and herpes simplex 
virus type 1 (HSV-1) (4, 5, 6). Extracts of P. maritimum 
plants growing in Turkey were proved to inhibit Plasmodium 
falciparum growth due to the presence of the alkaloids 
lycorine, hydroxyhaemanthamine and haemanthamine (7). In 
vivo and in vitro activities of lycorine have been assessed by 
many authors, whereas its in vitro biosynthesis was reported 
mostly as related alkaloid in cultures aiming at production of 
galanthamine (8, 9, 10).  
Previously, we studied the opportunity to apply 
biotechnological techniques for ex situ conservation of P. 
maritimum (11, 12). In consequence of the lost of its natural 
habitats and populations the species is endangered in 
Bulgaria (13) and under protection by the Biodiversity Act 
(2002). We succeeded in the enhancement of its in vitro 
multiplication by growing of directly regenerated bulblets in 
liquid culture followed by subcultivation on solid medium. 
Our present study was aimed at testing the ability of the 
liquid cultures of P. maritimum to produce alkaloids. We also 
tried to additionally enhance bulbing by indirect 
organogenesis. 
Materials and methods 
In vitro cultivation of P. maritimum 
Initiation of in vitro cultures from mature seeds of P. 
maritimum collected from natural populations along 
Bulgarian South Black Sea coast, subcultivation of seedlings, 
and shoot multiplication were described previously (11, 12). 
Regenerated bulblets of equal size (about 6-8 mm in 
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diameter) were cultured in parallel on solid and in liquid MS 
based media (14) supplemented with 3 mg/L BAP and 0.1 
mg/L NAA for 2 months in Magenta containers with 125 mL 
medium (3 bulblets per vessel, 2 repetitions). Fresh weight 
(FW) was measured after the first and the second month of 
cultivation. At the end of the second month plantlets were 
dried at 65˚C and dry matter was calculated as percentage of 
dry weight (DW) towards fresh weight of the biomass: 
DW/FW.100. To assess the rate of the biomass augmentation 
taking into account both the increase of the fresh weight as 
well as the percentage of the dry matter we inserted growth 
coefficient (GC) which allowed comparison of the cultures’ 
growth efficiency: GC = DW/FWinitial. 
Furthermore, cultures were maintained on medium with 
30 or 60 g/L sucrose and 2/0.15 mg/L BAP/NAA, solidified 
with 6 g/L Plant agar (Duchefa, NL) in Vitro Vent containers 
(Duchefa, NL). Medium with auxin predominance was used 
for callus initiation: 0.9/1.8 mg/L BAP/NAA. To compare 
direct and indirect bulblet regeneration 18 in vitro bulblets 
were used as initial source. They were subcultured by 
longitudinal cut to 4 sectors and explants were put on media 
with 30 g/L sucrose, supplemented with 2/0.15 and 0.9/1.8 
mg/L BAP/NAA, respectively. Once obtained calli were 
further grown on medium with 2/0.15 mg/L BAP/NAA to 
produce shoots. Propagation coefficient (PC) was calculated 
as number of bulblets obtained per initial in vitro bulblet. 
Medium with 0.2/1.0 mg/L BAP/NAA was used for rooting 
of the bulblets. All cultures were grown under 16/8 h 
light/dark period, 40.5 µmolm-2s-1, at 23±2°C and transferred 
to fresh medium every two months. 
Alkaloid determination 
Each sample consisted of three in vitro plantlets grown in one 
container. Each analysis was carried out in two repetitions. 
Alkaloids were extracted from dried (65˚C) and powdered 
plant material. Samples (50 mg DW) were macerated with 
methanol (3 ml) in ultrasonic bath, 3 times for 30 min every 8 
h at 25˚C. After filtration (FILTRAK 390∅), centrifugation 
at 9500g for 10 min and evaporation with liquid nitrogen, the 
total methanol extract was diluted with a mixture of 2.5% 
methanol and 1.7% acetonitril in water, and filtrated through 
a 0.45 µm filter (Waters). Chromatographic analysis was 
carried out on Waters HPLC system supplied with quaternary 
pump 600E and PDA 996 detector; Alltech Ultrashere-Octyl 
RP-C8 column (150 × 4.6 mm i.d., 5 µm) protected by a 
Symmetry guard column C8 (20 × 3.9 mm i.d., 5 µm). The 
mobile phase consisted of acetonitril/methanol/water 
(containing 7.5 mM triethanolamine, pH up to 6.9 with 
phosphoric acid) (20/15/65); column temperature: 35°C; flow 
rate: 1.0 ml/min; injection volume: 20 µl). Data acquisition 
and analysis were made by Empower chromatographic 
software. Determination was done for lycorine, 
homolycorine, galanthamine, norgalanthamine, ungiminorine 
and galanthaminone. 
The effect of liquid medium on lycorine biosynthesis was 
assessed by ANOVA single factor analysis. 
Results and Discussion 
Chromatographic analysis confirmed biosynthetic activity of 
all tested 3-year old in vitro cultures of Pancratium 
maritimum, grown on agar solidified and in liquid medium. 
Lycorine was detected as the only alkaloid produced under in 
vitro conditions. Its concentration was significantly higher in 
plantlets grown on solid medium (P < 0.001) (Fig. 1). 
However, the growth coefficient of the liquid cultures was 
three times higher due to the fast augmentation of the fresh 
weight related to slight decrease of the dry matter (Table 1). 
Finally, the amount of lycorine per container was higher in 
the case of cultivation of plantlets in liquid medium (Fig. 1).  
2.90
1.53
0.99
0.61
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
solid medium liquid medium
Ly
c 
[m
g/
g 
DW
]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
m
g 
Ly
c 
pe
r 
co
nt
ai
ne
r
Lyc [mg/g DW] Lyc [mg]
 
