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1.
Ottenschläger I Barinova I Voronin V Dahl M Heberle-Bors E Touraev A 《Transgenic research》1999,8(4):279-294
The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollenspecific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ßglucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFPexpressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollenexpressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a nondestructive marker for plant reproductive biology and development. 相似文献
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Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment 总被引:1,自引:0,他引:1
Shariatpanahi ME Belogradova K Hessamvaziri L Heberle-Bors E Touraev A 《Plant cell reports》2006,25(12):1294-1299
The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood 相似文献
4.
Induction of embryogenesis from isolated apple microspores 总被引:2,自引:0,他引:2
We report, for the first time, the induction of embryogenesis and plant formation from isolated apple (Malus domestica Borkh.) microspores in vitro. Different isolation techniques were tested and an optimized protocol was elaborated. Furthermore,
the influence of the induction medium and starvation treatment, using different starvation material, temperatures and time,
were studied. In addition to embryo induction, the number of multicellular structures per divided microspores was found to
be a suitable parameter of assessment and could be used in earlier stages during microspore culture. Although the number of
embryos induced in these first experiments is low, the best frequency of embryo induction was shown to be at least twice as
efficient as that obtained by anther culture.
Received: 9 September 1998 / Revision received: 22 December 1998 / Accepted: 12 January 1999 相似文献
5.
Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies 总被引:9,自引:0,他引:9
Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA
ethylenediaminetetraacetate
- DAPI
4,6-diamidino-2-phenylindole 相似文献
6.
Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis. 相似文献
7.
A. Touraev A. Indrianto I. Wratschko O. Vicente E. Heberle-Bors 《Sexual plant reproduction》1996,9(4):209-215
We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries. 相似文献
8.
Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis 总被引:7,自引:0,他引:7
Mehran E. Shariatpanahi Ugur Bal Erwin Heberle-Bors Alisher Touraev 《Physiologia plantarum》2006,127(4):519-534
Microspore embryogenesis is the most commonly used method to produce doubled haploids. It is based on the ability of a single haploid cell, the microspore, to de-differentiate and regenerate into a whole plant after being exposed to stresses, such as low or high temperatures, carbon starvation and colchicine. Some stresses such as temperature treatments and carbon starvation have been used with success in many plant species, whereas others such as colchicine had limited application in a few species. Reports on the application of whole plant treatments with feminizing agents on inflorescences and buds are scarce. Furthermore, the technical means to apply some stresses such as γ-irradiation are not readily available. Recently, novel stresses such as pH, inducer chemicals, carrageenan oligosaccharides and heavy metals were reported to induce microspore embryogenesis. It remains to be seen, however, whether these stresses are effective in a wider range of species. Finally, pretreatment of cultured cells with high concentrations of 2,4-D efficiently induces somatic embryogenesis in several species (carrot, alfalfa). However, reports on the use of this particular chemical stress are not available in microspore embryogenesis. The paper presented here gives an overview of various stresses and mechanisms of action of these stresses in inducing microspore embryogenesis. 相似文献
9.
Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo 总被引:11,自引:0,他引:11
The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking.
Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured
in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was
followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised
anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1
microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This
type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a
peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned
in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores
first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles
and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures.
Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct
classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric.
Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore
which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing
cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected
at the root pole of the embryo.
Received: 27 December 1999 / Accepted: 11 May 2000 相似文献
10.
Julia Hosp Alexandra Ribarits Katarzyna Retzer Yongfeng Jin Alisher Tashpulatov Tatiana Resch Christina Friedmann Elisabeth Ankele Viktor Voronin Klaus Palme Erwin Heberle-Bors Alisher Touraev 《Plant cell reports》2014,33(7):1187-1202