Concentrations Of Colchicine Research Articles

Increasing Ploidy Level of Garlic (Allium sativum L.) “Tawangmangu Baru” In-Vitro Using Colchicine


 
 
 “Tawangmangu Baru” garlic variety is known to have low productivity. The variety is still highly demanded due to its strong flavour and aroma; however, its production has not yet been able to fulfill the local needs of Central Java due to the small size and limited production area. This study aimed to determine the effect of concentration and time duration of colchicine treatment towards increasing the ploidy level of “Tawangmangu Baru” garlic variety for genetic variability. The experimental design used in this study was a complete randomized design with two factorials and 12 combinations. The first factor was concentration of colchicine, i.e. 0.00, 0.02, 0.04, 0.06, 0.08 and 0.10%, and the second factor was the immersion time, i.e. 24 and 48 hours. The result indicated that, 4.72% callus induction was obtained in BDS + 0.4 mg.L-1 2,4-D + 2.0 mg.L-1 kinetin; and 4.0% callus proliferation were obtained in both BDS + 1.5 mg.L-1 2,4-D + 1.0 mg.L-1 kinetin and MS +1.5 mg.L-1 2,4-D and 1.0 mg.L-1 kinetin. The untreated plantlets showed higher mortality rate compared to the explants with 48 hours colchicine treatment. Higher number of shoots were recorded in 0.1% colchicine at 48 hours and lower shoots in 24 hours, whereas 0.1% colchicine at 24 and 48 hours showed the highest ploidy level of total nuclear DNA analyzed by flow cytometry. The genetic diversity of the “Tawangmangu Baru” garlic was successfully enhanced by colchicine and immersion treatment. Mutant lines with tetraploid and mixoploid plants were obtained. The putative lines obtained at 0.1% colchicine treatment were subcultured to produce new mutants before testing the phenotype. The application of colchicine at 24 and 48 hours treatment improved the genetic potential of “Tawangmangu Baru” garlic variety in vitro. The application of colchicine increased the ploidy level and an increase in ploidy is expected to make the bulb size larger. Larger tuber size will increase the tuber weight, and also the overall garlic productivity and production in the future.
 
 

Haploid Culture and Double Haploid Induction in Medicago sativa L. cv. XinJiangDaYe

Background: Alfalfa (Medicago Sativa), a perennial cross-pollinated plant, is one of the most important forage crops in the world with commercial value and ecological significance. However, due to the complexity of its genome, varietal improvement is difficult. Therefore, generating genetically homozygous materials have greater significance for breeding. In the current study, we aimed to identify the best tissue culture conditions to obtain haploid plants and double haploid plants.Methods: In this study, the haploid plants of alfalfa were obtained by combining tissue culture regeneration system with Flow cytometry. Different concentrations of colchicine were applied to the haploid plants using solid and liquid cultivation methods to determine the optimum conditions to obtain double haploid plants of Medicago Sativa L. cv. ‘XinJiangDaYe’. Result: Among the two colchicine cultivation methods tested, the doubling rate of regenerated plants obtained by liquid cultivation method was higher and the leaves developed under this system have the best doubling effect among the three explants tested. Optimal doubling conditions for alfalfa haploid (Medicago Sativa L. cv. ‘XinJiangDaYe’) were identified. The double haploid plant material generated from the current study could serve as a genetic resource for developing the hybrid combinations and for analyzing genetic linkage in alfalfa improvement programs.

