Aqueous Solution Of Colchicine Research Articles

Some Observations on the Induction of Colchiploidy in <i>Solanum Melongena</i> Linn. (Brinjal)

The present investigation deals with the morphological, cytological and cytohistological studies between the normal and colchicine induced polyploids of Solanum melongena Linn. (variety Long Green).Treatments given to the growing tips of the young seedlings having two cotyledonary leaves with 0.2 percent aqueous colchicine solution for 12 hours gave best results from the point of view of survival of seedlings and the induction of polyploidy.The induced polyploid plants show thick, deep green and broad leaves with larger stomata and epidermal cells. The polyploid was characterised by larger flowers, pollen grains and seeds. The pollen fertility, fruit size and fruit setting in the polyploid plants were less than those of the control.The chromosome numbers of Solanum melongena variety long green (diploid) and the induced polyploid were determined as n = 12 and n = 24, respectively. Most of the possible chromosomal associations ranging from 12 quadrivalents to 24 bivalents at diakinesis and metaphase I were recorded in the polyploid. Abnormalities such as unequal distribution of chromosomes at anaphase, presence of lagging chromosomes were also noted in the polyploid.

Influence of the chemical mutagens on the seed germination, the growth and manifestation of decorative characteristics in the seed offspring of the representatives of the genus Pinus L.

The influence of various concentrations of chemical mutagens on the seed germination, the growth and manifestation of decorative traits in the seed offspring of mountain pine (Pinus mugo Turra), Scots pine (Pinus sylvesrtis L.), and Scots pine “witch’s broom” (“WB”) is assessed. A 0.1 % aqueous solution of colchicine, as well as 0.1, 0.5, and 1.0 % aqueous solutions of dimethyl sulfate (DMS) was used as mutagens. The species-specificity of the influence of chemical mutagens on the seed germination was established. Thus, the treatment of mountain pine seeds with aqueous solutions of colchicine and DMS had some stimulating influence on their germination. On the contrary, the germination of the Scots pine seeds and the seeds collected from the Scots pine “WB” decreased with increasing the DMS concentration in the solution. Among the offspring obtained using chemical mutagens, specimens were identified with changes in the growth strength towards dwarfism, the branching features of which manifested themselves in the form of apical dominance violation and shoot formation growth, with changes in the color of needles, including winter color changes of different-intensity color from green to yellow, bronze or bronze yellow. As a result of the research, 363 specimens with atypical traits were selected for further breeding work.

Colchicine Induced Polyploidy in Cassia tora (L.) Rox.

An Induction of artificial polyploidy by 0.4% aqueous solution of Colchicine has been successfully made in Cassia tora . The data obtained from the experimentally treated materials have been further analysed for all the implications to understand the effect of Colchicine on the Chromosome structure, and function. The tetraploids showed n = 26 bivalents and different combinations of univalent and multivalent, the percentage of quadrivalents being the highest Chromosome irregularities in PMCs of polyploids has been detected more frequently than normal diploid. The polyploids thus, raised did show autopolyploidy in nature. The chemical environment within the cell can change the meiotic behaviour of a species. The tetraploid plants showed a slower rate of growth, delayed flowering, thicker, darker and wider leaflet, and decreased stomatal frequency per unit area. This also showed larger stomata, increase in height, increase in number of lateral branching, bigger fruit, seed size and increase in the number of pollen grains with absence of exine. The pollen sterility increases as compared to diploid. It is thus clear that experimental induction by Chemical means (Colchicine) holds a clue for understanding the adaptability of the species in different environmental conditions.

In vitro Induction and Generation of Tetraploid Plants of Sophora tonkinensis Gapnep.

