Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture

In vitro chromosome doubling during ovule culture of sugar and fodder beets (Beta vulgaris L.) was studied with four anti-microtubule herbicides: amiprophos-methyl (APM), oryzalin, pronamide, and trifluralin at concentrations of 0–300 μM. Best chromosome doubling results were obtained by treatment of the ovules with 100 μM APM which produced 4.7 diploid plants per 100 ovules. Highest chromosome doubling was found with oryzalin using 1 μM, with trifluralin at 10 μM, and with pronamide at 10 μM producing 2.8, 2.0, and 2.0 diploid plants per 100 ovules, respectively. The APM treatments showed relatively low toxicity on embryo formation which in combination with a high chromosome doubling effect, resulted in up to 89 diploids per 100 plants regenerated. Oryzalin and trifluralin had more severe toxic effects, which reduced embryo formation, thereby lower percentages of chromosome doubled plants were obtained from these treatments. Pronamide had no significant toxic effect but it induced chromosome doubling at lower frequencies. Compared to colchicine, APM seems to be as efficient for chromosome doubling during beet ovule culture, but at molar concentrations 100 times lower than those used for chromosome doubling with colchicine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology

We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.     

Tetraploid bahiagrass hybrids: breeding technique,genetic variability and proportion of heterotic hybrids

Manipulating apomixis for fixing tetraploid bahiagrass (Paspalum notatum Flüggé) hybrids exhibiting superior agronomic characteristics would be a valuable tool for the genetic improvement of this species. The objectives were to create a second generation of hybrids by crossing sexual first-generation tetraploid hybrids (FGTH) and unrelated apomictic FGTH or ecotypes, determine the segregation for mode of reproduction, and estimate the resulting genetic variation and heterosis for several agronomic characteristics. The segregation for mode of reproduction was analyzed using mature embryo sac observations. Field measurements and visual ratings were used to evaluate the growth habit, production of inflorescences, cool-season growth and freeze resistance of hybrids. A ratio of 4.6:1 between sexual and apomictic hybrids was observed. Only 3% of the progeny was classified as highly apomictic. The low proportion of highly apomictic progeny restricts the feasibility of this breeding approach. Large genetic variation was observed among hybrids for growth habit, cool-season growth and freeze resistance. The proportion of hybrids exhibiting heterosis was high for plant height, cool-season growth, and freeze resistance. The tetraploid bahiagrass germplasm contains a large diversity for traits of agronomic importance that can be exploited for the genetic improvement of this species.     

Polyploidization and early screening of Rhododendron hybrids

Polyploid induction represents a useful tool for breeders of floral crops as larger flowers, longer flowering period and deeper colors can be achieved through chromosome doubling. This study aimed at testing the efficiency of colchicine and oryzalin in inducing polyploidy in three Rhododendroncultivars grown in vitro. The chemicals were used in two concentrations with 24 h and 48 h treatment durations. The survival of the plants was better in colchicine than in oryzalin solutions. The higher concentration of both chemical skilled more plantlets. The treatment duration in oryzalin did not affect the survival, but 48 h in colchicine was more destructive than 24 h. The low survival rate may not be a disadvantage, if the treatment induces desired ploidy. The ploidy levels were screened with flow cytometry. Oryzalin was more efficient than cochicine in inducing polyploidy, the treatment duration and the concentration did not have significant effects as main factors. The biggest proportion of solid tetraploids (18.2% of the survived plants) was obtained from the 24 h treatment in 0.005% oryzalin. Immediately after the treatment the polyploids grew very slowly, whereas most of the unaffected diploids were vigorous from the very beginning. More mixoploids than solid tetraploids were obtained in all treatments. Most of the mixoploids retained their chimerism, one third shifted todiploidy and one single plant to tetraploidy. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro chromosome doubling of Miscanthus sinensis

