Twenty field-grown genotypes of diploid Italian ryegrass (Lolium multiflorum Lam., 2n = 2x = 14) were tested for their ability to induce callus and regenerate plants. Callus cultures were initiated from segments of immature inflorescences cultured on the MS medium supplemented with 4.0 mg L-1 2,4-D. The calluses were subcultured first on the maintaining medium (MS medium with 2.0 mg L-1 2,4-D) and later on the rooting medium (MS medium with 0.2 mg L-1 2,4-D). The frequency of callus induction varied depending on the source of explant and the initial genotype. A total of 473 green plantlets were regenerated, of which 420 were established in the soil. All these plants had the morphological characteristics of Italian ryegrass. Among 372 regenerants analysed cytologically, 302 (81.2%) had the expected diploid chromosome number (2n = 2x = 14), 65 (17.5%) were tetraploid (2n = 4x = 28); several aneuploids and mixoploids were also observed. All diploid and tetraploid regenerants were male and female fertile. However, a great variation of female fertility within and between both groups of regenerants was observed.
Ten Polish red clover cultivars were tested in vitro for their capability to produce callus and somatic embryos. A three-step tissue culture protocol (callus induction, embryo induction and plant development) based on Gamborg's B5 basal salts, NAA and 2,4-D as auxins, and kinetin and adenine as cytokinins was utilized. Explants source were petiole and hypocotyl sections. All clover varieties under study have showed high efficiency of callus production, and low ability for plant regeneration from the callus. The highest percent of genotypes which developed embryos was observed on variety Parka (3,6). Single embryogenic genotypes were selected from nearly all varieties. Hypocotyls showed the greatest ability for callus regeneration comparing to leaf petioles.
Experiments were carried out on (cvs. Wat and Hetman).L.luteus (cvs. Topaz and Iryd), (cv, Remik), and . For culture initiation 5- and 14-day-old seedling explants were used. The of the was stimulated by various media but the universal one appeared to be Gamborg's B5 medium solidified with Gelrite. For each investigated species a medium assuring 100% rooting of explants was developed. The most effective combination of growth substances for multiplication of L. hispanicus was found. Cotyledonary node explants, cultured on medium supplemented with benzyladenine and with or without naphthaleneacetic acid were stimulated to undergo in 4 and 2, for L. hispanicus and L. albus, respectively. Plantlets obtained from all experiments were easily potted.
The effect of sucrose and maltose in culture media PII, C17 and MN6 on androgenetic embryo formation was investigated in seven F2 triticale hybrid progenies. In all genotypes, the highest number of androgenetic embryos (89.8-320.6/100 anthers) was formed on medium C17 containing maltose. Also the highest number of green plants (6.17/100 embryos) was regenerated from embryos obtained on PII with sucrose. The effect of the physical environment (temperature, light) in the first week of embryo culture on the regeneration medium was tested. The highest rate of green plants per 100 embryos (2.5-11.8) was obtained from incubation at 22oC in the dark. Key words: androgenetic embryos, incubation temperature, media, plant regeneration, triticale.
The aim of the studies was to estimate the effect of cytokinins on the production of callus and shoots from different seedling explants of sugar beet. Two diploid multigerm genotypes were used in the experiment. The seedling explants (hypocotyls, cotyledons and leaves) were cultured on MS medium supplemented with cytokinins: BAP, 2iP, TDZ in concentrations 0,2; 1,0 and 2,0 mg/l. Hypocotyls, cotyledons and leaves showed different abilites to form callus and shoots. After two weeks of culture, the induction of callus was mainly observed on the explants of hypocotyls and cotyledons, whereas adventitious shoots formed on leaf explants. Frequency of callus and shoots induction was dependent on the origin of the explants, concentration and type of the cytokinins. The highest percentage of regeneration was obtained on the explants of young leaves, on medium containing TDZ (51,5%). The optimal concentration of cytokinins was 1 mg/l. Plant regeneration on seedling explants came only under direct organogenesis.