Background:Sophora tonkinensis Gapnep. is an important medical plant in China. Early researches of S. tonkinensis were focused on rapid propagation and quality analysis of in vitro tissue culture plantlet, and still no research focuses on the plant breeding of and there were no excellent varieties for artificial cultivation of S. tonkinensis.Objective:To set up a method to generate and select the best varieties of S. tonkinensis by polyploid breeding after induction by colchicine treatment.Materials and Methods:The adventitious buds were submerged in different concentrations of aqueous colchicine solution for different lengths of time to induce polyploidy in the plants, and the induced buds were identified by root-tip chromosome determination and leaf characteristics comparison. The contents of matrine and oxymatrine of radix ex rhizoma in 13 selected tetraploid lines were collected after 90 days in vitro rooting culture and were evaluated to provide evidence of good qualities of tetraploid S. tonkinensis.Results:The results showed that the highest percentage of tetraploid induction was 23.33% and occurred in the 0.2% (w/v) colchicine treatment for 30 h. Fifty lines of tetraploid plants were obtained and 12 of the 13 selected tetraploid lines exhibited higher productivity of total contents of matrine and oxymatrine when compared to controls.Conclusion:The data demonstrate that polyploidy induction can be beneficial for improving the medicinal value of S. tonkinensis.SUMMARY Colchicine has a good in vitro induction effect on the tetraploid plants of Sophora tonkinensisThe leaf indices and stomatal apparatus of tetraploid plants were slightly larger than diploid plantsThe total content of matrine and oxymatrine of some tetraploid lines for 90-day-old in vitro rooting culture was higher than the diploid. Abbreviations used: MS medium: Murashige and Skoog medium; BAP: 6-benzylaminopurine; NAA: A-naphthaleneacetic acid; IAA: Indole-3-acetic acid; KT: Kinetin; IBA: Indole-3-butyric acid; ABT: Rooting power.

Factors Affecting Doubled Haploid Plant Production Via Maize Technique in Bread Wheat

Abstract The effect of two in planta factors (growth conditions, genotype) and two in vitro factors (time of embryo rescue, embryo rescue medium) on doubled haploid (DH) plant production in bread wheat via maize technique was investigated in nine F1 hybrids produced after crossing four bread wheat cultivars. During the first year one group of F1 plants was grown in a field and at the proper stage pollinated with maize pollen (sweet corn popu-lation). In parallel, a second group of F1 plants was grown in a growth chamber and pollinated as in the former group. In the second growing season the experiment was repeated but only field-grown plants were used. All the produced haploid embryos were cultured in three different media and the resulting 146 haploid plants were sub-sequently treated with aqueous solution of colchicine. Finally, 86 doubled haploid plants were obtained. We noted that the growing conditions of the parental plants and the intervening time between day of pollination and day of embryo rescue influenced the percentage of haploid embryo production. Culture medium also influenced haploid and doubled haploid plant production. The two media (MS/2, B5) were found equally effective. Most of the haploid embryos originated from the Penios × Acheloos cross, whereas most of the doubled haploid plants were produced from the KVZ × Penios cross. Doubled haploid plants were produced from all crosses.

POLYPLOIDY INDUCTION OF MUTATION BY USING COLCHICINE ON TUBE SEEDLINGS OF VICTORIA GRAPE

Stem segments of the tissue culture-grown seedlings of the Victoria grape were soaked 24 or 48 h using an aqueous solution of colchicine (0.05% or 0.1%). The best treatment for chromosome duplication efficiency was the use of the third and fourth buds treated for 48 h in 0.05% colchicine or 24 h in 0.1% colchicine. The doubling rates of primary generation cells were 33% and 31%, respectively. The polyploid cell percentage increases about 10% by subculture of the chimeras. The polyploid cell percentage could reach 75.8%-96.6% by subculturing the chimeras for four to six generations. Through root tip cell chromosome observations, there was little effect on the division cycle of root-tip cells in terms of the cultured seedlings with or without illumination in the short term, and the optimal sampling time of root tips for chromo-some observation was at 9:30 am.

Production and Identification of Colchicine-derived Tetraploid Vaccinium darrowii and Its Use in Breeding