The aim was to develop an efficient chromosome doubling method for Miscanthus sinensis to enable the production of triploids and so avoid seed dispersal to the environment. Antimitotic treatments with colchicine or oryzalin were tested in M. sinensis cl. MS‐88‐110 on: (1) in vitro shoots and plants established in soil; (2) during propagation of embryogenic callus; and (c) during the initial stages of callus induction. All systems produced chromosome‐doubled plants. A higher percentage of tetraploids was found after antimitotic treatment at the explant or callus level compared with treatment of in vitro shoots or plants established in soil. In general, oryzalin was more toxic to plant material than colchicine. A higher frequency of chimeras was found among plants with altered ploidy level when the target was formed shoot buds compared with adventitious shoot formation from callus. Antimitotic treatment of embryogenic callus from shoot apices also resulted in a high degree of albinism.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

Evaluating different bahiagrass cytotypes for heat tolerance and leaf epicuticular wax content

Summary Bahiagrass (Paspalum notatum Flugge) is a polymorphic species indigenous to South America which has become naturalized in the southeastern U.S. The most common form in the United States is Pensacola bahiagrass,P. notatum var.saurae Parodi., which is a valuable forage. Pensacola is a sexual diploid, while most other bahiagrasses are apomictic tetraploids. Pensacola bahiagrass is considered to have greater heat tolerance (based on anin vitro heat tolerance test) than a number of otherPaspalum species, but has less leaf epicuticular wax (a drought avoidance mechanism) than other species. Both heat tolerance and leaf epicuticular wax are desirable characteristics for species grown where periodic drought occurs. We measured both characteristics over two years in a collection of 23 bahiagrass accessions, many of which had been collected in South America near the center of origin of the species. The collection included various ploidy levels. For both years, no accessions ranked statistically lower in damage in the heat tolerance test than Pensacola, although eight had significantly higher damage. Two entries in addition to Pensacola had very low damage in the heat tolerance test. Pensacola was high in leaf wax, with 16 accessions rated significantly lower in wax. The accession having the lowest wax content was a triploid, which also exhibited considerable leaf death in the field in response to drought and heat. The diploid entries tended to be higher in leaf wax than the other ploidy levels. This study has identified additional bahiagrass germplasm which may be of use in a breeding program.     

Role of mitotic inhibitors and genotype on chromosome doubling of <Emphasis Type="Italic">Rosa</Emphasis>

Dinitroanilines represent a class of compounds that are widely used in herbicide formulations as they depolymerise plant microtubles, causing chromosome doubling. The potential of microtubule depolymerising herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling of Rosa was studied. Five concentrations (0, 3, 6, 12 and 24 μM) and three exposure periods (12, 24 and 48 h) for each of the compounds were compared. Oryzalin, trifluralin and APM were not significantly different in their ability to induce chromosome doubling of R. hybrida cv Iceberg. At concentration of 6 μM and exposure period of 24 h, chromosome doubling of R. hybrida cv Iceberg was not significantly different with each of the polyplodising agents. At higher concentration (24 μM) and longer exposure period (48 h), 66.7% and 62.5% chromosome doubling was achieved with APM and trifluralin, respectively. However, the application of 6 μM oryzalin to R. persica (2n = 2x), R. hybrida cv Iceberg (2n = 3x) and R. hybrida cv Akito (2n = 4x), resulted in 60.0%, 6.3% and 0% chromosome doubling, respectively, which suggest that chromosome doubling is genotype dependent and plants with lower ploidy level have a higher propensity for chromosome doubling. Flow cytometry results at 18 and 24 weeks after herbicide treatment, indicated that the best time to test the treated plants was after 24 weeks.     

