Effects of carbon dioxide in anther cultures

In anther cultures of Anemone canadensis L., Anemone dichotoma L., Anemone hupehensis Lemoine, Clematis viticella L. and Papaver setigerum DC. a positive relationship between incubation in 2% CO₂ and the production of microspore-derived embryos was observed. In anther cultures of Nicotiana tabacum L., Anemone hupehensis and Clematis viticella a combination of cold treatment (7°C) and incubation in 2% CO₂ increased embryo production. In Anemone canadensis cold treatment increased the number of proembryos, whereas incubation in 2% CO₂ had no effect. In Anemone hupehensis 5% CO₂ increased embryo production by more than 2%. In Anemone dichotoma and Papaver setigerum 2% CO₂ was the more efficient level. CO₂ had no significant effect on pH in the culture medium in anther cultures of Anemone canadensis.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

Correlations between activated charcoal, Fe-EDTA and other organic media ingredients in cultured anthers of Anemone canadensis

The effects of activated charcoal and organic substances on embryogenesis in anther cultures of Anemone canadensis L. were studied. Embryogenesis was independent of the presence in the culture medium of glycine, nicotinic acid, pyridoxine, thiamine, folic acid, d -biotin or myoinositol. Absence of Fe-EDTA totally inhibited embryogenesis. Activated charcoal (AC) adsorbed Fe-EDTA, pyridoxine, folic acid and nicotinic acid in a double-layer medium almost completely within 24 h. If petri dishes according to the souble-layer method were stored overnight or more, embryogenesis was totally inhibited, probably due to adsorbtion of Fe-EDTA by AC. It was shown that AC itself released some yet unidentified substance(s) that stimulated embryogenesis. The addition of polyvinylpolypyrrolidone (PVPP) to the culture medium stimulated embryogenesis, but PVPP was not as efficient as AC. Embryogenesis was totally inhibited when AC and PVPP were applied together. Minor additions of ethanol to the culture medium stimulated embryogenesis when AC was present, but no such effect was obtained when AC was absent.     

In vitro segregation of marker genes in anther cultures of rice

Summary Segregation of genes controlling expression of anthocyanin pigmentation in rice (Oryza sativa L. subsp.indica) leaf blade and leaf sheath was examined in the microspore-derived plants. The segregation pattern of marker genes was found to fit closely the expected gametic segregation ratios among microspore-derived green as well as albino plants. Microspore-derived in vitro regenerated plants expressed genetic traits similar to seedlings. The results indicate that the germ cell culture technique can be of significance while monitoring gene action, i.e. anthocyanin synthesis at monoploid phase of plant development.     

Effects of incubation temperature on microspore callus production and plant regeneration in wheat anther cultures

Anthers of wheat cultivars Orofen and Pitic 62 were incubated for 8 days at 15, 20, 25, 30, 35 and 40°C before transfer to 25°C. Compared with anthers cultured at 25°C constantly, anthers treated at 30°C produced 40% more microspore callus and green plants in both cultivars whereas those treated at 35°C produced 2–3 fold more green plants. Treatment at 40°C was deleterious. Possible modes of action of high temperature on callus production and albinism were discussed.     

桑树花粉愈伤组织的诱导与花粉细胞学研究

讨论影响桑树花粉愈伤组织诱导频率的问题 ,描述花粉细胞早期发育和愈伤组织的分化过程     

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal

Summary Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.     

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.     

Phenotypic and cytological variation among plants derived from anther cultures ofLilium longiflorum

Summary Anther culture of the Easter Lily (Lilium longiflorum; 2n=2x=24) was attempted in order to evaluate its potential in generating haploids for the production of hybrid cultivars. The effects of genotype, temperature (low temperature treatment of buds and high temperature treatment of cultures), sucrose concentration and growth regulators were tested. The most important factors for callus induction were the genotype and the presence of 2,4-dichlorophenoxyacetic acid. Pre-treatments at low or high temperature had no apparent effect, while high sucrose concentration was inhibitory. Callus was derived from 28 of the 108 genotypes tested and plants were regenerated. Phenotypic variations were observed among these regenerants. Somatic chromosome numbers were determined in 42 plants derived from 10 donor genotypes. Thirteen plants were diploid and 29 were mixoploid with chromosome numbers ranging from 11 to 26. Four of the mixoploid plants had a high proportion of cells with haploid chromosome numbers, particularly at early stages of development. Meiosis was examined in plants with flower buds. Most plants had 12 bivalents at Metaphase I, but also aneuploids were observed. Other irregularities included bridges and laggards at Anaphase I. The occurrence of high frequencies of haploid cells (up to 80%) in root tips suggests that some plants may be of gametic origin. Research was supported by the Easter Lily Research Foundation, the Ohio Floriculture Foundation, the Gloeckner Foundation and the Oregon Agricultural Experiment Station (technical paper no. 8398).     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Beneficial effects of activated charcoal on plant tissue and organ cultures

Summary Addition of activated charcoal to the medium for plant tissue cultures improves growth by adsorbing toxic metabolites. This research was supported in part by the National Science Council, Republic of China     

Effects of donor plant environment and light during incubation on anther cultures of some spring wheat (Triticum aestivum L.) cultivars

Four different growth environments (field, two phytotron greenhouses and one growth chamber) were compared, using two genotypes of spring wheat, one recalcitrant and one responsive. Field-grown plants gave inferior results. Large improvements could be made by improving the conditions, embryoid frequencies in the two genotypes reaching 77.1% and 183.9% per 100 anthers, respectively. High light intensity during the induction phase strongly suppressed induction in both genotypes, but stimulated regeneration of green plants in the recalcitrant genotype, which had the lowest regeneration ability. Weak, diffuse light did not inhibit induction while the positive effect on regeneration was maintained. Also, another recalcitrant genotype was grown in the field, together with two F₁-hybrids (recalcitrant x recalcitrant and recalcitrant x responsive). Evidence for a three-factor system was obtained.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum

Production of bulblets of Muscari armeniacum through tissue culture is enhanced when 1 g/l activated charcoal is added to a modified Murashige and Skoog (MS) medium. Bulblet regeneration is direct from bulb scale explants with no intermediate callus growth. Bulblets can be transferred successfully to a greenhouse environment directly from aseptic culture.     

Effects of exogenous application of polyamines on wheat anther cultures

Androgenesis of wheat genotypes was evaluated by pretreating anthers or embryo-like structures (ELS) with polyamines. Anthers of the genotype DH were pretreated with different concentrations of putrescine, spermidine, and spermine for 1, 3, and 6 h, and those of drought-tolerant International Center for Agricultural Research in the Dry Areas (ICARDA) wheat accessions were treated for 1 and 3 h. ELS of two genotypes were also treated for 30 and 60 min with the same polyamines and evaluated for green plant regeneration. The pretreatment of anthers with polyamines enhanced the development of ELS in all genotypes. The formation of ELS varied significantly with genotype. Pretreated anthers showed that four treatments improved significantly green plant regeneration with the genotype ICR 17. However, two treatments (1 mM putrescine or spermine for 1 h) significantly improved green plant regeneration per 100 ELS of only two ICARDA genotypes. ELS treated with polyamines for 30 min were greener and formed more adventitious roots. The chloroplasts of these greener ELS examined with a transmission electron microscope had agranal to grana thylakoids, while those of the control had plastids with mostly starch globules. Although exogenous application of polyamines to anthers improved the production of ELS and green plants, the effects of putrescine, spermidine, and spermine was dependent on genotype and the duration of pretreatment of anthers with the polyamines.