In Vaccinium L., most tetraploid hybrids between tetraploid cultivated highbush blueberry (V. corymbosum L.) and diploid darrow's evergreen blueberry (V. darrowii Camp) have been produced by exploiting V. darrowii's tendency to produce 2n gametes and a strong triploid block, which greatly reduces the number of triploid hybrids produced. Colchicine-derived tetraploids offer an alternative method of producing V. darrowii plants that will easily cross with tetraploid Vaccinium species. V. darrowii (2n = 2x = 24) seeds were imbibed in 0.2% aqueous colchicine solution for 24 h. The seeds were germinated and seedlings whose morphology suggested colchicine effects were selected at various stages of development. No macromorphological changes were consistently associated with chromosome doubling. However, stomatal guard cells and pollen size increased substantially as a result of chromosome doubling. Several types of plants were identified after colchicine treatment: 1) plants with a doubled LI (epidermal tissues) and LII (internal tissues) plant layers; 2) periclinal chimeras with a doubled LI layer and a normal LII; and 3) periclinal chimeras with a doubled LII layer and a normal LI. Of ≈4000 seedlings that emerged from colchicine-treated seeds, 200 were selected for further examination based on leaf and stem morphology. Of the 200, five appeared to be tetraploid in LI, LII, or both layers based on stomatal guard cell size and pollen size. Crosses between colchicine-derived V. darrowii (4x) plants and southern highbush blueberry (V. corymbosum) cultivars (4x) were successful compared with 4x–2x and 2x–4x crosses using diploid V. darrowii and tetraploid southern highbush blueberry. Stomatal guard cells and pollen screening of the colchicine-treated plants were used as indicators of doubled V. darrowii plants and periclinal chimeras. The results from the crosses between colchicine-treated V. darrowii plants and tetraploid highbush blueberry cultivars confirmed the information obtained by stomata and pollen screening.

English

The mutagenic effects of 0.2% aqueous solution of colchicine on gross and micromophological features of seedlings of Vigna unguiculata (L) Walp. and Cucumeropsis mannii (Naud.) were investigated. Terminal buds of two-week old seedlings were assaulted with 0.2% colchicine by the cotton plug and micro syringe methods and performances were monitored until maturity. Colchicine-treated V. unguicu-lata seedlings were observed to grow slower, had malformed leaves, flowered late and produced less number of seeds per pod than the control. Mean values of features like stomatal indices on both the abaxial and adaxial surfaces, terminal leaflet dimensions and trichome distribution between the treated and control were found to be significantly different. In contrast, treated seedlings of C. mannii flowered and fruited earlier than the case of control material. However, growth was also slowed down by the treatment while features like stomatal indices and trichome distribution were not significantly different. The results here have shown that apart from doubling of chromosomes, colchicine can also be used to induce other mutagenic changes which may be of agronomic utility.

A method to induce polyploids from adult strawberry plants, Fragaria spp., avoiding tissue culture in vitro

AbstractA method is described of inducing polyploids in detached stolons using an aqueous solution of colchicine, DMSO and glycerol. The donor plant remains uncontaminated and can be grown further to produce more stolons. The putative colchiploids can be chosen on a morphological basis and confirmed cytologically. About 10% of the treated stolons, starting with diploid and tetraploid strawberry, are converted, but 50% nonviability is caused by the treatment. The method can be applied to some other species and shoot types, using other chemicals with chromosome doubling or reducing effect, or even mutagens. The principle can be used without colchicine treatment to obtain strawberry plants from stolons without contamination of the soil around the main clone.

Colchicine-induced heritable variation in cell size and chloroplast number in the leaf cells of inbred ryegrasses (Lolium perenne, L. multiflorum)

A 3 hr period of treatment with a 0.2% aqueous solution of colchicine was given to one week old seedlings of 3 inbred lines of perennial ryegrass (Lolium perenne) and 4 lines of Italian ryegrass (L. multiflorum). The surviving plants developed as mixoploids and were self-pollinated. In the following generation (CT1) the seeds were grown without further treatment, and were screened cytologically at the seedling stage to separate out diploids from tetraploids. Only the diploids were kept, and in this CT1 generation comparisons were then made between controls (2x) and colchicine-treated diploids (C2x) for leaf mesophyll cell size and chloroplast number variation. Heritable differences were found such that the cell plan areas and/or the numbers of chloroplasts per cell were significantly greater in the C2x compared with the 2x treatment in 3 and 5 out of the 7 lines. In each case there was enhancement of chloroplast numbers independently of the variation in cell size.

Induced Autotetraploidy in Ageratum conyzoides L.

