Genetic analysis of a polyembryonic mutant in rye

We have obtained one plant regenerated from rye tissue culture which showed a high percentage of polyembryonic seeds in its progeny. The mutation inducing the development of extra embryos is also influencing erroneous cell division, mitosis and meiosis. The genetic analysis indicated that the aptitude for polyembryonic seed formation is a heritable trait controlled by a dominant gene. However, for expression of the phenotype the female parent should have a specific cytoplasm.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto-oncogene expression and the behaviour of myeloid leukemia cells in vitro

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c-fms, c-fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c-fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Root culturing ofFaidherbia=Acacia albida as a source of explants for shoot regeneration

Root culture ofFaidherbia=Acacia albida (Del.) A. Chev. can be maintained over several months by successively subculturing excised roots in modified Bonner and Devirian medium, which was found to be the best of the three media compositions tested. Adding auxins had an inhibiting effect on root growth characteristics. Mesoinositol at 0.05 mM slightly enhanced the overall elongation rate, and sucrose at 59 mM significantly increased root elongation. The effect of sucrose could not be replaced by glucose. Subcultured roots showed progressively less elongation in successive transfers. Shoots were regenerated in vitro from root segments beginning with the first passage on 1/5 strength MS medium.The large variability in the root elongation rate indicates that this technique provides an effective way to select clones with good potential for tap root growth.     

一种早期胚胎体外培养新方法的建立

建立一种不依赖于液体石蜡的早期胚胎体外培养方法——套皿法,并比较了4种不同处理方法体外培养胚胎的效果。结果显示,采用套皿法进行胚胎培养,盖液体石蜡和不盖液体石蜡皿中的胚胎在各阶段的发育率差异不显著。与套皿法相比,用单皿微滴覆盖法(Brinster法)培养的胚胎在各阶段的发育率显著降低。不覆盖液体石蜡单皿中的胚胎则阻断于二细胞阶段。实验设计的套皿法是一种有效的早期胚胎体外培养方法,为早期胚胎体外培养提供了一种新方法。     

Defined nutrient medium for the in vitro maintenance ofXenopus laevis oocytes

Summary A procedure is described for the isolation and culture of large numbers of follicle cell-freeXenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenance of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morphological and functional characteristics of oocytes for periods up to 3 weeks. Research supported by grant BMS 74-18790 from the National Science Foundation to JJE. Operated by Union Carbide Corporation for the U.S.E.R.D.A.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Use of Refrigeration as a Practical means to Preserve Viability of in Vitro-Cultured IDE8 Tick Cells

In vitro cultivation of the IDE8 cell line, derived from embryonic Ixodes scapularis ticks, constitutes an important system for the study of tick-borne pathogens, as these cells support growth of rickettsial species which are not normally transmitted by this tick. However, since cryopreservation of IDE8 cells is not always successful, there is a need to develop alternative ways to preserve these cells. In the present study, a suspension of IDE8 cells in culture medium was kept under refrigeration at 4°C for up to 60 days. Every 15 days, the suspension was mixed and aliquots were re-cultured in 2-ml tubes, under standardized conditions. In addition, three techniques for cryopreservation, using two different cryoprotectants (DMSO and glycerol), were evaluated. Medium changes were carried out every week and subculturing every 2 weeks. The development of cultures and their respective subcultures, after returning to standard culture temperature, was evaluated by percentage viability and by cellular morphology evaluated in Giemsa-stained cytocentrifuge smears. All cultures and subcultures appeared healthy, showing growth rates comparable to cultures that had not been kept under refrigeration. The results demonstrated that storage under refrigeration at 4°C is an efficient method for preservation of IDE8 cells for up to 60 days and that refrigeration may be preferable to cryopreservation for short-term preservation of IDE8 cells.     

Production of normal,germinable and viable pollen from in vitro-cultured maize tassels

Summary Immature tassel meristems (1.0–1.5 cm long) of Zea mays L. inbred, Oh43, and single cross hybrid, Se60, cultured on a nutrient liquid medium underwent extensive development through to maturity and produced normal, mature, trinucleate pollen grains. The grains germinated on nutrient agar and on receptive silks and also produced viable kernels. No differences were observed between in vitro-produced pollen and in vivo pollen (pollen from greenhouse-grown plants) in characteristics such as pollen size, in vitro and in situ germination, and pollen tube growth in vitro. The kernels produced with in vitro pollen grew into mature plants (in vitro plants) which were similar to in vivo plants (plants produced with in vivo pollen), with no significant differences for all the morphological characteristics measured, and no phenotypic and cytological abnormalities. Gel electrophoresis of polypeptides revealed no major differences between in vitro and in vivo seedlings. This demonstration of fertilization and production of normal, uniform plants with pollen from cultured tassels has significant potential in basic and applied research studies.     

Chromosomal variability in a tissue culture of rare species of the genus <Emphasis Type="Italic">Gentiana</Emphasis> L.

A cytogenetic analysis of plants and tissue cultures of Gentiana lutea, G. punctata, and G. acaulis is performed. It is discovered that in vitro culturing leads to changes in the chromosome number in the calli of the species. The species specificity of the variability of the genomes of the cultured cells is demonstrated. It is established that the cytogenetic structure of a tissue culture depends on the genotype of the initial plants. It is explained that in callus tissue of Gentiana (other than an in vitro culture of G. punctata, derived from a plant of the Breskul population), diploid cells and cells with near-diploid chromosome complements constitute the modal class.     

