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Hydroponic culture of Catharanthus roseus (L.) G. Don and studies on seed production

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Under climatic conditions of Poland, Catharanthus roseus may be grown in the open field only as annual crop, using transplants raised in a greenhouse or in a plastic tunnel. An effective method of hydroponic culture has been elaborated and tested. Herbal material obtained in hydroponics showed considerable concentration of alkaloids in different plant parts. Seeds of C. roseus may also be produced under these conditions. Characteristics of seeds as well as the effect of different methods of their storage are presented. Seeds stored in paper bags at room temperature may retain germinability for 7years and those stored in a refrigerator, at 5°C, up to 15years. Storage in a freezer, at -10°C, resulted in a quick and sharp reduction of germination rate.
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Hydroponic culture of Catharanthus roseus (L.) G. Don
and studies on seed production
Waldemar Buchwald Æ Irena Dedio Æ
Jan Kozłowski Æ Barbara Łata
Received: 21 December 2005 / Accepted: 1 February 2007 / Published online: 21 March 2007
Springer Science+Business Media B.V. 2007
Abstract Under climatic conditions of Poland,
Catharanthus roseus may be grown in the open
field only as annual crop, using transplants raised
in a greenhouse or in a plastic tunnel. An
effective method of hydroponic culture has been
elaborated and tested. Herbal material obtained
in hydroponics showed considerable concentra-
tion of alkaloids in different plant parts. Seeds of
C. roseus may also be produced under these
conditions. Characteristics of seeds as well as the
effect of different methods of their storage are
presented. Seeds stored in paper bags at room
temperature may retain germinability for 7 years
and those stored in a refrigerator, at 5C, up to
15 years. Storage in a freezer, at -10C, resulted in
a quick and sharp reduction of germination rate.
Keywords Apocynaceae Seed germinability
Seed storage Temperate climate Vinca rosea
Introduction
Madagascar periwinkle (Catharanthus roseus (L.)
G. Don, syn.: Lochnera rosea (L.) Rehb.f., Vinca
rosea L.), is a subshrub or shrub, up to 80 cm high
(Engler 1964). Under climatic conditions of Po-
land it may be grown in the open ground only as
an annual crop (Sadowska 1991). Then plants
develop flowers, but seldom set fruits with seeds
(Dedio 1983). As perennial, C. roseus may be
grown under conditions of Poland only in green-
houses or plastic tunnels, where heating may be
provided in the wintertime. Under such growing
conditions it has the habitus of a small shrub,
permanently blooming and bearing ripe fruits and
seeds (Narkiewicz and Sadowska 1991).
Within the species Catharanthus roseus (L.) G.
Don different forms or varieties exist with respect
to growth, development, morphogenesis and
alkaloid related characters (Mishra et al., 2001).
The colour of flowers could be from white to dark
pink and the height of plants varies from 1 to
2.5 m. Leaves are ovale-eliptical or lanceolate
with an acute or dub-acute apex and they are
placed alternately in opposite position on the
shoots. Their upper surface is lustrous, with
clearly visible nervure. Pentatherous flowers
develop single, in the angles of leaves, most
abundantly in the apical parts of the shoots. The
basal part of petals is blended into a tube. Sepals
are lanceolate, sharply ended and several times
W. Buchwald I. Dedio J. Kozłowski
Research Institute of Medicinal Plants, Libelta 27,
61-707 Poznan, Poland
B. Łata (&)
Laboratory for Basic Natural Sciences in
Horticulture, Warsaw Agricultural University,
Nowoursynowska 159, 02-776 Warszawa, Poland
e-mail: barbara_lata@sggw.pl
123
Phytochem Rev (2007) 6:413–417
DOI 10.1007/s11101-007-9060-6
shorter than petals and also blended at the base.
The flower has five stamens and one pistil,
composed of two carpels. The stigma is covered
with gluey hairs. Fruits of periwinkle have an
elongated form, usually slightly curved pouch, up
to 4.5 cm in length. They are green, but at
ripening get dark blots at the surface. Fully
ripened fruits drop easily, pouring out the seeds.
Depending on growing conditions, from several
to several tens of fruits are developed on a single
plant. One pouch contains about 20 seeds, on the
average. Seeds are black, with verrucose surface.
The root system of periwinkle is composed of a
strong main root and numerous lateral roots,
usually thin and curved. Roots are yellow.
The Research Institute of Medicinal Plants in
Poznan
´
elaborated a method of hydroponic cul-
ture of periwinkle and clarified the biology of
seed germination. The hydroponic method of
culture is mainly aimed at production of seed
material for the future possible periwinkle plan-
tations. Studies on seeds had to provide an
information concerning biology of seed germina-
tion, laboratory assessment of seeds and their
vitality after different storage conditions. The
investigations were carried out in the years 1982–
1991.
