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Microspores were cultured on the modified B₅ liquid medium containing 2.4D (0.1 mg L⁻¹), NAA (0.1 mg L ⁻¹), L-glutamine (500 mg L⁻¹), L-serine (100 mg L⁻¹), and sucrose (100 g L⁻¹). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B₅ regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.
The research concerned of the regeneration of plants from embryos obtained from anther cultures of seven carrot (Daucus carota L.) cultivars. The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. The optimization of the adaptation of the obtained plants was also carried out. Embryogenesis occurred on four of the tested media: B₅ and MS without hormones, MS with charcoal, and MS with 1 mg dm⁻³ BA and 0.001 mg dm⁻³ NAA. Embryos obtained from the anther cultures produced secondary embryos, from which the regenerations of plants was observed. Secondary embryos were formed most extensively on the B₅ medium without hormones. The efficiency of the regeneration process depended on the cultivar. Most of the secondary embryos were formed by androgenetic embryos of the cultivar ‘Feria F₁’. The highest number of plants (102) regenerated from one embryo during 12 weeks of culture was also obtained in case of the cultivar ‘Feria F₁’. Secondary embryogenesis and plant regeneration from embryos allow to omit the difficult stage of root induction applied when plants are regenerated form shoots’ explants. This makes the plant regeneration process quicker and easier. The plants regenerated by the conversion of embryos are better adapted to the ex vitro conditions than those obtained in the two-stage organogenesis involving the regeneration of shoots and in second stage roots induction.
Doubled haploid lines of carrot can be obtained through androgenesis in anther cultures and in isolated microspore cultures. The two methods were compared using three carrot cultivars (‘Kazan F1’, ‘Feria F1’, and ‘Narbonne F1’) at the androgenesis induction stage, during plant regeneration from embryos, and during acclimatization of androgenetic plants as well as their characterization. It was found that cultivar was the main factor affecting the efficiency at each stage of plant production in both anther and isolated microspore cultures. The efficiency of androgenesis in anther cultures of ‘Feria F1’ was considerably higher in comparison with isolated microspore cultures, and more plants were obtained from the embryos of androgenesis-cultured plants. In ‘Kazan F1’ and ‘Narbonne F1’, more acclimatized androgenetic plants were produced from anther cultures. Ploidy assessment of acclimatized plants of ‘Narbonne F1’ showed that the majority of the plants in the population derived from anther cultures had a doubled chromosome (DH) set. On the other hand, the majority of plants obtained from isolated microspore cultures were haploids. When assessing homozygosity, it was found among plants obtained in anther cultures that the percentage of homozygotes for phosphoglucose isomerase (PGI) and aspartate aminotransferase (AAT) depended on the cultivar. In contrast, the majority of plants derived from isolated microspore cultures were homozygous regardless of cultivar.
Określono wpływ odmiany oraz pożywki na regenerację roślin z zarodków androgenetycznych marchwi. Badania prowadzono na zarodkach odmian ‘HCM’, ‘Splendid F1’, ‘Norbonne F1’, ‘Feria F1’. Zbadano 3 pożywki: MS-1 z 0,5 g∙dm-3 węgla aktywnego i MS-2 oraz B5-1 bez hormonów zawierające 20 g∙dm-3 sacharozy każda. Część uzyskanych po 12 tygodniach kultury, roślin typu „siewka” wysadzano w podłożu z wełny mineralnej oraz torfu i piasku (1:1) i umieszczono w pokoju fitotronowym. Po upływie 1 miesiąca liczono rośliny, które przeżyły i przeniesiono do szklarni. Na pożywkach bez hormonów wystąpiła wtórna embriogeneza. Pojedyncze zarodki androgenetyczne wytwarzały grudy zarodków wtórnych, które następnie przekształcały się w rośliny. Stosowana pożywka miała wpływ na proces regeneracji roślin z zarodków androgenetycznych marchwi. W pierwszym pasażu uzyskano na wszystkich pożywkach tylko kompletne rośliny. W drugim pasażu pojawiły się na pożywkach o bazie MS rośliny zniekształcone, nieukorzenione rozety oraz rośliny z kalusem. Regeneracja zarodków androgenetycznych marchwi na pożywce B5-1 przebiegała zależnie od odmiany. Z zarodków odmiany ‘Feria F1’ otrzymano tylko kompletne rośliny, a z innych odmian również rozety bez korzeni i grudy kalusa. Po 30 dniach od wysadzenia w podłożu z wełny mineralnej przeżyło 80% wysadzonych roślin, a w podłożu torf:piasek 1:1 około 40%. Rośliny w podłożu z wełny mineralnej nie rosły, a w torfowo piaskowym podjęły wzrost. Ze względu na to, że po przeniesieniu do szklarni rośliny zamierały, metoda wyprowadzania regenerantów ze szkła wymaga dopracowania.
