ABSTRACT:The aim of this study was to develop a protocol for production of polyploid M1-generation plants of Viola × wittrockiana Gams. Two variants of colchicine treatment were compared for their efficiency. Early detection of novel ploidy levels was achieved by screening of stomata size, leaf index value (leaf blade length/width), and other morphological characteristics of the M1-generation. Secondary screening for novel ploidy levels was performed by flow cytometry (FCM). Hexadecaploid, aneuploid, and mixoploid plants were successfully identified by FCM.Keywords: flow cytometry; polyploidisation; colchicine; hexadecaploids; polyploidisation efficiency; morphological characteristics; Viola × wittrockiana Gams. × wittrockiana Gams. group of Pirna. The first method was achieved by soaking or dipping swollen seeds in colchicine aqueous solution (0.1%, 0.3%, 0.5%), for three treatment periods (7, 14 and 21 hours). The second one by treatment of the apex of the young seedlings. One drop of colchicine aqueous solution (1%, 1.5%, 2%) was applied to the apex of the seedling every day during the treatment period (3, 4 and 5 days). 5,400 of germinable seeds and 1,800 of young seedlings were treated in the experiment which was replicated three times. Immediately after the treatment, the plants were checked for the presence of different morphological characteristics. Some plants of the second treatment (swollen seeds) were different, compared to other seedlings in the same variant and to control. The cotyledons leaves were thicker, greener and smaller. The hypocotyls were thicker and shorted. The true leaves of these plants also, were thicker, greener and relatively were rounded. Also, most plants of the first treatment (of the apex) were different. The true leaves were thicker, greener and relatively were rounded. These different plants of both methods were selected and sampled for stomata size, leaf index value and hypocotyls length measurement.Screening of mentioned characteristics (primary selection) revealed that 232 plants (135 plants of the cv. Pure White and 97 of the cv. Light Blue) were presumably polyploids with higher ploidy-levels, and they were selected for FCM analysis. The chromosome number of the starting material was determined (2n = 48) in root-tips of germinated seeds as follows: root-tips were pre-treated for 4 hours at room temperature (RT) in 8-oxyquinoline 0.002 M solution, fixed in acetic acid-ethanol (1:3) and kept in the refrigerator (5°C) for 24 hours. Afterwards, they were hydrolyzed in 5N HCl for 30 min in RT, stained by Schiff reagent for 60 min in RT, macerated in 45% acetic acid for 2 min, and squashed in a drop of 45% acetic acid. FLOW CYTOMETRIC PLOIDY SCREENINGSamples were prepared according to GALBRAITH et al. (1998). Briefly, a small portion of leaf-stalk tissue (about 20 mg) was chopped with a sharp razor blade in a Petri dish containing 0.5 ml of Otto I buffer (0.1M citric acid, 0.5% Tween 20). The homogenate was filtered through a 50-µm nylon mesh to remove fragments and large tiss...

Jadrná P., Plavcová O., Kobza F., 2010. Morphological changes in colchicine-treated Pelargonium × hortorum L.H. Bailey greenhouse plants. Hort. Sci. (Prague), 37: 27-33.Polyploids were effectively pre-selected in colchicine-treated plants of the desirable brown-leaved cultivar Black Velvet Scarlet F1 of the species Pelargonium × hortorum L.H. Bailey to obtain the basic breeding material for creating new brown-leaved tetraploid cultivars. The green-leaved cultivar Gizela F1 was used for comparison of quantity and quality of response to colchicine treatments. Water solutions of colchicine in the range from 0.1% to 2.5% induced polyploidy in seedlings with treatments repeated each day for 2, 3, 5 or 7 days. Polyploid plants were pre-selected according to their morphological changes and stomata length and density and verified using flow cytometry. Some morphological changes (leaf coloration, flower shapes) in colchiploids differed between the genotypes, others were the same in both cultivars (loss of coloration in mixoploids, failure of blooming).