Hydroponic culture
The experiment was carried out in a heated
glasshouse, using the method of culture elabo-
rated by Gumin
´
ska (1966). The glass roofs were
at 2.5 m from the surface of hydroponic contain-
ers in the low glasshouse and at 4–5 m in the high
glasshouse. The temperature of the greenhouses
varied from 10 to 25C in autumn and winter and
from 20 to 40C in spring and summer. The
mixture of peat moss and walnut-size slug (in
proportion of 3:1 by volume, 10 cm thick), served
as a substrate in hydroponics. The substrate was
placed in the raisers above the containers with the
nutrient solution, separated from it by the metal-
lic net, on which another, plastic net was laid. The
air layer between the nets and the surface of the
nutrient solution was also 10 cm thick. The
containers with nutrient solution had the dimen-
sions of 1.5 · 1.5 or 2 · 2 m. The Gumin
´
ska
nutrient solutions (1966), partly modified by
Dedio (1983), were used, namely:
1. Gumin
´
ska solution, containing 0.3 g KNO
3
,
0.7 g of Ca(NO
3
)
2
, 0.22 g NH
4
NO
3
, 0.42 g
KH
2
PO
4
, 0.28 g (MgSO
4
.7 H
2
O), 0.12 g
Fe(SO
4
)
3
.5 H
2
O as well as 0.6 mg of
H
3
BO
3
, MnSO
4
, ZnSO
4
, CuSO
4
and
(NH
4
)
6
Mo
7
O
24
.4 H
2
Oin1dm
3
of water;
2. Gumin
´
ska solution with the increased (by
50%) Fe(SO
4
)
3
.5 H
2
O, CuSO
4
and potassium
in the K
2
SO
4
form;
3. Gumin
´
ska solution with addition of 2.84 mg
of KI per 1 dm
3
.
Every 10 days the pH of nutrient solutions was
checked and adjusted to 6.5 by adding the
adequate amount of H
3
PO
3
or NH
4
OH. The
nutrient solution was completed four times during
the vegetation period.
Seeds for the first series of cultures were
received from the Botanical Garden of Izmir
(Turkey). For the other experiments an own seed
material, obtained under hydroponical condi-
tions, was used. All plants had pink flowers.
Sowing was carried out at the beginning of March,
directly to the substrate placed on hydroponic
containers. Then seeds were covered with a thin
layer of peat moss. Seedlings emerged after 8–
14 days, the emergence was usually uniform.
Under hydroponic conditions plants developed
correctly, albeit their growth was slow at the
beginning. The earliest bloom, in the second
decade of May, was noted in hydroponics with the
Gumin
´
ska solution 2. In other treatments the
bloom time was delayed till the middle of June. In
the first year of culture, fruits started to ripen in
August and ripening continued till December.
Beyond this date plants were still blooming, but a
leaf drop took place and no fruits developed. In
December, plants were cut back, leaving stems
10 cm long. In January, when the new sprouts
appeared, the surface of hydroponic substrates
was cleaned and nutrient solutions were com-
pleted. Few weak plants declined and they were
removed. The remaining plants, in the second
year of culture, started to bloom already in April
and first ripe seeds were harvested at the turn of
May and June. The yield of seeds varied from 15
to 20 g per m
2
.
414 Phytochem Rev (2007) 6:413–417
123
It was found that addition of potassium, iron,
copper and iodine to the nutrient solution
resulted in acceleration of the development of
plants and in earlier blooming in particular. It did
not affect yield of seeds, however.
Plants of periwinkle, grown under hydroponic
conditions, reached the height of 2–2.5 m at the
end of the vegetation period. Cutting back of
plants after every growing season is a necessary
treatment in the hydroponic culture. Plants left
without cutting, branched intensely, which re-
sulted in insufficient illumination of the lower
branches and made harvesting of seeds nearly
impossible. In order to estimate the amount of
herb, possible to obtain under hydroponic condi-
tions, the plant biomass cut at heading back was
weighed. The yield of fresh herb mass obtained
after the first year was 2–3 kg, after 2 years 3–
4.5 kg and after 3 years 4.5–6 kg per m
2
. After
three years, the culture was terminated and yield
of fresh mass of roots was also recorded. It
reached 500 g m
-2
, on the average. Dry weight of
herb and roots made about 30% and 40% of dry
weight, respectively.
In order to compare the hydroponic culture of
periwinkle and its culture in open ground, some
plants raised in glasshouse were planted in the
field, in the first half of May. They were growing
well, reaching the height of ca 20 cm. The bloom
period started at the turn of July and August,
depending on the atmospheric conditions of the
year. Generally, till the first frosts, which
appeared at the turn of September and October,
plants were not able to set fruits. After a frost,
plants lost leaves and declined. Periwinkle is very
sensitive to low temperatures. Already the tem-
perature of 0C causes some damage to plants
and frosts destroy them completely.
