Wide hybridization is a useful tool in plant breeding, but little is known about its possible range. For the cereals, wheat, barley and rye, this was tested with 15 different species of the Poaceae and Panicoideae. Embryo formation could be obtained with Agropyron repens, Alopecurus agrestis, Dactylis glomerata, Festuca glauca, Hordeum bulbosum, Lolium perenne, Pennisetum americanum, and Zea mays. As well, haploid as diploid embryos occurred. New embryo culture techniques should enable these embryos to grow to plants.
Haploidisation is a biotechnological method used to obtain plants with improved traits that are of use to humans. Lettuce (Lactuca sativa L.), a well-known and popular leafy vegetable, is consumed worldwide. Its haploid form would provide a good basis for producing a pure line of plants (doubled haploids) allowing new varieties to be regenerated. The main aim of this work was to develop an effective haploidisation method for this economically important species. In order to stimulate the development of haploid embryos of lettuce based on our previous experience, we conducted in vivo distant pollination with fresh pollen grains of Helianthus annuus L. or H. tuberosus L. Because the haploid proembryos obtained after pollination did not develop further (despite the presence of cellular endosperm), we implemented the technique of in vitro culture of an isolated embryo sacs (surrounded by endothelium) with parthenogenetic embryos on various, modified Murashige and Skoog media. During the in vitro culture, we observed the formation of callus tissue and, after subsequent cultures of calluses, 23 haploid L. sativa plants were regenerated. The haploid status of the regenerated plantlets was confirmed by estimation of the genome size by flow cytometry, chromosome counting in root tips, stomata cell size and by disturbances in pollen formation resulting from abnormal microsporogenesis. This paper contains the complete protocol for obtaining haploid plants of L. sativa.
Intergeneric hybridisation between Salix viminalis L. as the female and four Populus species (Populus trichocarpa, P. tremula, P. × canadensis and P. simonii) as male pollen donors was performed by in vitro stigma pollination. To overcome postzygotic barriers, transfer of hybrid embryos to new medium is necessary. We carried out detailed ultrastructural analyses to establish: (i) at which stage of embryo development the first signs of programmed cell death (PCD) could be detected; and (ii) at which stage the lack of serious or irreversible changes guaranteed that advanced development of hybrid plants could occur after embryo rescue. Transmission electron microscopy and confocal laser scanning microscopy revealed the presence of both developing and degenerating embryos. Developing globular, heart-shaped, and early cotyledonary embryos contained cells of correct ultrastructure. The only sign of intergeneric hybridisation was a delay in development for a few days, in comparison with control embryos. The earliest indicators of embryo degeneration were noted at 9 days after pollination (DAP). The most common indicators were excessive embryo vacuolisation, which was characterised by a large number of vesicles and formation of small vacuoles, as well as enlarged central vacuoles. Extended plastid thylakoids, folding of the cell wall, and autophagosomes were observed. Our detailed investigation of PCD in hybrid embryos enabled us to conclude that the embryo rescue technique was most effective in intergeneric willow × poplar crosses if applied between 9 and 16 DAP.
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