In vitro induction of tetraploid plants from callus cultures of diploid bananas (<Emphasis Type="Italic">Musa acuminata</Emphasis>, AA group) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’

Tetraploid plants were induced successfully from diploid bananas Musa acuminata (AA genome) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’ (2n = 2x = 22) with in vitro oryzalin treatment. Calluses from in vitro-grown shoot tips of both cultivars were treated with oryzalin at concentrations of 1.5 or 3 mg l⁻¹ for 24, 48 and 72 h, respectively. The oryzalin treatments produced tetraploids at a frequency of 15.6% in ‘Kluai Leb Mu Nang’ and 16.7% in ‘Kluai Sa’ as detected by flow cytometry. Chromosome counting showed that the tetraploid plant chromosome number was (2n = 4x = 44). The selected tetraploid plants were transplanted in the field and variations in the morphological characteristic of leaf shape and fruit bunch compared to normal diploid plants were found under the same growing condition even after 3 years of cultivation.     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages—prior to and after meiosis—but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.     

不结球白菜同源四倍体苏州青的诱导鉴定

为获得高产优质的不结球白菜四倍体苏州青,本研究使用浓度分别为0.1%,0.2%及0.3%的秋水仙素溶液,点滴处理二倍体苏州青幼苗植株的子叶生长点,进行5次处理后,通过形态学和细胞学方法筛选鉴定四倍体苏州青植株,并比较二、四倍体苏州青的农艺性状及品质差异。结果表明：秋水仙素浓度为0.2%时加倍效果最好。形态解剖学鉴定结果表明：四倍体在株型、叶片、花、种荚、种子大小都有较明显的变大趋势;四倍体植株叶片气孔较二倍体变大,气孔密度变小;同时四倍体植株花粉粒比二倍体更膨大,更不规则;细胞学鉴定结果表示,四倍体植株染色体数为40条,二倍体植株为20条;通过流式细胞仪鉴定,四倍体植株荧光强度约为二倍体的2倍。通过品质比较分析,结果显示四倍体苏州青的各项生理指标含量较于二倍体均有不同。通过农艺性状比较,单株质量、叶宽、叶厚都有一定程度的增加。本研究利用秋水仙素操作技术,抑制有丝分裂后期纺锤丝的形成,实现染色体加倍,创制不结球白菜新种质,为选育四倍体不结球白菜提供理论基础和种质资源。     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

Efficient in vitro Chromosome Doubling During Beta vulgaris Ovule Culture

The effect of in vitro colchicine treatment of sugar beet ovules, after 7 days culture, on embryo formation, regeneration and ploidy of regenerated plants was studied with 5 concentrations of colchicine and 5 durations of treatment arranged as a 5 × 5 factorial in incomplete blocks. The best results were obtained with the shortest duration of treatment (5 hours) and the highest concentration of colchicine (0.4 %) giving 5.0 diploid plants per 100 ovules with 62.1 % of regenerated plants being diploid. Statistical analysis revealed that treatment effects could be separated into a toxic effect reducing embryo formation and a chromosome doubling effect affecting percentages of diploid regenerated plants. Toxic effects on embryo formation could be explained by simple exponential decay models, toxicity of the drug (decay constant) increasing linearly with duration of treatment. Duration of treatment had no effect on chromosome doubling percentages. The effects of colchicine concentration on chromosome doubling were explained by an exponential saturation model with spontaneous chromosome doubling of 8.1 % and saturation at 51.4 % diploid plants at 0.2 % and higher colchicine concentrations. In addition, treatments increased percentages of 4N and 6N plants from 0 % without colchicine to 10 % on average for treated ovules. A response surface model fitted to the total yield of diploid plants per ovule indicated that shorter durations of treatment and higher colchicine concentration may improve results.     

Induction of <Emphasis Type="Italic">Anthurium andraeanum</Emphasis> “Arizona” tetraploid by colchicine in vitro

Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7 h, and then were transferred into Murashige and Skoog medium containing 3 mg/l BAP + 0.2 mg/l 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.     

