Haploid and mixoploid cucumber (Cucumis sativus L.) protoplasts – isolation and fusion

This paper reports on the isolation of haploid and mixoploid protoplasts in the genus Cucumis. The cucumber mixoploid plants (C. sativus L., 2x/4x; 2x = 14) were obtained after oryzalin treatments and the mesophyll protoplasts (2x/4x/8x) were isolated and cultivated by well known in vitro methods. The influence of oryzalin pretreatment on the average viability and density of  protoplasts was tested. The average viability as well as the density is signifi cantly influenced by the oryzalin concentration, whereas the time-span of the treatment doesn’t have significant impact on the density and the viability. Callus formation was the highest level of regeneration in the experiments described in our study. Furthermore the isolation and cultivation of the cucumber and muskmelon (C. melo L.; 2x = 24) haploid protoplasts from young-stage pollen grains were improved. Subsequently, somatic hybridization between mixoploid cucumber protoplasts and muskmelon mesophyll and callus protoplasts, and kiwano (C. metuliferus E. Meyer ex Naudin; 2x = 24) mesophyll protoplasts, by chemical fusion with polyethylene glycol (PEG) 6000 was performed for the first time. Heterofusants were observed and developed into micro colonies. Additionally, the gametosomatic hybridization between mixoploid cucumber protoplasts and pollen muskmelon protoplasts was performed for the first time. Heterofusants and the first cell division were observed, however, the regeneration stopped in this stage. In conclusion, the different ploidy, especially the mixoploid character of isolated protoplasts, has positive influence on protoplasts isolation and the following fusion as represented by a higher regeneration capacity. In addition, both types of protoplasts, haploid and mixoploid, represent a unique systems for biochemical, molecular and genetic experiments. Especially, the haploid protoplasts could be used during in vitro fertilization

New chromosome counts and genome size estimates for 28 species of Taraxacum sect. Taraxacum

The species-rich and widespread genus Taraxacum F. H. Wiggers, 1780 (Asteraceae subfamily Cichorioideae) is one of the most taxonomically complex plant genera in the world, mainly due to its combination of different sexual and asexual reproduction strategies. Polyploidy is usually confined to apomictic microspecies, varying from 3x to 6x (rarely 10x). In this study, we focused on Taraxacum sect. Taraxacum (= T. sect. Ruderalia; T. officinale group), i.e., the largest group within the genus. We counted chromosome numbers and measured the DNA content for species sampled in Central Europe, mainly in Czechia. The chromosome number of the 28 species (T. aberrans Hagendijk, Soest & Zevenbergen, 1974, T. atroviride Štěpánek & Trávníček, 2008, T. atrox Kirschner & Štěpánek, 1997, T. baeckiiforme Sahlin, 1971, T. chrysophaenum Railonsala, 1957, T. coartatum G.E. Haglund, 1942, T. corynodes G.E. Haglund, 1943, T. crassum H. Øllgaard & Trávníček, 2003, T. deltoidifrons H. Øllgaard, 2003, T. diastematicum Marklund, 1940, T. gesticulans H. Øllgaard, 1978, T. glossodon Sonck & H. Øllgaard, 1999, T. guttigestans H. Øllgaard in Kirschner & Štěpánek, 1992, T. huelphersianum G.E. Haglund, 1935, T. ingens Palmgren, 1910, T. jugiferum H. Øllgaard, 2003, T. laticordatum Marklund, 1938, T. lojoense H. Lindberg, 1944 (= T. debrayi Hagendijk, Soest & Zevenbergen, 1972, T. lippertianum Sahlin, 1979), T. lucidifrons Trávníček, ineditus, T. obtusifrons Marklund, 1938, T. ochrochlorum G.E. Haglund, 1942, T. ohlsenii G.E. Haglund, 1936, T. perdubium Trávníček, ineditus, T. praestabile Railonsala, 1962, T. sepulcrilobum Trávníček, ineditus, T. sertatum Kirschner, H. Øllgaard & Štěpánek, 1997, T. subhuelphersianum M.P. Christiansen, 1971, T. valens Marklund, 1938) is 2n = 3x = 24. The DNA content ranged from 2C = 2.60 pg (T. atrox) to 2C = 2.86 pg (T. perdubium), with an average value of 2C = 2.72 pg. Chromosome numbers are reported for the first time for 26 species (all but T. diastematicum and T. obtusifrons), and genome size estimates for 26 species are now published for the first time

The pattern of genetic variability in apomictic clones of Taraxacum officinale indicates the alternation of asexual and sexual histories of apomicts.

Dandelions (genus Taraxacum) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis (clonal reproduction through seeds) in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts

Multilocus estimates of <i>F-</i> and <i>R-</i>statistic for all six microsatellite loci for studied apomictic <i>Taraxacum</i> accessions.

***<p>– significant value, P<0.001.</p

Character compatibility test for studied apomictic <i>Taraxacum</i> accessions.

<p><i>N</i>, number of samples; <i>NG</i>, number of genotypes; <i>MIC</i>, matrix incompatibility count; <i>E</i>, number of genotypes left at <i>MIC</i>  = 0. For taxon abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041868#pone.0041868.s001" target="_blank">Table S1</a>.</p

The Pattern of Genetic Variability in Apomictic Clones of <em>Taraxacum officinale</em> Indicates the Alternation of Asexual and Sexual Histories of Apomicts

<div><p>Dandelions (genus <em>Taraxacum</em>) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis (clonal reproduction through seeds) in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts.</p> </div

Distribution of molecular variance (AMOVA) compared among molecular markers and different hierarchy of studied groups of apomictic <i>Taraxacum</i> accessions.

<p>Values of <i>F</i>_ST correspond to the “Among apomictic accessions” differentiation; *, significant value, P<0.05.</p