Anthers of Feria F1 and Narbonne F1 carrot cultivars were cultured in vitro to induce androgenic embryos. To confirm the microspore origin of developed embryos, chromosome counts of root tip meristematic cells were made for each carrot plant obtained in anther culture. Using phase contrast technique and fluorescence microscopy, cytological changes of microspores during culture leading to proembryo formation were documented in the first days after anther placement on the induction medium. More than 90% of the carrot plants obtained in anther cultures had no haploid chromosomes.
The purpose of the present work was to evaluate carrot plants obtained from anther cultures with respect to their ploidy and homozygosity. Ploidy was determined using flow cytometry. Homozygosity of analyzed plants was determined using isoenzymes, glucose-6-phosphate isomerase (PGI) and aspartate aminotransferase (AAT). The cytometric tests revealed that more than 90% of the carrot plants obtained from anther cultures were doubled haploid. In the initial assessment of polymorphism of the two enzymatic systems in selected androgenetic carrot plants of the cultivars: Berjo, Kazan F₁, and Splendid F₁, it was proven that 100% of those plants were homozygotes in respect to PGI and also with respect to AAT. In the second experiment, the obtained androgenetic progeny of the heterozygous donor plant of cultivar Narbonne F₁ was found to be 94% homozygotic with respect to PGI and 100% homozygotic in the case of AAT. For the androgenetic plants of the cultivar Kazan F₁, 89% of them were homozygotic with respect to AAT but PGI enzyme system did not differentiate the homozygotic and heterozygotic androgenetic progeny. These results indicated that enzyme polymorphism depends on carrot genotype, therefore, analysis of some (more than one) isozymes was necessary to confirm homozygosity of plants. PGI and AAT can be a useful tool for determining homozygosity in androgenetic carrot plants.
The study was conducted to determine the effect of elevated concentrations of copper in the medium on the regeneration of androgenetic embryos of the carrot cultivar ‘Kazan F1’ obtained in anther cultures and to determine the level of soluble phenols produced in the regenerates under copper stress. Green embryos were laid out on 4 regeneration media based on B5 medium (G a m b o r g et al. 1968) without hormones, containing 0.1 – control, 1, 10, and 100 μM CuSO4×5H2O. The plant material was passaged 3 times, after 4, 9 and 15 weeks. During these passages the emerging structures were examined; they were classified in terms of growth and development in vitro, weighed and counted. The levels of soluble phenols in the freeze-dried regenerates were determined. The elevated concentrations of copper in the regeneration media affected positively the formation of complete plants (rooted rosettes) and secondary embryos during the first 4 weeks of culture. After a longer regeneration time (9, 15 weeks), the elevated concentrations of copper caused negative effects: deformation of rosettes. After 15 weeks, the number of rooted rosettes decreased. The 9-week culture subjected to copper stress brought about an increase in the amounts of soluble phenols. The highest values were recorded in the rosettes treated with 10 μM CuSO4. Prolonged exposure to media containing elevated concentrations of CuSO4 caused a reduction in the accumulation of phenolic compounds in the rosettes.
Badania prowadzono w celu określenia wpływu podwyższonych stężeń miedzi w pożywce regeneracyjnej na powstawanie roślin z zarodków marchwi odmiany ‘Feria F1’ otrzymanych w kulturach pylnikowych. Zarodki wykładano na 5 pożywek na bazie B5 bez hormonów zawierających: 0,1 - kontrola, 1, 10, 100 i 1000 μM CuSO4∙5H2O. Wykonano trzy pasaże po 4, 9 i 15 tygodniach kultury, obserwując powstające struktury, które klasyfikowano, liczono i ważono. Stężenia 1, 10 μM CuSO4w ciągu pierwszych 4 tygodni stymulowały organogenezę. Koncentracja 1 μM CuSO4 indukowała powstawanie największej liczby zawiązków rozet, a stężenie 10 μM CuSO4 korzystnie wpływało na tworzenie się wtórnych zarodków. W kolejnych pasażach wpływ podwyższonych stężeń jonów miedzi nie był już tak korzystny. W trzecim pasażu wyższe stężenia CuSO4 hamowały powstawanie prawidłowych ukorzenionych rozet. Najwyższe ze stosowanych stężeń miedzi okazało się toksyczne dla zarodków marchwi. Zamierały one nie rozwijając się w pierwszych 4 tygodniach kultury.
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