Egyptian henbane (Hyoscyamus muticus) is one of the most important medicinal plant of the Solanaceae family. It contains valuable tropane alkaloids, such as hyoscyamin and traces of hucin and atropine (Lavania, 1986).
Tropane alkaloids, especially hyoscyamin and scopolamine, are widely used in
medicine for their mydriatic, antispasmodic, anticholinergic, analgesic and
sedative properties (Zehra et al., 1998). These alkaloids are synthesized
in roots and then transported to aerial parts of the plant (Pal Bais et al.,
2001). The synthetic production of these alkaloids is more expensive than their
extraction from plant materials and they are, therefore, currently industrially
extracted from various Solanaceous plants belonging to the genera Atropa,
Duboisia, Datura and Hyoscyamus.
Polyploidy manipulation has been successfully used in plant breeding to facilitate
the production of superior cultivars in many crop species. Polyploids often
show novel phenotypes or exceed the trait range of their diploid ancestors in
characteristics such as increased drought tolerance, apomixis, pest resistance,
enhanced biomass and increased concentration and/or qualitative changes in active
plant principles-thus enhancing their chances of being selected in agriculture
The most widely applied and best studied chemical that induces polyploids is colchicine, an alkaloid extracted from seeds or corms of the autumn crocus (Colchicum autumnae) or Colemid, its synthetic equivalent (Hassawi and Liang, 1991; Poehlman, 1987).
Colchicine disrupts mitosis by binding to tubulin, inhibiting the formation of microtubules and the polar migration of chromosomes, resulting in cells with doubled chromosome number (Panda et al., 1995).
Colchicine has been used for chromosome doubling in many crops including banana
(Hamill et al., 1992), Zizyphus jujube (Gu et al., 2005),
mulberry (Chakraborti et al., 1998) and few cases of medicinal plants
(Gao et al., 1996). Tropane alkaloids, terpenoids and isoflavonoids are
among the most important medicinal compounds found in plant roots. Thus, induction
of artificial polyploidy may prove useful in increasing the production of these
important medicinal compounds (Dhawan and Lavania, 1996). It has been reported
that the tetraploid plant, Hyoscyamus muticus, had nearly 1.5 times higher
economic production potential compared with that of its diploid counterpart
(Lavania, 1986). In addition, the leaf, stem and root which can be useful parts
in most medicinal plants are usually bigger in polyploidy plants than in diploid
plant. Thus, the polyploid plants may increase biomass or product yields (Gao
et al., 1996).
Since, the plant meristem consists of many cells, in addition to tetraploids, it is possible to obtain mixoploids (chimeras consisting of diploid and tetraploid tissue) after colchicine treatment (Koutoulis et al., 2005). On the other hand, high chromosome numbers in tetraploid H. muticus (2n = 4x = 56) (Lavania, 1986) make microscopic cytological analysis of this species unfeasible. Therefore, flow cytometric analysis of the DNA content of leaves in Egyptian henbane was applied in the present experiment to determine ploidy level. Flow cytometry is a rapid and exact method for estimating nuclear DNA content (Galbraith et al., 1983). It can be efficiently used for ploidy determination in plants growing in fields or in the greenhouse (Dolezel et al., 1989; Joachimiak et al., 2001).
The aim of this study is to present a reliable protocol for obtaining tetraploid plants in the commercial exploitation of Hyoscyamus muticus by colchicine treatment. A further objective of the present study is to produce raw material for future study on different ploidy levels in transgenic hairy root culture of this species. Furthermore, this study reports on the DNA ploidy level of plantlets treated with colchicine using flow cytometry and finally on finding reliable morphological markers to identify putative tetraploids in these species.
MATERIALS AND METHODS
Plant material and polyploidy induction: This study was conducted for two years (2007, 2008) in the agricultural research glasshouse at Ferdowsi University of Mashhad-Iran. Diploid seeds of Egyptian henbane (Hyoscyamus muticus), which were imported from Finland were used as a source for polyploidy induction. The seeds were first surface sterilized with 1% sodium hypochlorite for 10 min, washed two times with sterile water and placed in filter paper. Afterwards pre-germinated seeds with newly emerged radicles were transferred to small pots containing vermiculite-perlite-peat mixture. The cultured plantlets were incubated in a growth chamber at a photoperiod of 16/8 h light/dark and irrigated with 50% Hogland solution.
