Polyploidization Facilitates Biotechnological In Vitro Techniques in the Genus Cucumis

Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed

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

Changes of DNA methylation and hydroxymethylation in plant protoplast cultures

Cytosine methylation patterns in higher eukaryotes are important in gene regulation. Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms. In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs. of protoplast cultivation. In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation. It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture

Impact of Artificial Polyploidization in <i>Ajuga reptans</i> on Content of Selected Biologically Active Glycosides and Phytoecdysone

Polyploidization in plants, which involves doubling or further multiplying of genome, has the potential to improve the constituents that make medicinal plants, like Ajuga reptans, attractive to the pharmaceutical, cosmetics, and food production industries; botanical pesticide effects could also be derived. The aim of this study was to determine how artificial polyploidization in A. reptans plants affected the composition and quantity of biologically active substances from the glycoside and phytoecdysone families. Diploids and artificial tetraploids of A. reptans were analyzed. Changes in the contents of trans-teupolioside, trans-verbascoside, and 20-hydroxyecdysone were evident in the aboveground parts of the cultivated plants (e.g., leaves and flowers). The tetraploid lines of Ajuga plants displayed variability in, and increased levels of, trans-teupolioside and trans-verbascoside content. The 20-hydroxecdysone content was slightly higher in tetraploids. These findings indicated that Ajuga tetraploids could be used in breeding programs to enhance the yield of substances with potential medicinal and industrial applications

Biotechnologické in vitro metody u ohrožených druhů vrb =Biotechnological in vitro methods with endangerred willow species

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