Plant protoplast fusion and growth of intergeneric hybrid cells

Kao, K. N.; Constabel, F.; Michayluk, M. R.; Gamborg, O. L.

doi:10.1007/bf00390290

Cited by 277 publications

(35 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…6 b and c and mosomes in squashed preparations reflects quite correctly their spatial arrangement in vivo. From our work and that of others (6,8,19), it is evident that the parental genomes in regenerating protoplast fusion products enter the first cell division almost simultaneously, and the chromosomes of each parent form their own spatially separated cluster within the common metaphase plate.…”

Section: Resultssupporting

confidence: 65%

Spatial separation of parental genomes in hybrids of somatic plant cells

Gleba¹,

Parokonny²,

Kotov³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Chromosome spatial arrangements on metaphase plates of intergeneric intertribal cell hybrids ofNicotiana chinensis and Atropa belladonna as well as interspecific somatic hybrid plants of Nicotiana plumbaginifolia and Nicotiana sylvestris were analyzed. In the metaphases of the first divisions of protoplast fusion products, chromosomes of the two parents were spatially separated (segmented metaphase). In long-term cultured somatic hybrids, the topology of genome separation pattern in both callus cells and plants showed changes in form from "segmental" to "radial." Growing the hybrid cells in the presence of colchicine resulted in random chromosome arrangement both in cells directly exposed to different colchicine concentrations and in colchicine-treated cells grown in colchicine-free media. The degree of genome separation calculated for different cell clones remained constant during in vitro propagation of cells but was significantly lower for subclones derived from colchicine-treated cells. Therefore, it is concluded that spatial chromosome arrangement in metaphase is epigenetically controlled.As early as 1925, chromosomes were observed to be positioned on the metaphase plate in a specific manner (1). Nevertheless, up till now the spatial arrangement of chromosomes at interphase and at mitosis and, in particular, the arrangement of the parental genomes in a hybrid cell still remained poorly understood. It is known that the arrangement and orientation of the parental (maternal and paternal) chromosomes are nonrandom in the zygote and the early stages of cleavage in some animal species (2-4). In the case of sexual hybrids between barley and rye, it was shown that the spatial separation of the parental genomes tends to persist for a large number of regular mitoses in somatic tissues (5). These facts and other information suggest that spatial separation of parental genomes in the somatic tissues of a sporophyte is of general occurrence, which has probably remained unnoticed due to the fact that in somatic cells, obtained by sexual crosses, maternal and paternal genomes are morphologically identical and, hence, difficult to distinguish. The hybridization of somatic cells permits the combination of cells of phylogenetically remote species which may have morphologically distinguishable chromosomes. Segmental disposition of the parental genomes at first metaphases after fusion (6-9) has a simple explanation and does not necessarily mean that this separation would persist through a large number of subsequent mitoses. Previous studies on the chromosome arrangement of progeny cells derived from somatic hybrids were limited almost exclusively to preparations treated with colchicine, a treatment known to result in distortion of chromosomal arrangements (7, 9-13). One early investigation (14) indicated that, within the hybrid metaphase plate of Nicotiana-Atropa somatic hybrids, the chromosomes of the two parents were unevenly distributed. This prompted a study of the chromosome arrangement in somatic cell hybrids of A...

show abstract

Section: Resultssupporting

confidence: 65%

Spatial separation of parental genomes in hybrids of somatic plant cells

Gleba¹,

Parokonny²,

Kotov³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Changes in the ESR signal from the 5-NS probe gave evidence of changes in the membrane structure which occurred under conditions (10 mm Ca) conducive to protoplast fusion. Plant protoplast fusion is generally enhanced by Ca treatment and transient exposure to PEG (5,6). Although the role of PEG in protoplasts fusion is unclear, evidence suggests that PEG alone will not enhance fusion but rather promotes phagocytosis (16).…”

Section: Discussionmentioning

confidence: 99%

Effects of Divalent Cations and Polyethylene Glycol on the Membrane Fluidity of Protoplast

Boss¹,

Mott²

1980

Plant Physiol.