Polyploidy was induced in A. conyzoides plants with 0.25% aqueous colchicine solution. Autotetraploid plants were dwarf, with more number of branches. Leaves were smaller and thicker. Increase in the size of stomata and pollen grain was observed. Pollen and seed sterility also increased. Expected number of quadrivalents were never observed. Meiotic studies confirmed the induction of polyploidy in the plant.

Colchicine-induced heritable variation in cell size and chloroplast numbers in leaf mesophyll cells of diploid ryegrass (Lolium perenne L.)

A 3 hr treatment period with a 0.2% aqueous solution of colchicine was given to one week old seedlings of imbred lines of perennial ryegrass (Lolium perenne L.). The surviving plants developed as mixoploids and were subsequently split down into single tillers and then classified as either diploid or tetraploid. The undoubled diploids of the treated material (C2x) were then self-pollinated and the seeds grown in the following generation (CT1) without any further treatment. In the CT1 generation comparisons were made between the C2x and the control 2x treatments within the same inbred lines, and heritable differences were found for leaf mesophyll cell plan areas and chloroplast numbers. The cell areas were significantly less in the C2x compared with the 2x treatment in four out of the five lines studied, and the chloroplasts numbers were likewise lower in two out of the five lines. In one line there was a significantly higher mean number of chloroplasts per cell in the C2x material compared with that of the 2x.

Cytomixis in Agropyron cristatum

An Agropyron cristatum plant (CB-9-41), crested wheat grass, and its vegetative clones have been identified that contain pollen mother cells that have a gain or a loss in chromatin (DNA). CB-9-41 was identified during the course of an experiment to determine the effectiveness of colchicine on the doubling of the chromosome complement. The seeds that produced this plant were presoaked and then treated with a 0.1% aqueous solution of colchicine for 12 h. All stages of meiosis were studied in the original colchicine-treated plant and three vegetative clones that were obtained 17 years later. Approximately 40% of the pollen mother cells had meiotic irregularities. These irregularities were caused by multipolar meiosis (23%), precocious separation of bivalents at metaphase (8%), inversions (6%), and cytomixis (11%). The gain or loss of chromatin occurred as a result of cytomixis. Key words: crested wheat grass, extragenomic chromatin, multipolar meiosis, colchicine.

Colchiploidy in Lindernia crustacea (L.) F. Muell.

Autotetraploid has been raised in Lindernia crustacea, a medicinal weed, by treating the seedlings with 0.2 percent of aqueous solution of colchicine for 20 hours. Morphological and cytological characters of colchiploid and diploid were studied in detail. Cytology of colchiploid was highly non-synchronized and one interesting phenomenon was observed that the frequency of multivalents were very low whereas the sterility of pollen grains was very high. Other anomalies like clumping of chromosome at various stages, precocious separation of chromosomes, formation of univalents and unequal distribution of chromosomes at anaphase I and II were also observed and discussed. Pollen grains were variable in size.

Colchicine induced interchanges in chillies (Capsicum annuum L.)

Seeds ofCapsicum annuum L. cultivar cerasiformies were treated with 0.4 and 0.2% aqueous colchicine solution for 24 and 72 h respectively. Tetraploids were not realized; instead, interchange heterozygosity was observed in several plants in 0.4% treatment. The interchanges varied from 1 to 3 per plant. It is presumed that colchicine has induced chromosome breaks.

THE EFFECT OF COLCHICINE ON CHROMOSOME PAIRING

Beginning at 120 hours prior to first metaphase of meiosis (MI) a 0.03% aqueous solution of colchicine was injected into the boot of pentaploid (hexaploid triticale × tetraploid wheat) hybrids developing at 20 °C ± 1° under continuous illumination. Colchicine applied 40 h or less prior to MI had no effect on chromosome pairing, while its application 40 h or more prior to MI induced a steady decline, culminating in a 40% reduction in chromosome pairing at about 80 h from MI. Between 48 and 35 h before MI (late premeiotic interphase to early zygotene) meiocytes underwent a period of active nucleolar fusion. The time, therefore, at which the colchicine sensitive aspects of chromosome pairing were completed coincided with the completion of nucleolar fusion. From comparison with other findings it was concluded that there is a colchicine sensitive bouquet stage which appears in leptotene and early zygotene; this bouquet is responsible for active nucleolar fusion and final close association between homologous chromosomes.