马铃薯高效染色体加倍方法建立与抗寒资源创制

In this work, different treatment combinations including colchicine concentrations and treatment methods as well as different time points was used to investigate the survival rate and chromosome doubling efficiency of potato tissue culture plantlets. The potato chromosome doubling by using colchicine had been successfully optimized. The potato plantlet stems treated with 0.1% colchicine for three days shaking at 120 r min^-1 showed the highest doubling efficiency due to its better contact to the colchicine solution. Compared with other potato chromosome doubling techniques, this method has much higher chromosome doubling rate, shorter time treatment and easier to operate, so that it could provide a higher efficient method for potato ploidy operation. In the meantime, compared with the diploid, the tetraploid interspecific hybrids showed differences in the morphological characteristic, which had higher plants, thicker stem, bigger petals and pollen grain. In addition, no significant difference was found between diploid and tetraploid interspecific hybrids in terms of cold resistance, but both significantly enhanced cold resistance compared with the common potato cultivar. Taken together, the doubled interspecific hybrids could sever for improving cold resistance of potato cultivars.     

Chromosome doubling in Tripsacum: the production of artificial, sexual tetraploid plants

A collection of embryogenic diploid calli of Tripsacum was established and treated with colchicine to induce chromosome doubling. Sections containing duplicated cells in calli were identified using flow cytometry and ploidy level was determined in the regenerated plantlets. Tetraploid plants from several origins were obtained. In contrast to wild polyploid plants, which show apomictic development, the regenerated tetraploid plants reproduced sexually. By hybridizing these plants with wild tetraploid apomicts, various populations were established; these will allow a study of the inheritance of apomixis in Tripsacum.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid Alstroemeria aurea × A. caryophyllaea, resulted in no seeds. Backcrosses of the hybrid with parent A. aurea did not produce any seeds. In an attempt to restore the hybrid fertility, an efficient in vitro procedure has been developed and applied effectively in the chromosome doubling of the diploid hybrid. Forty-one percent of the treated plants were proven to be truly tetraploid by chromosome counts and stomatal measurements after applying 0.2 to 0.6% colchicine for 6 to 24 hours. Over 87.5% of these colchicine-induced tetraploids were stable and retained their tetraploidy after one year of growth. The fertility of the hybrid was not restored although the pollen stainability was increased from 0 to 12% after chromosome doubling. Cytological studies on the pollen mother cells (PMCs) of the sterile diploid hybrids revealed abnormal meiotic behaviors. In addition, aneuploid chromosome numbers, ranging from 2n = 1 to 2n = 18, were observed in over 45% of the PMCs examined. PMCs of the colchicine-induced tetraploids showed that meiotic chromosome pairings were normal in most cases (1.59_I + 15.07_II + 0.05_III + 0.03IV). These results indicate that the sterility of this hybrid is not only caused by parental chromosome differences, but other complex fertility/sterility-regulating mechanisms are involved too. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of sexuality in induced tetraploids of Brachiaria brizantha (Poaceae)

Difficulties in obtaining new breeding lines of Brachiaria (Trin.) Griseb., an important forage grass in Brazil, are mostly related to differences in ploidy among the accessions, and to apomixis, an asexual mode of reproduction. Usually, sexual accessions are diploid while apomicts are polyploid. Induced tetraploids of Brachiaria brizantha (A. Rich.) Stapf have been successfully obtained and this paper presents the results of a study of their reproductive modes and fertility. Despite frequent meiotic aberrations during microspore development, the induced tetraploids produced viable pollen and produced progeny after controlled self-pollination. Similarly to the original diploid sexual progenitor, embryo sacs of the Polygonum type with confirmed meiotic origin were present in the induced tetraploids suggesting chromosome doubling did not alter the reproductive mode. The embryo sac of the Polygonum type was also observed in progenies obtained after self and open pollination. Nevertheless, embryo sacs of the Polygonum and the Panicum types within the same ovule were observed in some progenies obtained after open pollination, probably having resulted from hybridization with tetraploid, apomictic plants. Indeed, the compatibility of the progeny with tetraploid, apomictic B. brizantha was confirmed by the formation of mature caryopses after controlled pollination. Evidence is presented that the induced tetraploids and their progeny are sexual plants and that they are compatible with natural tetraploid B. brizantha. The induced tetraploids will be useful for analyses of apomictic inheritance as well as in the development of sexual tetraploid lines in Brachiaria breeding programs.