Regeneration and characterization of plants obtained from anther cultures in Medicago sativa L

Summary The androgenic ability of four Medicago sativa L. genotype (Boynitza 5, Byala, 494, and 3815) was tested. Callus and organogenesis were induced in all lines studied. The percentage of anthers producing calluses and organogenesis showed wide variation (calluses—from 11% up to 77%; organogenesis—4.8% to 15.2%). It has been established that genotype, nutrient medium composition, and stage of pollen development considerably affected both callus production and organogenesis. Androgenesis in M. sativa could be achieved via callus and direct embryogenesis. About 500 morphologically different regenerants were obtained. Wide variability in chromosome number of regenerated plants was observed by cytological studies. Haploid, dihaploid, as well as mixoploid plants were obtained.     

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).     

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.     

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.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

In vitro induction of haploid,diploid and triploid plantlets by anther culture of Iochroma warscewiczii Regel

Pollen of Iochroma warscewiczii Regel (Solanaceae) produced embryogenic calli or embryos inside anthers cultured on Nitsch & Nitsch medium. Two distinct pathways could be recognized in this process, one involving mainly the vegetative cell, and the second starting with two equal cells in the pollen grains.In all media tested, androgenesis initiation was highest when anthers contained pollen at the first mitosis, or close to it, at inoculation. High sucrose (7%) and calcium (11.3 mM) concentrations were found to be highly desirable for the induction of androgenesis in this species. Addition of benzylaminopurine (0.5 mg l^–1) to the culture medium seems to slightly improve callus or embryo production. When all three factors were present at optimal concentrations as much as 13.9% of inoculated anthers were found to be embryogenic.Plantlet development from pollen embryos required lower sucrose (3%) and a combination of 0.1 mg l^–1 benzylaminopurine and 0.5 mg l^–1 gibberellic acid in the culture medium. Cytological analysis of 55 regenerated plantlets showed that about 49% were haploids, but diploid (ca. 49%) and triploid (ca. 2%) plants were also obtained.     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

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).     

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     

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) anther cultures

The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.     

The effects of KNO3 concentration in callus induction medium for wheat anther culture

KNO₃ concentration was found to significantly affect the anther culture of wheat (Triticum aestivum L.). When KNO₃ was increased from 0 to 15 mM (in cultivar Jinghua 1) or from 10 to 15 mM (in cultivars 2531-10, Xiaoyan 759 and Norin 10), the callus induction frequency increased significantly. When KNO₃ was increased further above 20 mM, the callus induction frequency decreased significantly in all the tested cultivars. The subsequent frequency of green plantlet regeneration increased significantly, and the ratio of green to albino regenerants increased sharply when KNO₃ concentration increased. Further experiments found that the decrease of callus induction frequency in the medium with too much KNO₃ might be caused by NO₃ ^- ion alone, while the effect of KNO₃ on green plantlet regeneration might be caused by both K⁺ and NO₃ ^- ions, and that the effects of NO₃ ^- concentration were independent of NH₄ ⁺ concentration in the medium.     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species

Crude methanol extracts from callus cultures of Nigella arvensis, N. damascena, N. hispanica, N. integrifolia, and N. sativa were investigated for their anti-microbial activity. Growth inhibition was determined in Gram-positive and Gram-negative bacterial strains as well as in yeast by using a broth-microdilution method. The results showed that the extracts of all calli tested exhibited significant anti-microbial activity, especially against Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis. Compared with other Nigella species, a callus culture of N. hispanica was the most effective against the microorganisms used in this study.     

Heterozygous diploid plants regenerated from anther culture of F1 rice plants

Summary Plants derived from anther culture are theoretically haploid, but diploid plants are also known to arise. Anther culture-derived diploid plants are usually homozygous and are believed to be due to spontaneous doubling of chromosomes in either microsporocytes or callus cells during the culture process. However, heterozygous diploid regenerants may also arise from a) regeneration from cultured somatic cells, b) mutation occurring during or after a spontaneous doubling event, c) fusion of unlike haploid cells in chimeric callus, and d) regeneration from diploid microsporocytes resulting from aberrant meioses. This study was designed to elucidate the frequency and origin of diploid regenerants from rice anther culture. Regenerants were obtained from 11 F₁ genotypes. Progeny testing detected heterozygosity in 7 out of 211 regenerants. Each of the heterozygous regenerants were from ‘Calrose 76’/waxy ‘M-101’, Half of the diploid regenerants from this cross were heterozygous. No heterozygous regenerants arose from the other 10 F₁ genotypes. Progeny testing indicated that two of the heterozygous regenerants were as heterozygous as the F₁ plants for three parental characters. The other five regenerants exhibited decreased levels of heterozygosity. One of the heterozygous regenerants exhibited evidence of mutation for a non-parental character. However, mutation is an unlikely cause of the observed high levels of parental-type heterozygosity. No evidence for the occurrence of chimeric callus was detected, making this an unlikely cause as well. The most likely origin of the observed partial heterozygosity is regeneration from diploid microspores, which could also produce plants exhibiting complete parental-type heterozygosity.     

The effect of starch and ineubation temperature in anther culture of potato

Three Andean tetraploid potato genotypes (2n=48) and 7 anther-derived dihaploids (2n=24) originating from two of the tetraploids were used in anther culture. Relative number of embryos/vial was significantly higher when the anther culture media was gelatinized with 3% potato starch than when Gelrite or wheat starch (3%) were used as gelatinizing agents. The degree of anther culture response varied between tetraploids but also within a group of related dihaploids. Additionally, the embryo production of individual genotypes, tetraploids as well as dihaploids, was dependent on the incubation temperature (10, 15, 20, 25, 30°C) of the anther culture. The incubation temperature of the anther culture was also important for the regeneration rate. Direct regeneration was mostly stimulated when the anther culture was incubated at 20°C.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid     

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.     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet Banana) hypocotyl cultures during shoot formation

Summary The simultaneous presence of 6-benzyladenine (BA) and sucrose in a Murashige and Skoog medium (SIM) during the initial stages of shoot initiation have been found to be obligatory for high-frequency shoot formation in the Capsicum annuum L. var. Sweet Banana upper hypocotyl explants. The explants are determined for shoot formation following a minimum of 8 days of culture on SIM. Deprivation of exogenous sucrose from day 6 to day 20 of culture had no effect on the shoot forming response of the explants. BA and sucrose appear to act independently on different aspects of the competence of explants to respond to SIM during shoot initiation.Abbreviations BA N⁶-benzyladenine - MS Murashige and Skoog medium - SIM shoot induction medium - HFM hormone free medium - SUC sucrose minus medium     

The rediscovery of Capsicum lanceolatum (Solanaceae), and the importance of nature reserves in preserving cryptic biodiversity

After more than 50 years, the rare autochthonous species, Capsicum lanceolatum, of Mesoamerica was rediscovered in a virgin remnant of the Guatemala cloud forest, Mario D'Arcy Avila Biotopo el Quetzal, a nature reserve for the resplendent quetzal. At all previous known locations C. lanceolatum was extirpated. Specific variation in flower and seed traits of the C. lanceolatum from the Biotopo el Quetzal population suggests it is a heretofore undiscovered population of this species. The usefulness of nature preserves in saving designated and cryptic species is illustrated.     

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (g _s) and transpiration (E), and an increase in stomatal resistance (r _s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g _s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, g _s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth. This revised version was published online in June 2006 with corrections to the Cover Date.