Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.
Embryo-rescue was used to facilitate interspecific hybridization of Cucumis anguria L. and C. zeyheri Sond. Embryos were excised from developing fruits at one week intervals for six weeks after hand pollination. Medium containing coconut water was the most suitable for initial germination, and a medium with ascorbic acid was the best for embryo development and plant recovery. Viable plants were obtained from embryos and these plants showed morphological characteristics different from both parents. The analysis of the leucine aminopeptidase (LAP) locus revealed three hybrid types, H1.1, H1.2 and H2.
In vitro pollination can be used to overcome crossing barriers in interspecific hybridization within the genus Cucumis. This technique offers a way to produce viable interspecific hybrids. We tested two types of media, designated CP and YS, for in vitro pollination in Cucumis sativus and C. melo. Pollen grains were isolated by centrifugation or directly from mature male flowers and were cultured with mature ovules. We assessed pollen grain viability, fertilization ability, and fertilized ovule development. The developing ovules (becoming enlarged and green) were transferred to media supporting embryogenesis (with ascorbic acid, caseinhydrolysate, coconut water and gibberellic acid). The highest level of regeneration after in vitro pollination was callus formation from ovules. We found caseinhydrolysate to be the most beneficial component during in vitro pollination (CP medium) and during development of fertilized ovules (ON medium). The hybrid character of fertilized ovules arisen from crosses between cucumber and muskmelon was checked but not confirmed by RAPD analysis, for reasons we suggest. The in vitro protocol needs to be optimized further to obtain a high yield of potential hybrid embryos.K Ke ey y w wo or rd ds s: : Cucumis spp., cucumber, muskmelon, intraspecific hybridization, interspecific hybridization, in vitro pollination, in vitro fertilization, RAPD analysis.
Navrátilová B., Skálová D., Ondřej V., Kitner M., Lebeda A., 2011. Biotechnological methods utilized in Cucumis research -A review. Hort. Sci. (Prague), 38: 150-158.Our biotechnological research on selected Cucumis species has encompassed interspecific hybridization via embryorescue, in vitro pollination, somatic hybridization and cytogenetics of protoplasts. Embryo-rescue and in vitro pollination are suitable in vitro techniques for production of hybrid embryos. These methods were tested and optimized for cucurbits. Protoplast culture is another valuable tool for biotechnological applications, such as somatic hybridization and genetic transformation. We study protoplast dedifferentiation not only as a biotechnological application in breeding systems, but mainly to describe mechanisms of obtaining totipotency. Protoplasts of cucurbits were studied cytogenetically to observe changes in nuclear architecture during protoplastization and regeneration and for comparison with the expression profile obtained using cDNA-AFLP techniques and reverse transcription for the specific genes involved.
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.
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