Mutants resistant to the herbicide N-(3-[1-ethyl-1-methylpropyl]-5-isoxazolyl)-2,6,dimethoxybenzamide (isoxaben) were recovered from an M2 population of Arabidopsis thaliana. Two of these mutants, DH47 and DH48, had a high level of resistance in the homozygous state. Crosses of these mutants to marker strains, and to each other, showed that each contained a mutation at a single locus tightly linked to lutescens, a marker on the fifth chromosome of A. thaliana. Growth curves of these mutants and of the Fl progeny of a cross with the wild type parent strain, in the presence of different concentrations of the herbicide, showed that both mutants display a semidominant phenotype. The two mutations differed in their degree of resistance, both as homozygotes and heterozygotes. This suggests that they are two different alleles. Callus cultures were established from plants homozygous, as well as heterozygous, for each of these mutations. Growth curves of these cultures in the presence of the herbicide mimicked the data obtained in vivo indicating that sensitivity to isoxaben is not dependent on a differentiated function.

SummaryPlant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded 35% wells containing single protoplasts and 15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.

An isoxaben resistant mutant of Arabidopsis thaliana is described whose locus, Ixr BI, is unlinked genetically to the previously described resistance locus Ixr A (DR Heim, JL Roberts, PD Pike, IM Larrinua [1989] Plant Physiol 90: 146-150). A cross of strains each homozygous for one of these two resistance loci gives rise to some isoxaben sensitive F2 progeny. Growth curves versus isoxaben of this mutant, its F, progeny and the wild-type parent strain showed that this locus displays a weakly codominant Mendelian phenotype. Callus cultures were established from plants homozygous as well as heterozygous for this locus. Growth inhibition curves done with these cultures mimic the data obtained in vivo.

No abstract

Agrobacterium tumefaciens strains, known to induce tobacco crown galls that spontaneously develop shoots, were used to induce galls on cultured shoots of a tetraploid potato cultivar (Solanum tuberosum cv. 'Maris Bard'). Shoots also appeared spontaneously from the induced potato galls, although only after 2-4 months. The shoots were excised and cultured separately. Some of these frequently developed side-shoots from their axillary buds. They did not form roots and they produced opines, a strong indication that they were transformed and carried T-DNA. Grafts of the transformed plants were still able to develop tubers. Most of the tumour-derived shoots, however, formed roots, did not produce opines and were indistinguishable from the parental plants on the basis of morphology and chromosome numbers (48 chromosomes per cell). The results are discussed in relation to the origin of previously described variation among protoplast-derived potato plants and with respect to genetic engineering of tetraploid potato cultivars.