Induced Polyploidy in Legumes

Seeds and seedlings of Glycine max variety Biloxi and C. max variety UPI have been treated with aqueous solution of colchicine. Seedling treatments have been found to yield tetraploid plants in both the varieties.In the tetraploids, deformed, thick and dark green leaves, stunted growth, delayed flowering, changes in size and frequency of stomata and pollen grains and reduced fertility have been observed and discussed with reference to previous observations. More than three leaf-lets have been observed in the tetraploids and the size of the leaf-lets has increased. C1 generation has also been studied.Meiotic studies in the tetraploid revealed different combinations of uni-, bi-, tri- and quadri-valents with tetraploid number of chromosomes. Exact doubling of chromosomes was observed in comparatively few cells. PMCs with complete set of chromosomes involving quadrivalent formation have not been observed. Occurrence of high frequency of bivalents has been discussed. Maximum number of quadrivalents has been found to be six.

Investigation of aqueous Tween 80 as a vehicle for intravaginal administration.

Tween 80 has been used for application of anti-inflammatory steroids to sensitive membranes (Johnson, 1959); it does not, however, appear to have been used for vaginal administration of oestrogens and antioestrogens which are often water insoluble. The effects of Tween 80 on the vaginal epithelium were therefore examined to determine whether it influenced the response of the tissue to oestradiol-17β. Biological assay. Mice of Tuck's No. 1 strain, ovariectomized when 6 weeks old, were primed with a single s.c. injection of 1 μg oestradiol-17β in 0·05 ml arachis oil 2 weeks before use. Groups of mice received single intravaginal injections of 0·01 ml of 0, 10, 20 or 40% aqueous Tween 80 or doses of oestradiol-17β (0·2 × 10−4 and 0·8 × 10−4 μg) in either 0·01 ml distilled water or 0·01 ml 10% Tween 80. An aqueous solution of colchicine (0·1 mg in 0·05 ml/mouse), an agent

Colchicine induced polyploidy in chickpeas (Cicer arietinum L.)

The most successful induction of tetraploidy was obtained with 2 hour treatment by 0.25% aqueous colchicine solution of 18-hour watersoaked desi chickpeas material. However, kabuli types needed only 1 hour treatment under similar conditions. Gigantism accompanied induction of polyploidy in desi as well as kabuli types but yield and fertility were greatly reduced. The meiotic abnormalities accompanying polyploidy were multivalent association of chromosomes followed by unequal disjunction, chromosome bridges, laggards etc. The percentage of stainable pollen, however, was at par between diploids and tetraploids. Gene control of percentage seed setting was observed in both levels of ploidy. A striking feature of the studies was the high seed setting percentage in 4n F 1 material resulting from diverse crosses, viz., desi×kabuli. A probable reduction in multivalent association coupled with yield increases in segregants from the later generation of tetraploids indicates the possibility of selection for higher yield and fertility from polyploids, particularly from some hybrid material.

Observations on Sterility of Induced Autotetraploid Watermelons1

Abstract Autotetraploid watermelons were produced by treating the growing points of diploid seedlings with aqueous colchicine solution or by soaking diploid seeds in colchicine solution. The soaking method was more effective. Diploid pollen was apparently more viable than tetra-ploid pollen. Examination of germinating tetraploid pollen revealed that while over two-thirds germinated normally a small percentage sent forth 2, 3 or 4 normalsized pollen tubes or a single bifurcated tube. Germinating diploid pollen always put forth only one unbranched pollen tube. Tetraploid plants did not set fruit when pollinated with pollen from tetraploids; however, they did set fruit when pollinated with pollen from diploids. Cytological observations were made of pollen mother cells of both diploid and tetraploid plants. Meiosis in diploids was regular; however, irregularities were observed in tetraploids. Examination of some pollen mother cells of tetraploids at the quartette stage revealed micro sporocytes in addition to the 4 microspores. The abnormal quartette formations and lower pollen viability of tetraploids were believed associated with irregularities observed at meiosis. Preliminary investigations of megaspore formation revealed no apparent differences between the diploid and tetraploid megaspores. Although meiotic irregularities were found, a sufficient amount of apparently viable pollen was observed. A physiological basis for the self-sterility is suggested.