Seeds sown directly in open field emerged very
slowly, after 3–5 weeks. The development of
seedlings was weak. First flowers appeared only
in September and no fruits and seeds were set.
The herbal material collected, from hydropon-
ics and from field culture, was analysed for the
content of alkaloids, using the method of Holar-
hen (Dedio 1983). Plants grown in the open field
were harvested in October and those from
hydroponic culture in November. The raw mate-
rial was dried in an air drying house and ground
to a fine powder. The total alkaloid content was
determined in the flowering apical 15-cm parts of
shoots as well as in leaves, stems and roots
(Table 1). In general, the total alkaloid content
reached 2–3% in roots and about 1% in leaves
(Kohlmu
¨
nzer 1968; He’Risset et al. 1972; Kohl-
mu
¨
nzer 1998; Paturde et al. 2000). According to
Babakhanyan (1991) yield of indole alkaloids
obtained from hydroponically grown plants was
considerably greater than that from soil grown
plants. In our experiment, plants grown in hydro-
ponics showed a higher alkaloid content in the
leaves and a lower content in the roots, compared
to the plants grown in the open field.
Characteristics of seeds and methods of their
storage
Under climatic conditions of Poland, hydroponic
culture is one of reliable methods for seed
production of periwinkle. This method was used
for obtaining seeds necessary for further studies.
It also enabled broader laboratory studies on the
biology of germination as well as elaboration of
the method of evaluation of seed quality and their
vitality after storage at different conditions.
Morphological features of seeds
Seeds were collected from the periwinkle hydro-
ponic culture, set up of transplanted seedlings
raised in a glasshouse or of plants obtained from
Table 1 Total alkaloid content in the overall herb mate-
rial and in particular plant parts of Catharanthus roseus G.
Don grown under different conditions
Kind of plant
raw
material
Total alkaloid content [% DM]
Plants grown in
hydroponics on
Gumin
´
ska solution
Plants grown
in the open
field
a
Overall 1.13 1.08
Apical 15-cm
parts
of shoots
1.42 1.67
Leaves 2.48 1.64
Stems 0.40 0.72
Roots 1.60 2.49
a
From transplants raised in the greenhouse
Phytochem Rev (2007) 6:413–417 415
123
tissue culture, according to Floriya (1981). Char-
acteristics of seeds did not depend on the source
of plants used for establishment of the plantation.
Seeds of Catharanthus roseus had the shape of an
elongated ellipse, were black, with a mat, verru-
cose surface. Their length varied from 1.85 to
2.63 mm and width or thickness from 1.25 to
1.42 mm. The mass of 1000 seeds varied from
1.401 to 1.767 g.
Laboratory tests for germinability of seeds
Based on the long-term experience in testing the
germinability of seeds of C. roseus, the following
optimum laboratory conditions have been estab-
lished: substrate––filter paper in the Jacobsen
apparatus or on Petri dishes; temperature––vary-
ing from 25 to 35C. Presence or absence of light
has no effect on germination. The first check
should be done after 4 days and the final one after
14 days. The tests should be carried out in four
replications, 100 seeds in each, according to the
general rules of germination tests.
Changes in germinability rate during the first
year after harvesting seeds
Studies on the germination of seeds of periwinkle
were carried out for eight years. Information
concerning this topic is divergent (He’Risset et al.
1972; Kartmazova and Lyapunova 1976; Floriya
1981; Babakhanyan 1991). Germination was
checked every year, in the first year at monthly
intervals. Seeds for tests were harvested in Sep-
tember and October. At that time the germina-
tion rate was 85–90%. After two-month storage in
paper bags, at room temperature and relative
humidity of 60–80%, seeds reached full maturity
and their germination rate was 97–100%. In the
next months, during the first year after harvest,
germination rate remained at the same, high
level; variations did not exceed 5%.
Changes in the germinability of seeds stored
for more than ten years
Using the seed material of C. roseus, obtained in
successive seasons, the studies on seed senescence
were conducted. The cleaned seeds (seed water
content was approximately 10%), were placed in
paper bags and those in polyethylene foil bags. In
that form seeds were stored under different
conditions. Storage at the room temperature
served as reference treatment. The second treat-
ment was the storage in a refrigerator, at 5C, and
the third one––in a freezer, at the temperature
–10C. The results of this study are presented on
Fig. 1. It was found that seeds stored at room
temperature retain a high germination rate for
seven years after harvest. Storage at 5C (in a
refrigerator), even for 15 years, did not reduce
the germination rate of seeds in our trials. The
temperature below 0C (–10C in our studies) had
a detrimental effect on the seed viability, result-
ing in a quick reduction of germination rate.