Methods of experimental polyploidization and their use in apple breeding

The article presents methods of experimental polyploidy of fruit, berry and ornamental plants. The purpose of this review is to highlight the problems and prospects of polyploidization of plants in the open ground and in vitro culture and the possibility of their application for apple trees. For the purpose of obtaining apple tetraploids as donors of diploid gametes, seed seedlings were treated with a solution of colchicine in concentrations of 0.1-0.4 % for 24 and 48 hours. Colchicine concentrations of 0.3 % and 0.4 % at 48 hours of treatment had a detrimental eff ect on their development. As a result, tetraploids and chimeras were obtained from seeds from free pollination of the varieties Orlik, Svezhest, Kandil Orlovsky, as well as from seeds obtained from crossing the varieties Svezhest×Bolotovskoe, Moskovskoe Оzherel’e×Imrus, Girlyanda×Venyaminovskoe. The optimal concentration of colchicine was 0.1 %. Methods of colchicine treatment have been studied: 1) adding to the nutrient medium, colchicine concentration: 0.01%, 0.02%, exposure time 24h-19 days; 2) applying amitotic solution to the growth point, colchicine concentration: 0.1 %, 0.2 %, exposure time 24h-7 days. To increase the penetration of colchicine through the cell walls, a 0.1 % dimexide solution was used. Studies have shown that high concentrations and prolonged exposure to colchicine reduce the viability of explants.

In vivo-induced polyploidy in Dendrobium ‘Sonia’ in a bubble bioreactor system using colchicine and oryzalin

Development of polyploidy in Dendrobium ‘Sonia’ using colchicine and oryzalin is described. Five-month-old plantlets derived from protocorm-like bodies produced in vitro were used. These plantlets were immersed into a bubble bioreactor containing different concentrations of colchicine (0.05, 0.10 and 0.15% for 3 days) and oryzalin (14.40 and 28.90 µM for 4 days) together with Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Tetraploidy was successfully induced by 0.10 and 0.15% colchicine. Polyploidy levels were estimated using flow cytometry and then confirmed by chromosome counting, and through anatomical, cytological and morphological studies. The highest percentage (26.60%) of polyploids was induced with 0.15% colchicine. Oryzalin treatment did not yield tetraploid plantlets. The relative fluorescence intensity emitted by propidium iodide (PI), a fluorescent intercalating agent or a DNA-binding dye, was almost twofold higher in tetraploids and nearly fourfold higher in octaploid as compared to diploids. Plantlets with changed chromosome number were obtained at 0.05–0.15% colchicine concentrations (tetraploid induction) and 14.40 µM oryzalin concentration (mixoploid induction). The chromosome number was 2n = 38 in diploids and 2n = 76 in the tetraploids. The tetraploid and diploid plantlets exhibited significant differences in morphological, anatomical and cytological parameters. Stomata of tetraploid plantlets were longer than diploids. Survival rates from treatments were above 70%. In all treatments with colchicine and oryzalin, 0.10 and 0.15% of colchicine proved the most effective for generation of polyploid plantlets. This is beneficial for further breeding purposes of D. ‘Sonia.’ Oryzalin was found lethal. New ornamental varieties with improved traits can be produced by polyploidy using colchicine.

Induction of Polyploidy in C35 Citrange through in Vitro Colchicine Treatments of Seed-Derived Explants

ABSTRACT Polyploidy can be observed in citrus species which often enables better adaptation to environmental stresses. In this context, this paper aimed to develop stable tetraploid C35 citrange by in vitro colchicine treatments. Seed-derived explants were obtained by a germination experiment using several concentrations of GA3. Culturing seeds of C35 without seed coat on MT basal medium with the addition of 4 mg l−1 GA3 resulted in the highest germination rate (84%). Various colchicine concentrations and exposure time were tested to induce polyploidization. Seed-derived explants of C35 citrange treated with 0.1% colchicine for 48 h demonstrated high rates of mutation for polyploidization and the highest tetraploid induction percentage (15%) was determined in this treatment. Higher colchicine concentrations and exposure time decreased the survival rates of the seedlings. Flow cytometry demonstrated that the nuclear genome size of tetraploids (1.598 pg/2 C) was significantly (P < .01) higher than diploid (0.794 pg/2 C) C35 seedlings. Recovered seedlings were identified with morphological and cytological variables, such as leaf area and stomata size. Generally, leaf area and stomata size significantly increased with the increasing ploidy level. The method used in this present study was relevant and an effective way to induce polyploidy in C35 citrange.

