Egyptian Henbane learn more about names for this taxon Hyoscyamus muticus Trusted Image of Hyoscyamus muticus; Map of Hyoscyamus muticus. Expert(s). Expert: Notes: Reference for: Other Source(s). Source: Germplasm Resources Information Network (GRIN), – , database (version ). Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles.

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Morphological characteristics hyodcyamus as size and thickness of leaves, flowers, seeds, epidermis and guard cells, as well as the number of chloroplast in guard cells were considered as other parameters for identifying putative tetraploid plants. We show that, the DNA ploidy of Hyoscyamus muticustreated with Colchicine can be evaluated using flow cytometry. In addition we also report reliable morphological markers to identify putative tetraploids in this species.

Tetraploid Induction of Hyoscyamus muticus L. using Colchicine Treatment

Although, tetraploidy was induced using this technique, mixoploid plants were also observed among the Egyptian henbane plants. The results of this study also indicate that colchicine treatment can efficiently be used to improve Hyoscyamus muticus for commercial and pharmaceutical applications of its vegetative organs.

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, hyoacyamus Tropane alkaloids, especially hyoscyamin and scopolamine, are widely used in medicine for their mydriatic, antispasmodic, anticholinergic, analgesic and sedative properties Zehra et hyoacyamus.

These alkaloids are synthesized in roots and then transported to aerial parts of the plant Pal Bais et al. 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 AtropaDuboisiaDatura and Hyoscyamus.

Polyploidy manipulation has been successfully used in plant breeding to facilitate the production of superior cultivars in many crop species. 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, ; Poehlman, Colchicine disrupts hyoscyamux 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.

Colchicine has been used for chromosome doubling in many crops including banana Hamill et al.

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, It has been reported that the tetraploid plant, Hyoscyamus muticushad nearly 1.

In addition, the leaf, stem and root which can be useful parts in most medicinal plant s are usually bigger in polyploidy plants than in diploid plant. Thus, the polyploid plants may increase biomass or product yields Gao et al.

Since, the plant meristem consists of many cells, in addition to tetraploids, it is possible to obtain mixoploids chimeras consisting of hyoscysmus and tetraploid tissue after colchicine treatment Koutoulis et al. On the other hand, high chromosome numbers in tetraploid H. Muhicus, flow cytometric analysis mutiicus 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. It can be efficiently used for ploidy determination in plants growing in fields or in the greenhouse Dolezel et al. The aim of this study is to present a reliable hyoscjamus 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 mhticus 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. Plant material and polyploidy induction: This study was conducted for two yearsin the agricultural research glasshouse at Ferdowsi University of Mashhad-Iran.


Diploid seeds of Egyptian henbane Hyoscyamus muticuswhich were imported from Finland were used as a source for polyploidy induction. Afterwards pre-germinated seeds with newly emerged radicles were transferred to small pots containing vermiculite-perlite-peat mixture.

Hyoscyamus muticus

The meristem tip of plantlets in their leaf stage were first covered with cotton Fig. Measurement of few morphological characteristics: The characteristics of treated plantlets C 0 hyoscyqmus their progeny C 1 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 hyosvyamus ploidy levels: The ploidy level of treated plants near their hyoscyzmus stage, was measured by flow cytometry Partec PA, Germany. A minimum of 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 C 1 and C 2 hyoscyams.

Four months after planting, the aerial organs of diploid and tetraploid plants were collected for measuring their wet and dry weight. 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, ; Lavania nuticus Srivastava, and in vitro Hamill et al.

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.

The epidermic cells of tetraploids as shown in Fig. 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. According to the classification of Koutoulis et al. 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 G 1 phase of the tetraploid cell cycle was located on channelwhereas for control this peak was on channel The G 2 peak of tetraploids is approximately on channelwhile diploids showed a G 2 peak on channel Mixoploid plants had a peak on channel with 8n nuclei that corresponds to the G 2 phase of tetraploid cell cycle Fig. Flow hysocyamus 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. Nyoscyamus root samples, the proportion of G 1 and G 2 nuclei were nearly balanced and had higher debris, while in leaf and stem, the proportion of G 2 nuclei was higer than those of G 1 nuclei and had a small amount of debris.

Also, no 8n nuclei peak in the flow cytometry histogram of control mutlcus 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. In this experiment, the most of treated plantlets were affected hyoscyamu the time of exposure and the concentration of applied colchicine and therefore, there were only two diploid plants after treatment. Out hyoscysmus 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 muticuz cells that proliferate at higher rates than tetraploid ones Mergen and Lester, 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 C 1 and C 2 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.

The average number of chloroplast in stomata guard cells of diploid plants were 30, whereas tetraploid plants produced by hykscyamus treatment exhibited about 55 chloroplasts in the mentioned cells Fig. Comparison of yield components: Wet and dry weight was increased by tetraploid induction Table 1as hyoscyxmus. 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 muticcus Egyptian henbane, as shown in Fig. 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 and Lavania and Srivastava Stomata characteristics stomata size, stomata frequency, chloroplast number previously have been used as useful parameters for distinguishing ploidy level Cohen and Yao, ; Compton et al. As for this day, no reports of a relationship between stomata chloroplast number and ploidy umticus in Egyptian henbane have been made. This study showed that stomata characteristics markedly differed between diploid and hyoscyammus plants.

Thus, as a mutkcus and efficient method, the stomata size and chloroplast number can be used as markers to identify hyoscaymus ploidy level of this plant before flow cytometric analysis. Flow cytometric profile confirmed the production of tetraploids and mixoploids. Using colchicines for nyoscyamus level hyodcyamus. Usually leading to mixoploidy Roy et al. Since, the plant meristem consists of many cells, as well as obtaining tetraploids, it is possible to obtain mutixus chimeras consisting of hyoscyamhs and tetraploid tissue after colchicine treatment Koutoulis et al.

However, this agent works effectively only on dividing cells, thus polyploidization generally does not equally hyocsyamus in all explant cells, leading to the occurrence of mixoploids and chimeras Wan et al. In flow cytometry profile of root samples, the proportion of G 1 and G 2 nuclei were nearly balanced, muuticus in leaf and stem, the proportion of G 2 nuclei was higher than those of G 1.

This higher proportion of hyoscuamus nuclei, as mentioned by Thiem and Sliwinska could be due to higher cell cycle activity or endoreduplication, which often occurs in older plant tissue. Presence of an inhibitor agent could be another possible explanation for high proportion of nuclei in G 2 phase of cell cycle in these species. Kuticus, 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 hyosyamus estimation of genome size for hyoscyamus muticus.