View full text Add to dashboard Cite

Calcium is often used to stabilize membranes and enhance membrane fusion. We have used the fatty acid spin label, 5-nitroxy steanc acid to measure fluidity changes in the plasma membrane of carrot suspension culture celi protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2Am.) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millinolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2Am.a and evidence ofexchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a lving system describe membrane structural changes under conditions similar to those used for protoplast fusion.ESR2 has played an important role in the understanding of molecular motion of lipids in model and biological membrane systems (2,4, 17). We chose the fatty acid spin label 5-NS to measure fluidity changes of the plasma membrane of carrot suspension culture cell protoplasts. The 5-NS probe has the nitroxide moiety on the fifth carbon from the carboxy terminus of stearic acid and the probe intercalates into the membrane bilayer so that the nitroxide is located in the region of the glycerol backbone of phospholipids (4,15). The ESR signal from the probe is thus sensitive to perturbations near the surface of the bilayer. Ca stabilizes membranes and enhances membrane fusion in both artificial and biological systems (3,7,14). Ca binds to the negatively charged phospholipids near the membrane surface resulting in altered membrane fluidity and phase separation of the bilayer (11-13). Reported here are the effects of Ca on membrane fluidity of living carrot protoplasts as determined through ESR using the 5-NS probe. were released enzymically from cells in the exponential phase of growth (3 to 4 days after transfer) (1). The protoplasts were filtered through glass wool and washed once by centrifugation (40g, 5 min) through 1% (w/v) dextran in osmoticum A, which consisted of 0.45 molal sorbitol and 0.5 millimolal Mes (pH 5.5). Debris was further removed by three additional washes in osmoticum A without dextran. Spin-Labeling of Protoplasts. A freshly prepared stock solution of 5-NS spin-label probe (SYVA Co. Palo Alto, CA) in methanol (1.0 mg/0.1 ml) was kept in a darkened tube on ice during an experiment. Aliquots (4 ,ul) were pipetted into 10-ml glass tubes; 0.1 ml settled volume of protoplasts (approximately 105 protoplasts) was added, and the tube was tapped gently to mix the protoplasts and the spin-label. Labeled protoplasts were immediately drawn up into a quartz capillary (1 mm i.d., Wilmad Glass Co...

show abstract

“…Protoplasts were washed in a glucose solution (17) and mixed in a 1:1 ratio at a density of 105 cells per ml. Fusion was induced with polyethylene glycol, combined with high-calcium and high-pH washing (18) in the presence of dimethyl sulfoxide (19). Both treated and control parental protoplasts were cultured in K75 medium (20 …”

Section: Introductionmentioning

confidence: 99%

Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants

Dudits

Maroy

Praznovszky

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

139

View full text Add to dashboard Cite

Transfer of methotrexate and 5-methyltryptophan resistance from carrot (Daucus carota) to tobacco (Nicotiana tabacum) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.protoplasts (11). In some of the resulting nitrate reductasepositive tobacco plants, barley enzyme was detected with immunological methods. Because of leakiness of selection techniques, based on a single recessive and unstable mutation, the authors did not conclude that cell fusion-mediated gene transfer had occurred. At present, available experimental data are insufficient to explore the potential of asymmetric cell fusion for widening genetic variability.In this paper we describe a fusion system that allowed the monitoring of two dominantly acting selectable markers in tobacco + carrot fusion products. Molecular evidence confirming the expression of carrot-specific dihydrofolate reductase (DHFR; EC 1.5.1.3) in regenerated hybrid plants with normal tobacco morphology is presented. Furthermore, sexual progenies are analyzed to test the inheritance of transferred genetic markers.Gene flow between incompatible species is strongly restricted by evolutionary boundaries in sexual crossings. The use of somatic cells as targets in genetic manipulation experiments, based on DNA transformation or cell hybridization, has opened new horizons for combining diverse genes from unrelated species. If the gene of interest has been cloned, advanced transformation systems may provide the most efficient way of introducing it into a foreign host genome (1, 2). When isolated genes are lacking, asymmetric hybridization mediated by protoplast fusion offers an alternative approach for alien gene transfer. The asymmetric nature of nuclear genomes in cell hybrids originates from spontaneous or induced genome instability, with preferential loss of chromosomes belonging to one of the parental species. Enforced chromosome elimination can be a prerequisite of hybrid plant production in several wide fusion combinations in which somatic incompatibility prohibits hybrid development (3-5). The transient coexistence of two diverse genomes in hybrid cells may provide the opportunity for recombination of chromosomes or a few genes from the eliminated partner. To test this hypothesis, experimental methods are needed to control chromosome elimination from fusion produ...

show abstract

Plant protoplast fusion and growth of intergeneric hybrid cells

Cited by 277 publications

References 15 publications

Spatial separation of parental genomes in hybrids of somatic plant cells

Spatial separation of parental genomes in hybrids of somatic plant cells

Effects of Divalent Cations and Polyethylene Glycol on the Membrane Fluidity of Protoplast

Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants

Contact Info

Product

Resources

About