Phenotype and ploidy evaluation of colchicine-induced Echeveria ‘Peerless’

The study was conducted to evaluate the phenotype and ploidy level of colchicine-induced Echeveria ‘Peerless’. Leaf cuttings of Echeveria ‘Peerless’ were treated with different concentrations of colchicine (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) for different durations (3, 6, 9, and 12 h). The survival and mutation rates following various treatments were determined, and the phenotypic characteristics were evaluated 12 months after the treatment. Putative succulent plants were subjected to stomatal evaluation and flow cytometry to confirm the ploidy level. Colchicine treatment produced more compact mutant plants with thicker and shorter leaves with a redder tone. In addition, colchicine-induced mutant plants showed obviously broader leaf apices than control plants. Treatment with 0.2–0.6% colchicine for 3–12 h achieved a survival rate of over 50% while producing the required effects. Moreover, treatment with 0.2–0.6% colchicine for 3–12 h or with 0.8% colchicine for 3–9 h successfully produced diploid–tetraploid mixoploids, which explains the altered phenotype, increased stomatal size, and decreased stomatal density of the succulents. This study established an effective method to induce polyploidy in Echeveria ‘Peerless’, which can be used to create novel genetic sources for developing and breeding new cultivars.

Effect of different in vitro and in vivo variables on the efficiency of doubled haploid production in Triticum aestivum L. using Imperata cylindrica-mediated chromosome elimination technique

Development of doubled haploid (DH) plants in wheat is an important technique for establishment of completely homozygous lines in the shortest time frame. In this study, we standardized the in vitro and in vivo variables for the production of wheat DH plants via crossing of three spring wheat genotypes with a wild grass (Imperata cylindrica). The experiment involves testing new treatments and protocol modifications for 2 years. Wheat florets were pollinated with I. cylindrica pollen after 2 days of emasculation, and next day pollinated spikes were injected with different auxin analogues: 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 100 mg/l 3,6-dichloro-2-methoxybenzoic acid (dicamba) or 50 mg/l 2,4-D + 50 mg/l dicamba. A combination of 50 mg/l 2,4-D and 50 mg/l dicamba was superior to other treatments having maximum haploid embryos and haploid plants developed per emasculated florets. The responsiveness of embryo formation to the addition of silver nitrate (AgNO3) (the ethylene inhibitor) with the auxin treatments showed that the presence of silver nitrate in the medium may not be essential for wheat embryo induction. Time of embryo rescue had a significant effect on embryo and haploid plant production. The most successful time for embryo rescue was 24 days after pollination (DAP) resulting in maximum haploid plants developed (40% in wheat variety AKYK92 and 25% each in variety HD2888 and HS542). The immature embryos were cultured in different media, i.e. Murashige and Skoog (MS), half-strength MS, modified MS media, Gamborg’s B5 and modified B5 media. Maximum number of haploid plants were found in half-strength MS media in all accessions (60.0%) followed by modified MS media (40.00%). For colchicine concentration, during 2016–2017, maximum numbers of DHs with less plant mortality were obtained at 0.08 and 0.06% dose. Taking into account the first year results, refinement of the dose in the second year showed that colchicine doses of 0.08 and 0.075% were the best dose for maximizing DH induction in wheat; however, in this case the number of examined plants was very small. Therefore, we can treat the two doses as a guide for further research on larger samples.

A study on enhanced production of 3-demethylated colchicine by a novel strain of Bacillus endophyticus isolated from rhizospheric soils of Gloriosa superba

A native strain of Bacillus endophyticus, PC was found to be effective in demethylation of colchicine. The strain was isolated from rhizospheric soils of Gloriosa superba from Erode, Tamilnadu, and was grown in optimised conditions of carbon and nitrogen. To intensify the demethylation, the strain was enriched and grown for 15 generations starting from low (0.5 mg/ml) to very high (10 mg/ml) concentrations of colchicine. Owing to enrichment, B. endophyticus showed an MIC of 25 mg/ml, and notable activity of demethylation of colchicine. Maximum production was observed at idiophase level, wherein PC produced around 71.97% of 3-DMC. Supplementary studies with the strain B. endophyticus PC proved the adverse effects of colchicine on the metabolic and structural portrait of cells. There was major variance in the cell shape and size, including the clipping of thickness of plasma membrane and cell wall. The number of polyhydroxybutyrate (PHB) rich inclusion bodies were found to be enhanced, suggesting their portrayal in activation of P450-BM3 enzyme which initiates demethylation.