Fig. 1. Lycorine concentration (mg/g DW) and amount per container (mg) of 
P. maritimum plantlets grown on agar solidified and in liquid medium with 
equal composition. 
 
Multiplication of P. maritimum was enhanced by indirect 
bulblet regeneration (Table 2). Numerous plantlets were 
obtained when calli were transferred on medium containing 
2/0.15 mg/L BAP/NAA, however they were slow growing 
and needed more time to reach size suitable for 
subcultivation. Bulblets were easily rooted on medium with 
0.2/1 mg/L BAP/NAA but this combination of growth 
XI ANNIVERSARY SCIENTIFIC CONFERENCE                                      BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 
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regulators also stimulated callus induction. Roots appeared 
spontaneously on the medium used for shoot formation and 
cultures maintenance as well, usually 2 to 4 roots per bulblet. 
In order to faster accumulate mass, small bulblets with 
average weight of 0.07 g were transferred in liquid medium 
after removal of their roots. Large bulblets were subcultured 
aiming further culture multiplication.  
TABLE 1 
Growth of P. maritimum plantlets on solid and in liquid media with equal composition (data per container) 
Medium FW in (g) FW 1m (g) FW 2m (g) DW/FW% DW (g) GC 
agar solidified  0.70 ± 0.02 1.29 ± 0.04 2.10 ± 0.15 10.1 ± 0.7 0.21 ± 0.00 0.30 ± 0.01 
liquid  0.65 ± 0.05 3.06 ± 0.09 8.00 ± 0.94 8.0 ± 0.4 0.64 ± 0.11 0.99 ± 0.09 
 
TABLE 2 
Comparison of direct and indirect organogenesis of P. maritimum: 8 months cultivation. 
Bulblets 
Total 
number 
PC 
% 
Rooted 
Large 
Middle 
size 
Small 
Directly regenerated 54 6.0 88.9 24 30 0 
Indirectly regenerated 286 31.8 100 27 83 176 
Average weight (g) 1.05 0.32 0.07 
 
