Asymmetric somatic cell hybridization in plants

Summary An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700–4200 J/m² and those of ⁶⁰Co gamma radiation over the range 0.15–1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m² UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

Regulation of somatic embryogenesis in celery cell suspensions

The regulation of somatic embryogenesis in celery (Apium graveolens L.) was studied to determine means of increasing its efficiency. Highly embryogenic cell lines were achieved by inducing cell cultures from in vitro plants which were previously regenerated from somatic embryos (secondary cell lines). The early detection of embryogenic potential of new cell lines was found to be regulated by 2,4-dichlorophenoxyacetic acid, mannitol and culture duration. Less frequent subculturing allowed embryogenic potential to be expressed earlier. Increased synchronization of celery somatic embryos was induced by two means: adding abscisic acid to the regeneration medium; and segregating the embryos by sieving them through serial metal mesh screens. When abscisic acid was removed from the growth medium, its effects became quickly transient. Embryos of 1400 µm in size provided the best growth rate and uniformity.     

Regulation of somatic embryogenesis in celery cell suspensions

Embryogenic suspension cultures of celery (Apium graveolens L.) were established from petiole and leaf callus. Suspensions were routinely subcultured in a maintenance medium (with 2.3 M 2,4-D and 0.88 M BA). Somatic embryogenesis occurred in this medium, but was considerably improved in a regeneration medium (2.3 M kinetin, without 2,4-D). Cultures thus maintained, contained embryogenic clumps, aggregated somatic embryos, and few free-floating singular somatic embryos. Addition of mannitol (3–4% w/v) prevented cell lysis, greatly increased the number of singular somatic embryos, improved their normal differentiation, and accelerated torpedo embryo development. Experiments to reveal the nature of the mannitol effect demonstrated that the decreased osmotic potential was an important factor, but not the only one: iso-molar solutions of sucrose alone were not as effective. The mannitol effect could be manifested after a short (2–3 days) exposure period, suggesting a trigger (induction) mechanism. Several pathways of somatic embryogenesis in celery and its regulation by subculturing, with the addition of mannitol, are outlined. Cultures thus maintained resulted in a high rate of normal somatic embryogenesis and production of normal transplantable celery plants.     

Genetic analysis of dexamethasone resistance in L cells by somatic cell hybridization

Stable dexamethasone resistant and receptor-containing (R+) variants of L cells have been characterized by somatic cell hybridization. Neither of the variants had a clearly dominant phenotype in hybrids with dexamethasone-sensitive fibroblast lines, i.e. the resistance of the variants was not due to transdominant factors. Somatic cell hybrids formed between one of the R+-resistant clones and an independent resistant fibroblast cell line showed complementation--the hybrid clones were as sensitive to the steroid as the sensitive parental lines. Complementation, however, disappeared after continued culture of the clones. The return of the dexamethasone-sensitive phenotype was not always linked with similar changes in the responsiveness to another steroid, e.g. progesterone. Our clones can be considered to be resistant variants, designated death-less (d-), where the cells are defective in a non-receptor component involved in the hormone response. The fact that complementation can occur indicates the existence of at least two such steps in the pathway.     

Spontaneous chromosome aberrations in human somatic cells

Conclusion In conclusion it is necessary to say that at present, we cannot consider whether there may be a geographical difference in frequency of spontaneous chromosome aberrations in somatic cells. For that purpose, a very abundant experimental material is required, as well as an improvement in methodology: what causes the difference in the results of various investigators; what methodical principles should be used for collection of data.Important factors in the differences of frequencies of spontaneous chromosome aberrations are likely to be the conditions of cultivation and making the preparations, as well as the methods of scoring the chromosome aberrations. The international standardisation of the cultivation conditions and of the estimates of chromosome aberrations is needed for the further study of the rate and reasons of the spontaneous mutation process in somatic cells.     

植物体细胞不对称杂交研究进展

介绍了近代的植物体细胞杂交研究发展历程,进一步综述了近年来植物不对称体细胞杂交和植物配子一体细胞杂交方面的研究进展以及存在的困难。     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.     

