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.     

Selective elimination of fibroblasts from cultures of normal human melanocytes

The main obstacle to establishing pure normal human melanocytes in vitro is contamination of the cultures by fibroblasts. The obstacle can be overcome by selective destruction of fibroblasts with geneticin ( G418 sulfate). Treatment of mixed cultures with this drug at a concentration of 100 micrograms/ml for two days results in pure cultures of normal human melanocytes.     

Inheritance of callus formation ability in anther cultures of rice,Oryza sativa L.

Summary Inheritance of ability to form callus in rice anther culture was studied using the diallel technique. Anthers containing uninucleate microspores from two japonica cultivais (Minehikari and Taipei 309), two indica cultivars (Mingolo and Suweon 290), and 12 F₁'s of the diallel crosses involving these four parents were cultured on Chaleffs R₂ medium and evaluated for callus induction. The parents showed significant differences in anther callus formation, from 41.9% (Taipei 309) to 0% (Suweon 290). Callus induction ability was inherited as a recessive character conditioned by a single block of genes. Additive gene effects were predominant. The japonica types seemed to be good combiners for callus induction. The order of dominance among the four parents was Suweon 290, Mingolo, Minehikari and Taipei 309.     

Effects of activated charcoal in anther cultures

Embryogenesis in anther cultures of Anemone Canadensis L., Anemone hupehensis Lemoine and Nicotiana tabacum L. was shown to be inhibited by abscisic acid added to the medium. However, this inhibition was reduced in the presence of activated charcoal (AC).
The presence of AC in the culture medium strongly promoted embryogenesis in anther cultures of Anemone Canadensis compared with other media combinations. Treatment of the agar medium with AC, which was removed before inoculation of the anthers, also stimulated embryogenesis, but treatment of the water constituent did not.
The number of embryos produced in anther cultures of Anemone Canadensis and Nicotiana tabacum was shown to be positively related to the length of lime of incubation on medium supplemented with AC. In the case of Anemone Canadensis the stimulating effect of AC was most pronounced when the first visible embryos had emerged.
The presence of anther-derived embryos from Anemone Canadensis in anther cultures of Anemone Canadensis and Nicotiana tabacum was shown to inhibit embryogenesis. It was also demonstrated that embryos from anther cultures of Anemone canadensis, Papaver setigerum DC and Clematis viticella L. produced phenolic substances, and that the concentration of these substances was higher in culture medium lacking AC. Treatment of such medium with AC could reduce the concentration of phenolic substances by more than 80%.     

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.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.     

Large-scale deletions of rice plastid DNA in anther culture

Summary Plastid DNA (ptDNA) in albino rice plants regenerated from pollen by anther culture was investigated by Southern blotting. Of the 20 albino plants investigated, 7 contained ptDNA that had suffered large-scale deletion. The size and location of the deletions differed among the plants. In all cases about 30 kbp of the region containing the PstI-2 fragment (15.7 kbp) had been retained. The deleted ptDNA molecules were retained in calluses derived from the roots of each albino plant.     

Pollen embryogenesis in anther cultures of Solanum carolinense L.

The direct differentiation of bicellular pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and plantlets was induced by culturing whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency of embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was similar to that of zygotic embryos. In a previous study, it was show that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. It appears then that there are two distinct pathways of androgenesis in this species that are determined by the type of auxin present in the medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BA benzyladenine - KIN kinetin - MS Murashige and Skoog     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

Androgenesis in vitro in anther cultures of chlorophyll mutants ofNicotiana tabacum

The course of androgenesisin vitro was investigated with anther cultures of two chlorophyll mutants of Nicotiana tabacum. A different sensitivity to the hormonal composition of the medium was revealed between the cultures White Seedling and Sulfur; the stimulatory effect of kinetin on the frequency of androgenesis was observed only in White Seedling cultures. In addition to green plants, “aurea” (mutation Sulfur) or “albino” (mutation White Seedling) phenotypes also differentiated in both cultures. The possible causes of variability in the participation of green and mutant forms are discussed.     

Chromosomal behavior of anther culture derived plants of rice

Cytological examination of anther-culture-derived plants of rice showed that the regenerants were predominantly diploids and haploids. Haploid meiosis indicated that the earlier hypothesis of rice being an ancient polyploid is unlikely. Diploids generally were normal and fertile. The low frequency of polyploids (1.5%) probably was due to rapid regeneration of plants from short term callus cultures.     

Haploid embryogenesis in anther cultures of pigeon-pea (Cajanus cajan)

Summary Pollen embryogenesis and callus showing a wide range of ploidy is induced in the in vitro cultured anthers of pigeon-pea. A suspension of pollen from such anthers incubated in drop cultures on agar medium develops further to form embryoids and colonies of callus.     

Molecular characterization of rice genes specifically expressed in the anther tapetum

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5-upstream region was found to regulate -glucuronidase expression in the tapetum of transgenic tobacco. A set of 5 deletions was also generated and a 1095 bp 5 region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.     

Production of haploid plantlets in anther cultures of Albizzia lebbeck L.

Anthers of Albizzia lebbeck on B₅ medium (BM) supplemented with kinetin (2 mg/l) and 2, 4-D (0.5 mg/l) showed callus initiation from microspores. Differentiation of embryoids and shoots was obtained on BM + BAP (1 mg/l) + IAA (0.5 mg/l) and of roots on BM. Root tip squashes of the regenerated plantlets showed the haploid chromosome number (n=13), confirming the microspore origin of the regenerants.     

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.     

Cytological analysis and genetic control of rice anther development

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte and haploid gametophyte generations.Rice (Oryza sativa) is the world's major staple food,and manipulation of pollen fertility is particularly important for the demands to increase rice grain yield.Towards a better understanding of the mechanisms controlling rice male reproductive development,we describe here the cytological changes of anther development through 14 stages,including cell division,differentiation and degeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form mature pollen grains through meiosis and mitosis.Furthermore,we compare the morphological difference of anthers and pollen grains in both monocot rice and eudicot Arabidopsis thaliana.Additionally,we describe the key genes identified to date critical for rice anther development and pollen formation.     

A GARP transcription factor anther dehiscence defected 1 (OsADD1) regulates rice anther dehiscence

Plant Molecular Biology - Anther dehiscence, one of the essential steps in pollination and double fertilization, is regulated by a complex signaling pathway encompassing hormones and environmental...