Effect of colchicine and oryzalin treatment on polyploid induction and morphogenesis of Anoectochilus setaceus Blume cultured in vitro

Jewel orchid (Anoectochilus setaceus Blume) is one of the important medicinal plants used for many common diseases treatment such as hypertension, diabetes, and heart, liver and lung diseases, as well as improving the health in general. Polyploid induction using colchicine and oryzalin has been widely performed in various types of crops and could be considered a valuable tool for plant breeding. In the present study, in vitro young shoots of A. setaceus Blume (1 cm in height) were treated with different concentrations of colchicine (0, 1, 2, 4, 6, 8, and 10 µM) and oryzalin (0, 10, 20, 40, 60, 80, and 100 µM) at different durations (0, 12, 24, 36, and 48 h). The results showed that shoots with length of treatment from 24 to 48 h and the concentrations from 2 to 10 µM of colchicine and from 40 to 100 µM of oryzalin appeared morphological differences (10.00% - 16.00%) but the tendency to return to normal of these shoots after several times of sub-culture was observed. Stomatal length had a close correlation with the level of ploidy. Diploid plantlets (2n = 2x) had the average stomatal length of 33.50 µm when observed under an optical microscope at 100x magnification and corresponding to the peak at 70 FL in flow cytometric DNA histogram. In all identified polyploid plantlets, the stomatal lengths were greater than 40.12 µm. Moreover, flow cytometric analysis indicated that plantlets with stomatal lengths ranged from 40.12 to 50.00 µm were diploid-tetraploid mosaicisms represented by two separate peaks at 70 FL and 90 FL. Finally, samples treated with 4, 6, and 8 µM colchicine with treatment time of 48 h, and 60 µM oryzalin with treatment time of 24 h that had stomatal lengths ranged from 52.03 to 71.25 µm were determined to be tetraploid (2n = 4x) represented by a peak at 120 FL position.

Phenotypic and molecular characterization of polyploidy Vicia faba induced by colchicine

To induce polyploidy in diploid control plant of Vicia faba, seeds were socked in various concentrations of colchicine (0.1, 0.2, 0.3 and 0.4 %) for 9 and18 h. Putative ploidy plants were confirmed by screening morphological characters, measuring DNA content via spectrophotometer and analyzing stomatal behavior. Polyploidy plants showed slower growth rate along with increased potency and thickened large leaves. Three types of polyploidy were achieved; triploid, tetraploid and mixoploid. In induced polyploid plants, the length and width of stomata were increased, while the index of stomata was declined, in comparison to the initial diploid plants. Treatment with 0.3 and 0.4 % colchicine for 9 and 18 h were efficiently induced polyploidy. The highest stomatal size (22.53 µm) and widest stomatal aperture (9.72 µm) and the lowest stomatal index (34.24) where obtained when 0.4 % of colchicine where applied at 18hr tested time with high significant differences at p<0.05 in comparison to dipliod plants. We recommend this method for induction of polyploids in other different crops and possibly will selected these tetraploid plants for further breeding purposes.

POLYPLOIDY INDUCED BY COLCHICINE IN Robinia pseudoacacia L. AND IT'S EFFECTS ON MORPHOLOGICAL, PHYSIOLOGICAL AND ANATOMICAL SEEDLING TRAITS

This study was aimed to induce and produce a polyploidy Robinia pseudoacacia seedling by different concentrations of colchicine (0.0%, 0.5%, 1.0%, 1.5%, and 2.0%) in various exposure times (12, 24 and 48 h), using in two experiments. In the first, were applied to the seeds by immersing method. In the second, were applied to shoot apical meristems by dropping method. The results showed significant effects of different interactions between colchicine concentration and exposure period on the seed germination, seedling survival rate and seedling characters. By increasing of the colchicine concentration the seed germination and seedling survival rate decreased significantly in colchicine concentrations (0.15 and 0.2%). Frequency of stomata decreased and on the opposite the stomata length and width increased with increases the colchicine concentrations. Polyploidy seedlings show vegetative growth superiority yield compared to diploids seedlings, leaf area in treated plants was larger and with deep green pigmentation as compared to the control seedlings. Seed treatment method was found to be more efficient than shoot apical meristems treatment method in producing tetraploid and mixoploid plants, colchicine solutions at 0.1% and 1.5% in 24 h. and 48 h. was found most effectively produced polyploidy and inducing variation in both investigations.