Pollination in vitro: effects on the growth of pollen tubes,seed set and gametophytic self-incompatibility in Trifolium pratense L. and T. repens L.

Summary Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor selfers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0- and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.     

Photosynthetic characteristics of Cymbidium plantlet in vitro

The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO₂ concentration inside and outside of the culture vessels. The CO₂ measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m^-2 s^-1, a chamber air temperature of 15, 25 and 35°C and a CO₂ concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO₂ concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m^-2s^-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO₂ concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C₃ plants grown outside under shade. The results indicate that CO₂ enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration outside the vessel (in the culture room) - PPF photosynthetic photon flux     

Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Deltacph1/Deltaefg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement.     

Micromanipulation of male and female gametes ofNicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture

This research is part of an attempt to establish an in vitro fertilization system in tobacco to aid in understanding mechanisms of fertilization. Fusions of isolated male and female gametes were induced in a polyethylene glycol solution. Fusion appears similar to that in maize. One nuclear division of both an unfertilized egg cell and a synergid was induced in KM8p medium with 1 mg/l 2,4-dichlorophenoxyacetic acid in a microchamber culture; one cellular division of the egg cell was also induced in the same medium in solid-drop culture. The osmolality of suspension culture feeder cells was critical for the development of these cells. These results indicate that in vitro fertilization is possible in tobacco, which would be the first such system in dicots.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - PEG Polyethylene glycol     

Early in vitro flowering and seed production in culture in <Emphasis Type="Italic">Dendrobium</Emphasis> Chao Praya Smile (Orchidaceae)

Plantlets of Dendrobium Chao Praya Smile maintained in vitro were induced to flower, which produced viable seeds within about 11 months. A two-layer (Gelrite-solidified layer topped with a layer of liquid medium of the same volume and composition) culture system containing benzyladenine (BA) at 11.1 μM induced the highest percent of flowering (45%) in plantlets within 6 months from germination. The percentage of inflorescence induction was increased to 72% by pre-selecting morphologically normal seedlings prior to two-layer culture. Plantlets in culture produced both complete (developmentally normal but smaller than flowers of field grown plants) and incomplete flowers. Pollen and female reproductive organs of in vitro-developed complete flowers were morphologically and anatomically similar to flowers of field grown plants. In addition, 65% of the pollen grains derived from in vitro-developed flower were tetrad suggesting that regular meiosis occurred during microsporogenesis. The percentage of germination of pollen grains derived from in vitro-developed flowers and flowers of field grown plants, incubated on modified Knops’ medium for 8 days, were 18.2 and 52.8%, respectively. Despite a lower percentage of germination of the pollen grains derived from in vitro-developed flowers, flowers induced in culture could be self-pollinated and developed seedpods with viable seeds. Nearly 90% of these seeds developed into protocorms on germination in vitro. These seedlings were grown in culture and induced to flower in vitro again using the same procedure.     

The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid lines in barley

Summary The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid (DH) lines during the stages of field evaluation was investigated. The results showed a positive correlation between coleoptile height of haploid plantlets in culture and final plant height of their DH progeny lines in the field. These results illustrate that in vitro selection for plant height and other linked quantitative or pleiotropic characters can be carried out at the initial stages of a DH breeding programme.     

The effects of medium composition and culture conditions on in vitro rooting and ex vitro establishment of jackfruit (Artocarpus heterophyllus Lam.)

A sucrose level of 20 g/l in the culture medium and a 12 h photoperiod were required for optimal in vitro rooting of proliferated jackfruit shoots and these culture conditions also promoted subsequent establishment of plantlets in the glasshouse. High agar levels of 10 and 20 g/l reduced the humidity in culture but also reduced in vitro rooting and had no effect on the establishment of plantlets in the glasshouse. A 12 h photoperiod in ex vitro conditions gave greater growth and development of plantlets than 8 h or 16 h photoperiods.     

Influence of agar on in vitro cultures: I. Physicochemical properties of agar and agar gelled media

Summary The success of in vitro culture is related to several factors. Beside factors associated with the plant material or the medium composition, the physicochemical characteristics of gelled media can play an important role. In this paper, the latter aspect has been considered and the nature of agar powders has been investigated. Moreover, the process of gel formation for three different media and the availability of water and minerals for the corresponding gels have been studied. Analysis of agar powders showed that they can contain different amounts of impurities and the dialysis of these powders suggested that the impurities might be available to the tissues. Thermal analysis on the hygroscopic properties of the agar brands suggest the importance of these data to obtain comparable and reproducible gelled media. The study on the process of formation of gelled media indicates that there is a critical temperature T_ss which can be used to control the gel processing. In fact, at this temperature, agar powders in water transform into a sol status through a rapid shift of electrical conductivity. Water potential of the medium, water loss from gels over the culture period, and the ease of releasing liquid from gels under pressure were shown to be different for different agar brands. A different availability of water and minerals in Murashige and Skoog medium was deduced from the gels prepared with three agar brands (Oxoid, Merck, and Roth).     

In vitro carcinogenesis of mammary epithelial cells byN-nitroso-N-methylurea using a collagen gel matrix culture

Summary Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of “microtumors” in collagen gels, induced by 7,12-dimethylbenz(a)anthracene. In the present study the direct acting water soluble, mammary carcinogen,N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiated cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these “tumors” was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.     

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.