No abstract

Abstract. Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)-fl-linked glucan with a few 6-1inked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3-/?-D-glucan 3-/?-D-glucosyl transferase, EC2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca 2+ or other divalent cations, and exhibited MichaelisMenten kinetics only between 0.25 mM and 6raM uridine-diphosphate glucose (K m 1.5 2.5 raM); it was activated by /?-glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca 2+, and in the presence of 2 mM Ca2+exhibited MichaelisMenten kinetics above 0.1 mM uridine-diphosphate glucose (K m 0.45 mM); it also required a/?-glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1, 6-1inked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non‐overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(‐4) and 10(‐6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin selection did not increase the number of recombinant kanamycin‐resistant (KanR) cell clones. DNA analysis of KanR clones derived from cells carrying multiple tandem recombination units suggested that these units have a tendency to undergo concerted recombination. Recovery and analysis of kanamycin‐sensitive seedlings with patches of KanR cells provided direct evidence for mitotic recombination in plant tissue.

We present here the application of protoplast technology in the selection and recovery of rare, spontaneous plant genome alterations. Using protoplasts as a cell cloning system allowed the detection and molecular characterization of intrachromosomal recombination events between genomic repeats. The mechanism, frequencies and the induction of intrachromosomal recombination are discussed as well as its application for genome mutagenesis.