&lt;i&gt;Zamioculcas zamiifolia&lt;/i&gt; novel plants with dwarf features and variegated leaves induced by colchicine

Zamioculcas zamiifolia (Lodd.) Engl. is an export-oriented, potted foliage plant used for indoor scaping. Since it shows high environmental and pest tolerability, it does not need much attention in cultivation. Thus, the aim of present study was to develop stable, novel mutants of Z. zamiifolia induced by colchicine (a mutagenic chemical) treatment. Three leaflet types (rooted leaflets with tuber, tuber initiating leaflets without roots and freshly harvested leaflets) were applied separately with four concentrations of colchicine ranging from 0 to 0.4% to induce mutations in a 70% shaded greenhouse. The experiment was conducted for three generations. Results showed that at 0.4% and 0.04% colchicine, plant size decreased in some leaflets, thus producing extreme dwarf and semi dwarf plants, respectively, which are suitable to be used as table-top, miniature ornamental plants. Some leaflets at 0.004% colchicine produced yellow and light green variegated leaflets. According to our knowledge, this is the first report of producing Z. zamiifolia plants with variegated leaves using colchicine. Other than molecular breeding, which is costly and takes a longer period, conventional breeding cannot be employed to generate new plants, since there is only one species under this genus. As such, use of chemical mutagens such as colchicine would provide a rapid means of developing new plant types of this species with attractive morphological features since there is a very narrow range of variants available in the world.

Effects of Gradient Concentration of Colchicine on Callus Induction and Differentiation ofRose rosea

To induce polypoid plants ofRosa rugosa tunb, the effect of the interaction between the concentration of colchicine and the treatment time on the callus induction and differentiation of R. rugosa tunb was studied using colchicine as the inducer. The colchicine immersion method was selected, and the callus of red Rosa rugosa tunb was immersed in colchicine of different concentrations for 2h, 4h, and 8h. The best culture medium for inducing callus was as follows: MS+1.0mg/L6-BA+0.1mg/L NAA+9.0 g agar+30.0g sucrose. After cultivation, the callus was dense in texture, light green and obviously granular. After the callus was immersed in colchicine, the combination of different colchicine concentrations and treatment time had an interaction with the callus. Therefore, the best treatment method for polyploid-induced Rosa rugosa tunb polyploid should be higher colchicine combined with shorter processing time. This experiment can provide experimental basis and theoretical basis for cultivating Rosa rugosa tunbpolyploid plant with excellent traits, thereby increasing the content of essential oil in the plant itself.

In vivo colchicine manipulation for enhancing DH production efficiency in Triticum durum using Imperata cylindrica-mediated chromosome elimination approach

Durum wheat × Imperata cylindrica hybridization was carried out utilizing two durum wheat genotypes as maternal parents (A-9-30-1 and PDW 314) and I. cylindrica as a pollen source followed by in vivo application of colchicine to establish a standard protocol for colchicine application aimed at enhancement of doubled haploid production in durum wheat. Various concentrations of colchicine ranging from 500 to 7000 ppm were injected to the pollinated spikes at different time interval viz., 24 h, 48 h and 72 h after pollination. Application of 5000–7000 ppm colchicine at 48 h after pollination was established as the optimum colchicine dose for chromosome doubling as confirmed through cytological studies in durum wheat using I. cylindrica-mediated chromosome elimination approach. PDW 314 was identified as a durum wheat genotype having more potential for doubled haploid induction via in vivo colchicine application than A-9-30-1. Identification of optimum dose of colchicine for application at in vivo level for induction of doubled haploids has opened new vistas in durum wheat improvement programme.

