Comparison of methods for obtaining doubled haploids of carrot

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

Effect of increased copper ion content in the medium on the regeneration of androgenetic embryos of carrot (Daucus carota L.)

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

Research on in vitro haploidization of heterozygotic breeding lines of tomato (Solanum lycopersicum L.)

Experiments were made in order to examin the influence of various factors on the induction of androgenesis in heterozygous breeding material of tomato. The factors like: length of buds the manner of sterilization, type and the composition of induction media, genotype and thermal shock were included in conducted experiments. Most of all sterile cultures with the highest number of anther-derived callus were obtain by applying 2,5% calcium hypochlorite for 5 minutes. Anther-derived calli was obtained in 9 breeding lines from 18 used genotypes. The induction medium with the composition based on B5 medium with the addition of 750 mg L-1 calcium chloride and 100 g L-1 sucrose proved to be the best for inducing androgenesis. The addition of thidiazuron and NAA to this medium in the following season and silver nitrate in the other experiminet improved the efficiency of this process, which was depended on the genotype. Despite the lack of statistically significant differences, the highest number of anther-derived calli was obtained when anthers were cooled for 2 days in +4oC in the refrigerating chamber.Przeprowadzono doświadczenia, w których zbadano wpływ różnych czynników na indukcję androgenezy u heterozygotycznych linii hodowlanych pomidora. Poddano analizie następujące czynniki: długość pąka, sposób wyjaławiania materiału roślinnego, genotyp, rodzaj i skład pożywki indukcyjnej oraz szok termiczny. Wykazano, że długość pylnika jest dobrym zewnętrznym markerem faz rozwojowych mikrospor pomidora. Stosując 2,5% podchloryn wapnia przez 5 minut uzyskano najwięcej czystych kultur i największą ilość kalusa pochodzącego z tych kultur. Z 18 użytych linii hodowlanych u 9 linii hodowlanych uzyskano kalus pochodzenia pylnikowego. Skład pożywki indukcyjnej B5 z 750 mg L-1chlorku wapnia i 100 g L-1 sacharozy okazał się najlepszy dla wywołania androgenezy. Dodatek thidiazuronu oraz kwasu naftylo-1-octowego do tej pożywki w następnym sezonie oraz azotanu srebra w innym doświadczeniu podniósł efektywność tego procesu, który jak wykazaliśmy, uzależniony był od genotypu. Mimo braku statystycznie istotnych różnic zaobserwowano najwyższą ilość kalusa, gdy pylniki chłodzono przez 2 doby w +4oC

Influence of Polyamines on Red Beet (<i>Beta vulgaris</i> L. ssp. <i>vulgaris</i>) Gynogenesis

The influence of polyamines (PAs), putrescine (Put) and spermidine (Spd) on the efficiency of gynogenesis in ovule cultures of red beet (syn. beetroot) (Beta vulgaris L. vulgaris) cultivar “Czerwona Kula” and breeding accessions no. 3/2010 and no. 7/2008 was investigated. The effect of Put on the process of plant regeneration from gynogenetic embryos was studied. The response to the applied PAs was strongly dependent on the genotype. In “Czerwona Kula”, an increase in the number of obtained embryos was achieved by using each of the two PAs in the B5 medium. The effect of Spd was stronger. Put added to the regeneration medium at the concentration of 0.5 mg L−1 increased the number of obtained plants. All shoots placed on the rooting medium supplemented with 160 mg L−1 Put formed roots. The distribution of ploidy and homozygosity of gynogenetic plants depended on the genotype. Of the tested genotypes, the highest number of haploid plants, 68%, was obtained in red beet “Czerwona Kula”. The highest percentage of homozygotes, 69% for the glucose phosphate isomerase (GPI, E.C.5.3.1.9) isoenzyme and 100% for the aspartate aminotransferase (AAT, E.C.2.6.1.1) isoenzyme, was obtained in the population of gynogenetic plants of cultivar “Czerwona Kula”

Development of embryoids by microspore and anther cultures of red beet (Beta vulgaris L. subsp. vulgaris)

So far there is no information about receiving red beet androgenic embryos by androgenesis. Several factors were tested which affected this process: starch accumulation in microspores, correlation between bud length and microsporogenesis course, induction and regeneration medium composition. Ploidy level of obtained regenerants were evaluated. Treating anthers with α-amylase or watering donor plants with gibberellin increased number of obtained androgenic embryos. The highest percentage (80%) of microspores at uninuclear stage appeared in buds with 1.3-1.5 mm. The B5 medium with 100 g·L-1 sucrose and 0.1 mg·L-1 2,4-D (2, 4-dichlorophenoxyacetic acid) proved to be better for inducing androgenesis than MS medium supplemented with 0.2 mg·L-1 BAP (6-benzylaminopurine) and 0.5 mg·L-1 IAA (indole-3-acetic acid). First androgenic embryos were placed on B5 medium without plant growth regulators and then on MS medium containing 0.2 mg·L-1 BAP and 1 mg·L-1 NAA (α-naphthaleneacetic acid). Androgenic embryos died on B5 regeneration medium without plant growth regulators. On MS medium first shoots and callus with and without roots were obtained. Rosettes withered during following passages whereas callus tissue developed further. The quantity of DNA in this tissue equivalent to 4X chromosomes