INTRODUCTION .. TRANSITION FROM CELL TO PROTOPLAST .. Ultrastructure . Hydrolases . RNA. Protein, and DNA Synthesis ..

Plant Protoplasts as Physiological Tools

The remarkable regeneration capacity of plant cells is based on their capability to dedifferentiate. We recently reported that cellular dedifferentiation proceeds through two distinct phases, each accompanied by chromatin decondensation: acquisition of competence for fate switch followed by a signal-dependent reentry into S phase. The purpose of this study was to (1) characterize changes in chromatin factors associated with chromatin decondensation, and (2) study the relationship between chromatin decondensation and transcriptional activation of pRb/E2F-regulated genes. We show that plant cells competent for fate switch display a disruption of nucleolar domain appearance associated with condensation of 18S ribosomal DNA, as well as modifications of histone H3 and redistribution of heterochromatin protein 1 (HP1). We further show that the pRb/E2F-target genes RNR2 and PCNA are condensed and silent in differentiated leaf cells but become decondensed, although not yet activated, as cells acquire competence for fate switch; transcriptional activation becomes evident during progression into S phase, concomitantly with pRb phosphorylation. We propose that chromatin reorganization is central for reversion of the differentiation process leading to resetting of the gene expression program and activation of silent genes.

Chromatin reorganization accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F-target genes

An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Techniques in Somatic Cell Genetics

Fusion of freely suspended protoplast mixtures (hypocotyl protoplasts of Brassica napus mixed with mesophyll protoplasts of either B campestris or Nicotiana plumbaginifolia) was induced by a solution containing 10% polyethylene glycol, 10% dimethyl-sulfoxide and 01M glycine-NaOH buffer (pH 100) The fusion products represented 15 to 17 percent of the surviving cells More than 50% of the fusion products divided within two days after fusion, indicating that the fusion procedure did not significantly affect the viability of fused cells The fusion products were not bound to the surface of the fusion vessel, so they could be isolated with a micropipette immediately after fusion

G. Lázár

Papers

Osmotic shock triggers an increase in ribonuclease level in protoplasts isolated from tobacco leaves

Increased frequency of polyethylene glycol induced protoplast fusion by dimethylsulfoxide