The Effect Of Colchicine on Phenotypes And Stomata Pakcoy (Brassica Rapa L) Hydroponically With The NFT (Nutrient Film Technique) System

Colchicine (C22H25O6N) is a toxic alkaloid compound that causing a polyploidy in the plants, so it produces a robust individuals. The aims of this research are to know the effect of kolchhisin against phenotypes and stomata pakcoy (Brassica rapa L) hydroponically with the NFT (Nutrient Film Technique) system. This research used experimental method with a complete random design (CRD), five treatments and five replications. The data were analyzed by using ANAVA with 5% of credibility. The sample of this research were used pakcoy red arrow F1 nauli with colchicine P0 (Control), P1 (250 mg / L), P2 (500 mg / L), P3 (750 mg / L), P4 (1000 mg / L). The result of this study showed that the influence of colchisin against phenotype and the stomata’s size of pakcoy plant (Brassica rapa L). The high concentrations of colchicine can caused the size of the phenotype (leaves’ number, leaf area, wet weight, dry weight) pakcoy smaller than the control. While the size of the stomata increased, the largest of stomata size was found in the P3 treatment (750 mg / L).

Genetic Improvement of Calendula officinal through Mutation Induction using Gamma Irradiation and Chemical Mutagens

Calendulais one of the basic plants of ornamentals. It is used in various purposes as cut flowers and in gardens. They are rich with active compounds such as chlorophyll a and b, phenols, flavonoid, and carotenoids. The present study focused on mutation induction in order to improve the morphology of the plant which can increase the turnout of its market. In order to examine which mutagen will make the most noticeable and permanent improvement of the plant. For the mutagens, seeds were treated with three doses of gamma ray (2500, 5000, 7000Gy) and two chemical mutagens; colchicine and EMS; which were applied at concentrations of 1000, 3000, and 10000 ppm. With the aim to measure the plant improvement, we measured the plant morphology, its active compounds and protein profiling before and after treatments with both γ-radiation and chemical mutagen to see which one has the most effective imprint on the plant. The morphological parameters data showed the highest plant high by the two doses; gamma dose of 7000 Gy and EMS concentration 10000 ppm, while the lowest dose of colchicine (1000 ppm) made the most impact on the plant height. As for the highest number of leaves/plant, it was obtained at 5000 Gy and 10000 ppm of EMS, while 1000 ppm of colchicine had the same effect. But the number of flowers/plant was not affected by the gamma radiation and colchicine but increased significantly by 10000 ppm of EMS. Surprise, the flower diameter was not affected by EMS while decreased at 2500 Gy and 3000 ppm of colchicine. Considering the importance of flower shape, all treatments either gamma radiation or chemical mutagens showed noticeable changes. Whereas, the biochemical parameters measuring the active compounds, both Chl-a, Chl-b and carotenoids contents increased at 5000 Gy, while flavonoid increased at 2500 Gy. Although, all doses of gamma ray eliminated the phenols content. While the lowest used concentration of colchicine (1000 ppm) increased both of Chl-a, Chl-b, flavonoid and carotenoids, even though the 10000 ppm of colchicine increased the phenol. On the other hand, the highest concentration of EMS (10000 ppm) increased Chl-a and Chl-b while carotenoids, flavonoid and phenol increased at 3000 ppm of EMS. The protein-profile analysis showed fluctuation in the gene expression. The highest performance appeared at 7000 Gy of gamma treatment,1000 -3000 ppm of colchicine, while the same result was obtained by EMS at 3000 ppm.

In vitro polyploid induction using colchicine for Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger

‘Fengtou’ ginger is a high quality ginger (Zingiber Officinale Roscoe) cultivar only planted in Laifeng County, Enshi, Huhei, China, and is a National Product of Geographical Indication originated. ‘Fengtou’ ginger yield is low compared with most ginger. Therefore, it is imperative to apply breeding methods to improve yield. One strategy to accelerate breeding is polyploid induction, which could improve yield and maintain quality. In this study, different concentrations of colchicine (50, 100, 150, 200 mg/L) and different durations of exposure (3, 5, 7 days) were applied to stem segments of adventitious buds in ginger. Among obtained polyploidy plantlets were identified by flow cytometric analysis. The optimal induction condition of tetraploids was that the concentrations of colchicine was 150 mg/L, induced for 7 days, the chromosome doubling rate reached 18%. The tetraploid plants achieved were larger than their diploid plants for leaf length, leaf width, leaf thickness, stem diameter and guard cell. The soluble sugar, soluble protein, proline and other substances were also significant difference, especially carotenoids concentration was 1.375 times in contrast with diploid plants, which showed that tetraploids ginger had potentiality to increase yield and better adaptability. We explored the polyploid induction system of Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger. Studies of morphological characteristics and secondary metabolites have shown that tetraploids ginger have potentiality to increase yield and better adaptability.