31Cultivars of Pelargonium × hortorum with attractive black leaves with a narrow green margin are relatively new. The first cultivar of this type was Black Velvet Rose F1, bred in Silva Tarouca Research Institute for Landscape and Ornamental Gardening (VÚKOZ) Průhonice in 1996 (the original name was Black Magic Rose F1; later the name of the whole series was changed to Black Velvet). The first cultivars of this diploid black-leaved F1 series were awarded for their originality and quality (Plavcová 2007).We aimed our work at induction of polyploidy in a cultivar from the Black Velvet series to obtain basic breeding material for creation of new black-leaved tetraploid cultivars with double or full flowers.Tetraploid cultivars of zonal pelargonium have larger flowers that are mostly double or full. The colours of flowers are often more interesting because of higher amount of genetic information in tetraploid cells.Nevertheless, the most important advantage of tetraploid cultivars (from a customer's point of view) is lower flower fertility, typical for autotetraploid plants due to disorders in segregation and dislocation of chromosomes in meiosis (Acquaah 2007). This means that the flowers remain blooming for much longer time, whereas after fertilization they would lose their petals and begin creating seeds, which is an unfavourable attribute for ornamental flowering plants. Thus, tetraploid cultivars of zonal pelargonium can be more interesting than diploid cultivars. This character however represents problems for plant breeders that they obtain fewer seeds for their work compared to diploid plants. Even so, it is profitable to have breeding programs for tetraploid pelargoniums because of their market importance (Hofmann 1992). MATERIALS AND METHODSTwo diploid F1 cultivars of Pelargonium × hortorum L. H. Bailey, a black-leaved cultivar with orangered flowers Black Velvet Scarlet F1 and a comparative green-leaved cultivar Gizela F1 with a standard type of leaf zone and bright-red flowers, were chosen to induce polyploidy in their seedlings. Seeds of these cultivars were sown into trays (each tray with Czech RepublicABSTRACT: This study is aimed at induction of polyploidy in the black-leaved cultivar Pelargonium × hortorum L. H. Bailey Black Velvet Scarlet F1 to obtain basic breeding material for creating new black-leaved tetraploid cultivars. The cultivar Gizela F1 was chosen as a control for the experiment. Tetraploidy was induced in seedlings in the cotyledon stage using various concentrations (from 0.1 to 2.5%) of colchicine water solutions; the treatments were repeated daily for 2, 3, 5 or 7 successive days. The first experiment, done in 2005, was very successful; 17.4% of treated Black Velvet Scarlet F1 plants and 23.7% of treated Gizela F1 plants were tetraploid, and other ploidy levels were also found. However, two other replications from 2006 (involving only five best treatments from the first experiment) were much less successful in comparison with the first one.

Pansies (Viola × wittrockiana Gams) are a commercially important ornamental plant, grown and marketed during autumn and spring (ADAMS et al. 1997).Two hexadecaploid (16x) genotypes were obtained by induced polyploidy in octoploid garden pansy cultivars (AJALIN et al. 2002). The hexadecaploids were selfed in advanced generations (LAGIBO, KOB-ZA 2004a,b) and used also for further breeding in order to exploit the induced desirable traits such as improved compactness and general vigour effect in plant height and in flower size. As a result, the genotypes with 2n = 10x, 2n = 12x, and 2n = 14x ploidy levels were produced from crosses and backcrosses which are included in this study.According to LEVIN (1983), polyploidy, induced or natural, may greatly alter the cytological, genetic and physiological characteristics, and it often alters resistance to cold. Moreover, the effect of polyploidy on frost tolerance may vary from species to species and in some plants it has been demonstrated to show an increase or decrease with higher ploidy level. For instance, in Brassica campestris, autotetraploids were more resistant to frost than their diploid counterparts (CHOUDHURY et al. 1968). On the other hand, GORAL et al. (1964) found lower resistance of tetraploids to frost in Trifolium repens and higher resistance of tetraploids in