The meristem tip of plantlets in their 6-8 leaf stage were first covered with
cotton (Fig. 1) and then 0.2% aqueous solutions of colchicines
(Sigma) was used as droplet on cotton for 24 h with interval times of 4 h. Afterwards,
the plantlets were completely washed with sterile distilled water and after
one month Colchicine treatment, plantlets with new organs emerged from the treated
section were transferred to big pots and grown in glasshouse for acclimatization
under a photoperiod of 16/8 h light/dark.
||Treatment of tip meristem by the cotton plug method
Measurement of few morphological characteristics: The characteristics of treated plantlets (C0) and their progeny (C1) were compared with non-treated ones (control). These characteristics included visual comparison of plant body size, leaf, flower and seed size. For the stomata measurements of the above plantlets, the upper epidermis of leaves was removed and stuck on microscopic slides. The number of chloroplasts in 8 guard cells of stomata was counted by removing the upper epidermis of leaves. For this purpose, three plantlets were chosen from both the diploid control and tetraploid groups.
Flow cytometry analysis of ploidy levels: The ploidy level of treated plants near their flowering stage, was measured by flow cytometry (Partec PA, Germany). Fresh leaves (nearly one month old) were first harvested and nuclei suspensions were prepared by chopping the leaf tissue (50 mm2) of colchicines treated and diploid (control) plants, with a fresh razor blade in 400 μL of nuclei extraction buffer (Kit A, suggested by Partec PA, Germany) for 30-60 sec. After filteration through a 30 μm Cell-Trice disposable filter Partec, 1600 μL of 4-6 diamidino 2-2 phenylindole (DAPI, provided by Partec PA, Germany as Kit B) was added. A minimum of 5000 nuclei were measured per sample and histograms of DNA content were generated using the Mode Fit software.
In this research to investigate the stability of the induced tetraploids, flow cytometry measurement was conducted again from leaves in C1 and C2 generations.
Biomass comparison: Four months after planting, the aerial organs of diploid and tetraploid plants were collected for measuring their wet and dry weight.
Statistical analysis: Where appropriate, statistical analysis was performed using student t-test for morphological and biomass comparison between tetraploid and diploid types (control).
Survival and growth of colchicine treated plantlets: The effects of colchicine on the growth of plantlets were assessed one month after treatment. The plant survival was not affected by the concentration and time of exposure of colchicines. Although, all of the plantlets were affected by the colchicines, small number of plantlets showed some browning and burning at the place of treatment.
An inverse relationship has been reported between colchicine concentration and explant survival with ex vitro (Lavania, 1986; Lavania and Srivastava, 1991) and in vitro (Hamill et al., 1992; Chakraborti et al., 1998) studies using other plant types.
The first visible effect of colchicines was the delayed growth of treated plantlets. The initiation of growth occurred about 10 days after treatment, whereas untreated plantlets continue to grow without any delay. After about 40 days of growth, all colchicines treated plantlets had significantly shorter shoots than untreated plantlets.
Morphological differences between diploid and tetraploid Egyptian henbane:
The morphological features of tetraploid and diploid plants were evaluated to
determine whether they could be used as markers for identifying putative tetraploids
in these species. In most cases, the first leaves of tetraploids had a distored
appearance, but the subsequent leaves appeared normal at the place of treatment.
The body size of diploids and tetraploids were significantly different, tetraploids
being stronger than diploids one (Fig. 2a-g).
The epidermic cells of tetraploids as shown in Fig. 3a-d
were bigger than diploids, so, that visual comparison was possible.
||Representative body size, flowers, seed sizes and leaves from
diploid and colchiploid plants of Egyptian henbane (Hyoscyamus muticus)
(a, b) body size, (c, d) relative size of flowers, (e, f) relative size
of seeds and (g, h) leaves of diploid and tetraploid, respectively
|| The stomata frequency and sizes of diploid (a, c) and tetraploid
(b, d) Egyptian henbane (Hyoscyamus muticus)
Flow cytometric profiles of colchicine-treated plants: All colchicine-treated
plants, subjected to flow cytometry were classified as diploid, mixoploid and
tetraploid, according to the peaks obtained by the flow cytometry (Fig.