Effect of colchicine on plant growth and leaf nutrient acquisition of sweet orange (Citrus sinensis (L.) Osbeck) cv. Mosambi

The present study was conducted on sweet orange cv. Mosambi mutants, developed by treating with different doses of colchicine (0.05%, 0.10%, 0.15% and 0.20%) and raised on Jatti Khatti rootstock. Two years after the field establishment of plants, the developed mutants were examined in terms of alterations in growth parameters and leaf nutrient acquisition. Colchicine had both inhibitory and stimulatory effects on the mutants developed at different doses. Reductions in stem girth and TCSA were recorded at 0.05% and 0.20% of colchicine treatment. Mutants developed at 0.10% and 0.15% colchicine concentration, had stimulatory effect on plant growth parameters. Higher nutrient acquisition was noticed in the leaves of mutants developed at 0.10%-0.15% doses, while lower macronutrient acquisition was recorded at 0.05% and 0.20% doses. Variation induced in the mutants by colchicine indicated that both lower and higher doses of mutagenic treatments may give rise to the mutants of economic importance and indicating its potential use as a mutagen.

Breeding dominant genic male sterility restorer line of Brassica napus L.

To facilate breeding process of Brassica napus, a microspore culture and molecular marker-assisted screening combined system were proposed in this research. At early flowering stage, F1 offspring of hybridized combination HY15A ​× ​HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore. Treatment with 50 ​mg/L colchicine resulted in embryogenic rate of 3.56 embryos/bud, which was substantially higher than control (0.78 embryos/bud). A total of 1,387 embryos and 862 single plants were obtained after induction culture. Ploidy detection was performed for the regenerated plants by flow cytometry. Diploid rates of microspores treated with 50 ​mg/L and 70 ​mg/L colchicine were 17.2% and 21.0% respectively, which was significantly higher than control (10.5%). Totally 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10. Approximately 54 of 108 plants generated a 305 bp amplification product, whereas the other 54 plants showed a 398 bp band, thereby satisfying 1:1 separation ratio (x20.05 ​= ​0.0093). These coincided with field identification results. Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time, thereby remarkably accelerate breeding.

Improving productivity of steviol glycosides in Stevia rebaudiana via induced polyploidy

The polyploidy induction using mutagenic chemicals is one of the breeding approaches to improve enhancing productivity of yield and secondary metabolites in medicinal plants. In the present research, to induce polyploidy in Stevia rebaudiana plants, the seeds were treated with four concentrations of colchicine (control, 0.05%, 0.1% and 0.2%) for 12, 24, 36 and 48 h in a factorial experiment based on randomized complete block design with three replicates. The morphological and phytochemical traits of plants were measured before the flowering stage. The results indicated that different concentrations of colchicine had a significant effect on the plant height, number of leaves, number of branches, mean of leaf length, stomata size and stomata density. The exposure times of colchicine also caused significant changes in the plant height, number of branches, leaf width and length (P ≤ 0.01). Enhancement of colchicine concentration, significantly increased the above mentioned morphological traits, along with stomata size and rebaudioside-A, while significantly decreased stomata density in comparison to the control (P ≤ 0.01). In contrast, the interaction of colchicine concentration and exposure time had a significant effect on leaf length. The findings of this study indicated that estimation of physiological changes and secondary metabolites amount is one of the effective methods in primary screening of polyploid plants in polyploidisation breeding program and flow cytometry is recommended to be used for accurate identification of ploidy level in Stevia rebaudiana.