The influences of genetic and environmental factors on the anther culture responses of wheat were investigated. Significant differences for callus induction, plant regeneration, and green plant percentages were observed when the nucleus of Triticum aestivum L. cv. Selkirk was transferred to ten alien cytoplasms by substitution backcrosses. In most cases, the alien cytoplasms decreased anther culture responses, but sometimes they were as good as or better than the T. aestivum cytoplasm. Significant within-genotype variation for anther culture responses were observed for wheat varieties Chris, Yecora Rojo, WA7176 and Edwall, indicating genetic heterogeneity in the present commercial cultivars, and potential for improving anther culture responses by in vitro prescreening. When five genotypes (Chris, Pavon 76, Butte 86, WA6916, and Edwall) were cultured across three (potato-4 liquid, 100 g L-1 ficoll-supplemented, and 6 g L-1 agar-solidified) induction media, the liquid and ficoll-containing media were 10 to 15 times more productive than the agar-solidified medium. Whereas, the ficoll medium was not significantly different from the liquid medium. Several low concentration starch media appeared promising to replace current induction media. The starch media sustained the high-callus-induction properties of the liquid medium, while improving callus aeration similar to that observed on solid media, resulting in markedly higher plant regeneration and green plant percentages.
In plant tissue cultures, somaclonal variation is often observed. It can be an effect of the changes in the individual chromosome number or in the ploidy level. Flow cytometry, a fast and accurate method for the estimation of the nuclear DNA content, can be applied to study these changes. The DNA content in differentiated tissues of Nicotiana tabacum cultured in vitro was estimated using Partec CCA flow cytometer, starting from explant, through callus, up to regenerated shoots. The explant constituted stem segments of N. tabacum plants, non-transformed and transformed with gfp gene. Flow cytometric analysis showed differences in the proportion of 2C, 4C, 8C and 16C cells in plant tissue in different culture stages. Among the regenerated plantlets originated from non-transformed and transformed plants, diploid, tetraploid and mixoploid forms were observed. The transformation did not influence the share of cells representing different ploidy levels in the investigated plant material.
The effect of cold pretreatment of spikes on somatic embryo induction and ather culture response of 25 F1 winter hybrids was investigated.The efficiency of androgenic embryos was the highest when spikes were incubated at 4 C degree for 6-9 days.A total of 2242 (73%) green and 885 (27%) albino plants were obtained from 9900 cultured anthers.Anther culture response in wheat wsa found to be markedly affected by the genotype of donor plants.This percentage of green plants varied from 0 to 115.7%.The great majority of anther-derived regenerants were haploids (82.35%), while the remaining plants were spontaneous diploids (13,73%) and aneuploids (3.92%).
In Arabidopsis biotechnology plants are regenerated in vitro via shoot organogenesis induced in callus derived from different somatic tissues. An alternative way of in vitro plant regeneration via somatic embryogenesis has not been applied in Arabidopsis so far. Recently, it was found that development of Arabidopsis somatic embryos can be induced in the culture of immature zygotic embryos and that the callus phase is not nocessary for the initiation of embryogenesis. The aim of the presented research was to determinate the in vitro culture conditions enabling high efficiency of somatic embryo induction and their conversion into plants. The influence of induction medium composition including liquid or agar medium, type and concentration of auxin, carbohydrates and ammonium sources as well as duration of auxin treatment of explants on DSE efficiency were evaluated. Advantages of described regeneration system via DSE are as follows: short time needed to induce somatic embryos (10-15 days), high efficiency of the process (up to 90% explants responded), numerous embryos produced per explant (on average 17) and high percentage of embryo conversion into fertile plants (70-80%).
The aim of the presented paper is to show the present status of in vitro studies of Gentiana kurroo (Royle). Experimental material originated from seedlings, organs of regenerants, callus tissue, cell suspensions and isolated protoplasts. Two years of experiments proved very high morphological potential of plant material originated from various system of cultures. The mostly used media were those based on Murashige and Skoog medium, but for protoplast cultures, Kao and Michaluk medium appeared to be most useful. Numerous plant growth regulators with various concentrations and combinations controled plant regeneration of studied gentiana. Plant regeneration via somatic embriogenesis was observed in the majority of the studied culture systems.
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