4a-c). According to the classification of Koutoulis
et al. (2005), mixoploids were graded base on the relative amounts of diploid
and tetraploid nuclei. In grade 1, the number of diploid nuclei is more than
tetraploids, whereas in grade 2, the number is equal. In grade 3, the situation
is completely opposite and so, they are most similar to tetraploid plants.
In this study, the peak that represents the G1 phase of the tetraploid
cell cycle was located on channel 180, whereas for control this peak was on
channel 90. The G2 peak of tetraploids is approximately on channel
360, while diploids showed a G2 peak on channel 180. Mixoploid plants
had a peak on channel 360 with 8n nuclei that corresponds to the G2
phase of tetraploid cell cycle (Fig. 4a-c).
Flow cytometry was also individually performed for stems and roots in few diploid
plants. The stem histograms showed no significant difference with those of leaves,
while the root histogram showed a higher difference. In root samples, the proportion
of G1 and G2 nuclei were nearly balanced and had higher
debris, while in leaf and stem, the proportion of G2 nuclei was higer
than those of G1 nuclei and had a small amount of debris. Also, no
8n nuclei peak in the flow cytometry histogram of control root samples was observed,
whereas leaf and stem samples of this plants, in some cases showed a small percentage
of 8n nuclei peaks. This result shows that endureduoplication possibly does
not occurred in the root of this species.
||Flow cytometric histograms representing Egyptian henbane (Hyoscyamus
muticus) seedling with (a) a diploid profile (2n), (b) mixoploid (2n
+ 4n) profile and (c) a tetraploid (4n) profile
In this experiment, the most of treated plantlets were affected by the time of exposure and the concentration of applied colchicine and therefore, there were only two diploid plants after treatment. Out of 50 plantlets, five pure tetraploid plants were seen and the rest of them were mixoploids which were mostly in grade 3.
Regarding to the mixoploidy production in colchicines treated meristems and remaining of diploid cells that proliferate at higher rates than tetraploid ones (Mergen and Lester, 1971) it is possible that selected plants, partially or totally reversed to the diploid condition after successive cell division cycles. For this reason, stability analysis by flow cytometry was conducted in C1 and C2 generations. It was also found that all of the peak values were nearly twice that of the diploid control, indicating the stability of tetraploids.
Comparison of chloroplast numbers: Increased numbers of chloroplast
in the stomata guard cells of tetraploid plants compared to diploid ones is
another consequence of polyploidy induction (Gu et al., 2005), which
was quite evident in the present study. The average number of chloroplast in
stomata guard cells of diploid plants were 30, whereas tetraploid plants produced
by colchicine treatment exhibited about 55 chloroplasts in the mentioned cells
||Yield comparison (wet weight, dry weight and percentage of
dry matter (measured as Mean±SE) between diploid and tetraploid Hyoscyamus
|Within each row Mean values followed by the same letter(s)
are not significantly different at the p = 0.05 level by students t-test
||Histogram comparison of average number of chloroplast between
diploid and tetraploid cells
Comparison of yield components: Wet and dry weight was increased by tetraploid induction (Table 1), as expected. Although, an increase in wet weight was noticeable in the present study, a 17% increase of dry weight in tetraploids compared to diploids is quite in agreement by Lavania (1986) Lavania and Srivastava (1991).