Seeds stored in a freezer for one year germinated
in 60%, for two years in only 10–20%. After 3 or
4 years only few seeds germinated and after
5 years in a freezer seeds completely lost the
ability to germinate.
Conclusions
1. Under climatic conditions of Poland, Catha-
rantus roseus may be grown in the open
0
20
40
60
80
100
0123456789101112131415
Storage period [years]
germination rate [%]
room temperature
temperature +5° C
temperature -10° C
Fig. 1 Seeds
germinability under
different storage
conditions
416 Phytochem Rev (2007) 6:413–417
123
ground only as an annual crop. Temperatures
below 0C are detrimental for this plant. For
production of herbal material or for growing
as an ornamental, transplants should be
prepared in a greenhouse or by tissue culture,
in order to plant them in the field after spring
frosts. Plants grown in the field showed the
highest alkaloid content in roots, whereas
those from hydroponic culture––in leaves.
Regardless of growing conditions, the lowest
alkaloid content was found in stems.
2. Seeds of proper quality for commercial plan-
tations can be produced in heated green-
houses. Very good results may be obtained in
hydroponic culture.
4. In result of the long-term studies on storage
of seeds was found that seeds of C. roseus
may retain high germinability for up to
7 years, if stored in paper bags at room
temperature and air humidity of 60–80%.
Germination rate decreases in successive
years and seeds completely loose ability to
germinate after 13 years. In a refrigerator, at
5C, seeds may be successfully stored even for
15 years, retaining a high germination rate.
Seeds stored in a freezer, at –10C, loose
viability after 4–5 years.
Acknowledgements The studies were partially financed
by Polish National Programme PR-6 ‘‘Control of Tumour
Diseases’’ co-ordinated by Maria Sklodowska-Curie
Memorial Cancer Center and Institute of Oncology.
The task concerning obtaining of Catharanthus roseus
crude material was conducted in co-operation with
Agricultural University in Warsaw, namely with the
Laboratory of Anticancer Plants supervised by Prof.
A. Sadowska co-ordinator of this project.
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Chapter
Cancer is a major disease around the world with high mortality rate. Cancer is caused due to internal and external factors. Currently, the available treatment methods are chemotherapy, radiotherapy and surgery. The current treatment methods cause severe side effects to the patient, and more over the treatment cost is highly expensive. Medicinal plants could be the better alternative to cut down these barriers. Here in this chapter, we discuss about the importance of the medicinal plant Catharanthus roseus. Vinblastine and vincristine are the two important bioactive alkaloids produced from this plant. These two are key compounds in treatment of various types of cancer. The potent anticancer compounds are synthesized in shikimate, mevalonate and methyl-erythritol phosphate (MEP) pathways. These two compounds have many biological properties, and it is discussed in detail in the following section. Recent years have seen nanotechnology-based synthesis, and these formulations have been considered to increase the efficacy of the bioactive compounds. This could be opening doors to a new era in the development of nanotechnology-based drugs. On the other hand, toxicity concerns of the prosperity of the vinca compounds were taken into consideration.
Article
Thirty two accessions of periwinkle Catharanthus roseus collected from different semitemperate to tropical geographical areas of Indian subcontinent, Madagascar, Singapore and Malaysia were characterized under field conditions for 53 growth, development, morphogenesis and alkaloid yield related characters over a few seasons at Lucknow, India. Large differences were observed among the accessions for each of the characters examined. The differences among the accessions ranged 3, 80 and 15 fold for the alkaloid yield related traits – leaf dry matter yield and leaf vincristine and vinblastine concentration, respectively. Strong correlations were observed between leaf area and leaf yield with leaf alkaloid, root and root alkaloid yields, contents of dimeric alkaloid in leaves with yields of respective alkaloids, leaf number and root alkaloid content with leaf vinblastine yield and leaf vinblastine content with leaf vincristine and vinblastine yields. Multi-variate analyses allowed classification of the accessions into 5 to 7 morphologically and presumably genetically distinct groups. Generally, the accessions coming from tropical agro-climates tended to get separated from those having origins in subtropical to semi-temperate environments. Three clusters were observed to bear complementary characters for possibly cross-parenting high alkaloid yielding transgressive segregants. One of the accessions of C. roseus in which the leaf vincristine content was 5 fold and vinblastine content one and a half fold of the respective all accession means appeared suitable for the domesticated cultivation. It was proposed that accumulation of high amounts of total alkaloids and one or more dimeric alkaloids in leaves of certain accessions may be a reflection of reproductive fitness achieved under the biotic and abiotic stresses imposed on their parent populations, sometime in their evolutionary pathway.
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Uprawy hydroponiczne roślin (Hydroponics in plant cultivation). Pań stwowe Wydawnictwo Naukowe
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