Peg-mediated fusion of Rubus idaeus (raspberry) and R. fruticosus (blackberry) protoplasts, selection and characterisation of callus lines

Abstract

ABSTRACT Cell suspension-derived protoplasts of two cultivated Rubus species, Rubus idaeus-raspberry (subgenus Idaeobatus 2n=2x=14) and R. fruticosus-blackberry (a complex species aggregate within the subgenus Eubatus, 2n=4x=28) were fused using different polyethylene glycol (PEG) fusion treatments. Duration of PEG treatment and choice of culture media influenced the rate of cell divisions and plating efficiency. Colony formation was initiated on solid media for the production of several callus lines. Cytological analyses were performed on selected callus lines with hexaploid chromosome number. Two hexaploid fusion callus lines, selected for their homogeneity in growth and ploidy level, were examined by molecular cytogenetic techniques of fluorescent in situ hybridisation (FISH) and genomic in situ hybridisation (GISH). GISH revealed the presence of the heterokaryon within the fusion callus lines. FISH probed with ribosomal DNA (rDNA) showed variable numbers and sizes of loci. Aberrant distribution and condensation of rDNA were common in interphase cells. FISH results suggest that large karyotype rearrangements occurred, including variation in chromosome number and rDNA loci translocations. Attempts to regenerate plants from the hexaploid callus lines following several applications of plant growth regulator combinations were unsuccessful. This may be attributed to the genomic reorganisation and instability of these long-term fusion callus cultures.