Solid, stable autotetraploids as well as mixoploids were produced by treating Egyptian henbane plantlets with colchicine. Increased size of stomata guard cells is one of the general effects of tetraploidy induction in the Egyptian henbane, as shown in Fig. 3. This is actually a genetic effect which can be seen in all organs, such as seeds, flowers, leaves and shoot. Visual observations also indicated strong correlation between increased polyploidy level and bigger vegetative organs, for those plantlets grown in greenhouse. Similar results also had been observed in other studies by Lavania (1986) and Lavania and Srivastava (1991). Stomata characteristics (stomata size, stomata frequency, chloroplast number) previously have been used as useful parameters for distinguishing ploidy level (Cohen and Yao, 1996; Compton et al., 1996; Gu et al., 2005). As for this day, no reports of a relationship between stomata chloroplast number and ploidy level in Egyptian henbane have been made. This study showed that stomata characteristics markedly differed between diploid and tetraploid plants. Thus, as a simple and efficient method, the stomata size and chloroplast number can be used as markers to identify the ploidy level of this plant before flow cytometric analysis.
Flow cytometric profile confirmed the production of tetraploids and mixoploids.
Using colchicines for ploidy level change. Usually leading to mixoploidy (Roy
et al., 2001; Koutoulis et al., 2005; Wan et al., 1989;
Thiem and Sliwinska, 2001). Since, the plant meristem consists of many cells,
as well as obtaining tetraploids, it is possible to obtain mixoploids (chimeras
consisting of diploid and tetraploid tissue) after colchicine treatment (Koutoulis
et al., 2005). However, this agent works effectively only on dividing
cells, thus polyploidization generally does not equally occur in all explant
cells, leading to the occurrence of mixoploids and chimeras (Wan et al.,
1989). In flow cytometry profile of root samples, the proportion of G1
and G2 nuclei were nearly balanced, while in leaf and stem, the proportion
of G2 nuclei was higher than those of G1. This higher
proportion of 4n nuclei, as mentioned by Thiem and Sliwinska (2001) could be
due to higher cell cycle activity or endoreduplication, which often occurs in
older plant tissue. Biradar et al. (1993) indicated that nuclear DNA
amounts vary in somatic tissues due to endoreplication resulting to polyploid
nuclei. Presence of an inhibitor agent could be another possible explanation
for high proportion of nuclei in G2 phase of cell cycle in these
species. Tambong et al. (1998) reported that colchicine-treated plantlets
had nuclei arrested at all the three phases suggesting that the cells were actively
dividing with significant amounts of the cell population at G2 phase.
Vera et al. (1994) reported that quiescent pericycle cells of transgenic
tobacco were developmentally arrested at G2 and rapidly progressed
into M phase upon mitogenic stimulation with colchicine. Since, key facts with
regards to the physiology of this plant are unknown, however, basic physiological
studies are still required to understand this stringent process in Hyoscyamus
muticus. To our knowledge, this is the first estimation of genome size for
hyoscyamus muticus. The information provided in this study may be helpful in
genome research as well as in study of the relationship between DNA content,
plant physiology and ecology.
A common model for explaining changes, originating from tetraploid induction, relies on the assumption that the lower ratio of nuclear membrane to chromatin (the volume of tetraploid cells is typically about double and their surface area is about 1.5 times), results in more chromatin coming into contact with the nuclear membrane, thereby enhancing gene activity, improving water relationships, hormonal status and photosynthetic rate on a per cell basis (Lavania, 2005), coupled with enlarged cell size, all of these factors could have a positive impact on secondary metabolism and the production of active phyto-pharmaceuticals (Dhawan and Lavania, 1996). Thus, the measurement of alkaloids and its possible raising should be considered in polyploidy induction.
Male sterility was a visible effect of tetraploid induction of H. muticus. Although, the seed is not the ultimate economic product in this species, it is still required for propagation. Therefore, if seeds are required for multiplication, it is possible to search those genotypes with high fertility as reported by Lavania (1986). The flower structure of the Egyptian henbane is as heterostylous and so open pollination leads to increased heterozygosity, resulting bivalent formation in meiosis and high fertility.
The overall conclusion is that the method used is reliable for routine in vivo production of polyploids in Egyptian henbane. And this study showed that flow cytometry can be used as an efficient method for screening of H. muticus plant with different ploidy level and the results of flow cytometry and morphological characteristics paralleled to each other. The positive and significant correlation between ploidy level and biomass production, is economically important for medicinal plants, such as the Egyptian henbane.
This study was carried out with the financial assistance of the Institute of Biotechnology and the Deputy Research of Ferdowsi University in Mashhad-Iran.