Interspecific somatic cell hybridization between asparagine-requiring and drug-resistant cell lines

Interspecific hybrids between Walker 256 carcinosarcoma rat cells which are asparagine requiring, and LMTK^t mouse cells which are drug resistant and asparagine independent have been isolated. The hybrids were selectively isolated by taking advantage of the asparagine requirement, or, in some cases, combining the asparagine requirement with an azaguanine resistance marker. The hybrids: (a) possessed a chromosome complement which was additive between the two parent lines; (b) showed two marker chromosomes; (c) possessed both rat and mouse forms of a number of different isozymes. The specific activities of asparagine synthetase was measured in the two parents and the hybrids. The enzyme level in the hybrids was found to be higher than the levels observed in the W 256 line, but only 10% of that observed in the LMTK⁻. The results are in agreement with, but do not prove, the hypothesis that asparagine requirement is due to a mutation in a structural cistron specifying the asparagine synthetase polypeptide.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

Establishment of an antigen-specific B cell clone by somatic hybridization

Splenic B cells of A/J mice immunized with 2,4,6-trinitrophenyl (TNP)-lipopolysaccharide were fused with 2.52M, a mutant of a B cell line, in the presence of polyethylene glycol and dimethyl sulfoxide. TP67.21, a subclone of a resulting hybridoma, expresses IAk, IEk, IgM, B220, P50, and receptors for C3 fragment of complement, the Fc portion of IgG, and interleukin 2 receptor on the cell membrane; it also possesses receptor molecules for TNP on its surface, derived from TNP-reactive B cells of A/J mice primed with TNP-lipopolysaccharide used for somatic hybridization, by a rosette-forming assay with TNP-sheep erythrocytes. In contrast, parental 2.52M lacks IAk and IEk on the cell membrane and does not bind to TNP-sheep erythrocytes under the same conditions. Thus, it is likely that TP67.21 is an antigen-specific B cell clone directed against TNP. The antigen binding of cells was markedly inhibited by the specific free hapten or anti-IgM antibodies. Interestingly, TP67.21 was induced to generate a significant amount of anti-TNP antibody when treated with TNP conjugates including T cell-independent and -dependent antigens, such as TNP-lipopolysaccharide, TNP-bovine serum albumin, TNP-ovalbumin, and TNP-keyhole limpet hemocyanine in the absence of T cell help, as well as polyclonal activators; this was followed by a marked decrease in the expression of B cell surface markers on the cell membrane. This suggests that the cross-linkage of receptor molecules on TP67.21 by antigen may directly provide a differentiative signal for maturation to a lineage of B cells, and consequently results in the generation of antigen-specific antibodies without T cell involvement.     

Rescue of marker phenotypes mediated by somatic cell hybridization.

The effect of irradiation prior to virus-induced cell fusion on the frequency of hybrid production has been measured as a function of radiation dose. The Chinese hamster line wg3h (HGPRT-) was crossed with the TK- mutants; Chinese hamster A23 or mouse 3T34E, and hybrids were selected in HAT medium. Irradiation of one (marker rescue) or both (mutual rescue) partners before fusion yielded qualitatively different results. After X-irradiation marker rescue curves were of single-hit type, with D0 values about five-fold greater than the irradiated parent cell. Mutual rescue curves were of the multi-hit type, with zero-dose extrapolation value (n) greater than that of the more resistant partner, but no significant alteration in D0. Qualitatively similar results were obtained after U.V.-irradiation, but the probability of rescue per surviving parent cell was higher after U.V. than after X-rays. With both forms of radiation, reciprocal marker rescue curves were not significantly different. Control experiments showed that mutual rescue was not an artefact either of sensitization of parent cells due to TK- or HGPRT- mutations, or of the enhancement of recovery by feeder layers resulting from high-density mutant populations killed with graded radiation doses and HAT selection. Analysis of heterokaryon frequencies within 18 hours of fusion demonstrated that radiation doses up to four lethal hits, given to one or both parents of the cross wg3h x A23, did not increase heterokaryon formation.     

Buffer capacity of cotton cells and effects of extracellular pH on growth and somatic embryogenesis in cotton cell suspensions

Summary This research was designed to: a) characterize the normal pH changes that occur when cotton cell are grown in culture; b) determine if cotton cells can regulate the pH of their extracellular medium; and c) explore the effects of starting pH on cellular differentiation in culture, including formation of somatic embryos. When an aliquot of cotton cell suspension culture (Gossypium hirsutum L. cv Coker 312) was inoculated into fresh Murashige and Skoog (MS) medium at pH 4.5, the pH stabilized near 5.5 during the log phase of growth and then rose to pH 7.25. Cotton cells actively adjust medium with initial pH between 3 and 8 to near pH 5.5 in the early culture period. By acid/base titration, it has been shown that living cotton cells increase the buffer capacity of water and MS medium. Therefore, the metabolic activity of living cells accounts for the adjustment and stabilization of pH during the log phase of growth. The starting pH of the culture medium affects longterm viability, growth, and differentiation of the cells; pH 3 to 5 is best for cell viability, pH 3 to 4 enhances cell elongation; and pH 4, 7, or 8 stimulates somatic embryogenesis. Cultured cotton cells and the pH of their extracellular medium are in a complex, interactive relationship. This study was supported by the Texas Advanced Technology Program and the USDA-ARS. The journal no. for this paper is T-4-280, The College of Agriculture, Texas Tech University.