Sucrose concentration in media influenced plantlet 
growth. Elongation of the leaves was typical when the 
medium contained 30 g/L sucrose. Increase of sucrose to 60 
g/L led to faster enlargement of the bulblets which shortened 
the period between two subcultivations. In vitro rooting was 
observed regardless the concentration of sucrose. 
Conclusions 
In vitro regenerated plantlets of Pancratium maritimum kept 
their viability and regeneration potential in 3-year old 
cultures. Moreover, they expressed biosynthetic activity 
producing lycorine in considerable amounts: 1.53 and 2.90 
mg/g DW in cultures grown in liquid and on agar solidified 
medium, respectively. We determined similar lycorine 
content (2.85 mg/g DW on agar solidified and 1.57 mg/g DW 
in liquid medium) in the shoot-clumps of one clone of 
Leucojum aestivum grown under conditions of long-term 
cultivation (8, 15). In vitro biosynthesis of lycorine was also 
reported in cell cultures of Ungernia victoris, with related 
alkaloids galanthamine and norgalanthamine, however in 
much lower concentration: 0.42 mg/g DW for the total 
amount of the three alkaloids (10). This was most probably 
due to the relationship between the biosynthetic activity and 
the degree of tissue differentiation that has been confirmed by 
several authors for different Amaryllidaceae species (16, 17, 
18). It should be interesting to determine the content of 
lycorine in the indirectly regenerated plantlets of P. 
maritimum in our future studies.  
The alkaloid concentration in P. maritimum plantlets 
obviously decreased in liquid culture. Similar effect was 
noticed for in vitro organ cultures of L. aestivum grown in 
parallel on agar and in liquid medium (15) as well as for 
shoot-clump cultures of Narcissus confusus (19). It should be 
related with some release of the alkaloids in the liquid 
medium. 
Lycorine was the only alkaloid we detected in P. 
maritimum cultures grown in liquid as well as on agar 
solidified medium. This alkaloid was found in the bulbs, 
leaves and roots of native plants collected from the Black sea 
cost of Bulgaria but as a minor component in the plant tissues 
(2). Authors considered that crinane type alkaloids 
haemanthamine and crinine were the main alkaloids in the 
Bulgarian P. maritimum. However, such conclusion seems 
premature taking into account the possible population 
heterogeneity, as well as the influence of the mother plant 
hemotype on the biosynthetic capacity of the corresponding 
in vitro cultures. Such determination of the alkaloid 
production was recently reported about 16 in vitro L. 
aestivum clones keeping the alkaloid profile of their plants of 
origin during several years of cultivation (8, 9). 
On the other hand, for the present we cannot conclude 
that lycorine is the only alkaloid typical of our P. maritimum 
in vitro cultures, although there were no other 
chromatographic picks. We observed clearly expressed 
dynamics of the content of lycorine and galanthamine, the 
main alkaloids of 12 L. aestivum clones, in the course of 30 
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months in vitro cultivation under continuous conditions (9). 
The dynamics of the galanthamine biosynthetic activity 
differed from that established for the plants of this bulbous 
species grown in situ and under field conditions. It this 
relation it should be of interest to study the in vitro 
biosynthesis of P. maritimum during longer period.  
Acknowledgment   
This research was financial sponsored by Operational 
Programme “Human Resources Development” at European 
Social Found. Authors are also grateful to Ch. Gussev and Y. 
Bosseva from the Institute of Botany, Sofia, for the supply of 
P. maritimum seeds. 
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45 YEARS FACULTY OF BIOLOGY 
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BIOSYNTHESIS OF LYCORINE BY IN VITRO CULTURES OF PANCRATIUM MARITIMUM L. (AMARYLLIDACEAE)

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BIOSYNTHESIS OF LYCORINE BY IN VITRO CULTURES OF PANCRATIUM MARITIMUM L. (AMARYLLIDACEAE)

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