Trifolium pratense.In this study, in addition to induced polyploidy, since pansies are commonly planted in autumn for spring flowering, frost tolerance is one of the most important factors to be assessed in new genotypes. Therefore, this study was conducted to identify ABSTRACT: This study was conducted to interpret the differences in frost tolerance and winter survival between 8x, 10x, 12x, 14x, and 16x ploidy levels of garden pansy (Viola × wittrockiana Gams) genotypes grown in the field conditions. Plants of each genotype were analyzed for their ploidy levels using flow cytometry. The chlorophyll fluorescence parameters were measured with portable chlorophyll fluorometer in the greenhouse and in the field at different time intervals. Increased frost stress generally reduced the fluorescence values in all genotypes. However, the genotypes differed significantly in their responses to frost as they were exposed to minimum temperatures of 1°C to -7.7°C in the field. Based on the percentage reduction in F V /F M values against -7.7°C temperature the hexadecaploids were ranked as sensitive to intermediate followed by 12x (sensitive), and genotypes with 10x and 14x ploidy levels were tolerant as the controls. The winter survival rate of hexadecaploids was by 7 to 9% lower than in the controls followed by the genotype with 12x and both genotypes with 10x and 14x ploidy levels were about equal to the controls. On the other hand, the content of photosynthetic pigments (chlorophyll a, b and total carotenoids) was the highest in hexadecaploids and tended to increase with increasing ploidy level. Further, the results gave insight that chlorophyll fluorescence could be applied directly in the field conditions ...

Dalbato A.L., Kobza F., Karlsson L.M., 2013. Eff ect of polyploidy and pollination methods on capsule and seed set of pansies (Viola × wittrockiana Gams). Hort. Sci. (Prague), 40: 22-30.Pansy, Viola × wittrockiana, is a popular ornamental plant. Eff ects of polyploidy on phenotype and four pollination methods on capsule and seed set were studied using ten octoploid (2n = 8x) and two hexadecaploid (2n = 16x) genotypes, originating from induced mutagenesis. Principal component analysis, using 19 phenotypic, phenological and physiological characteristics, revealed that hexadecaploids showed larger diff erences to the corresponding standard cultivars than octoploids. Number of seed per capsule was similar among genotypes. Capsule set with open pollination was 32-64 %, with self-pollination by hand 18-49% and with cross-pollination by hand 14-43%, while no plant successfully set capsules with seeds under isolators if not pollinated manually. Th us, Viola × wittrockiana is self-compatible but requires an agent-mediation for successful pollination. Th e induced phenotypes were found stable over four generations. Hexadecaploids had more attractive phenotypes but fewer seeds than octoploids. However, variation in seed set enabled selection of plants with high fertility, and average seed set increased over generations. Th us, new varieties, fulfi lling aesthetic criteria as well as economic and agronomic traits, can be bred from induced mutagenesis. Pansies (Viola × wittrockiana Gams) are popular bedding plants. Whilst perennial by nature they are usually grown as an annual or biennial in garden beds, pots, borders, in hanging baskets or in landscapes (Horn 1996;Bailey 1998). Th ere is a seemingly unlimited need for new varieties. Th e planned pansy breeding through purposeful hybridization began in 1862 (Horn 1956). Breeding goals focused on selecting plants for unusual fl ower colours and increased fl ower size from the initial cross made between the small fl owered heartsease (V. tricolor L.) and yellow large fl owered V. lutea Huds. (Wittrock 1895). Hybridization between an alpine perennial, V. cornuta L., and V. altaica (Ker-Gwal) Pall. -a perennial with large and varied fl ower colours gave hundreds of pansy cultivars (Wittrock 1895;Horn 1956). Garden pansy (V. × wittrockiana Gams) is an octoploid (n = 6, 8x = 48) which is thought to be derived from cross combinations among V. tricolor (2n = 26), V. lutea (2n = 48), V. cornuta (2n = 22) and V. altaica (Clausen 1927;Horn 1956;Yockteng et al. 2003). Garden pansy is larger than its ancestors in plant height and fl ower size (Wittrock 1895).