Some theoretical and practical possibilities of plant genetic manipulation using protoplasts

Protoplasts capable of division and plant regeneration are now available for a large number of vegetable, oil and forage crops. However, routine hybrid production is not possible due to methodological limitations in selection and culture of hybrid cells. Recent improvement in techniques are the use of a double mutant as a universal hybridizer and the use of fluorescence activated cell sorter to recover hybrid cells. Interest is also centered on limited gene transfer by protoplast fusion. We propose a model of generating triploid plants by somatic cell fusion to transfer limited genomic information from an alien plant to a crop plant. Somatic hybridization has some novel features but, in practice and conception, it is an extension of the methods of sexual hybridization. By contrast, genetic transformation is a radically different approach to plant genetic manipulation. The success of this approach will depend upon how readily genotype can be related to phenotype in a tangible way so as to ascertain what biochemical and developmental activity is controlled or modulated by a DNA sequence

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6-12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background

Xylem colonization of the legume Sesbania rostrata by Azorhizobium caulinodans

A novel pathway of invasion of the legume Sesbania rostrata by Azorhizobium caulinodans is described that involves colonization of the root xylem, possibly following entry into the natural fissures created during emergence of lateral roots. Azorhizobia were detected microscopically, and their presence confirmed by the expression of a lacZ reporter gene. We have shown that rhizobial Nod factors are not required for either xylem colonization or for crack-entry of lateral roots. We discuss the extent to which this discovery of xylem colonization by azorhizobia is likely to improve our understanding of both symbiosis and of pathogenicity in plant–bacterial interactions