INTRODUCTION . 519 NOMENCLATURE 520 METHODS FOR THE ISOLATION OF VARIANT CELL LINES 521 BASIS OF PHENOTYPIC VARIATION IN SOMATIC CELLS 522 St�ies of SeJ;�1 Transmission to Confirm Mutation 522 Epigenetic Varrants.. . . ..... ... .. . . . ..... . . .. ...... . . .. ........ 525 CHARACTERIZATION OF DOMINANCE-RECESSIVITY RELATIONSHIPS BY CELL FUSION 526 MUTANTS AND VARIANTS 526 Auxotrophic Lines , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . .. . . . . . . . . . . 526 Chlorate Resistance-Nitrate Reductase Deficiency 527 Resistance to Amino Acids and Amino Acid Analogs 528 Res!s�an�e to "!ucleic Acid Base Analogs 530 AntibIOtIC ReSistance 530 Developmental Variants 532 Herbicide Resistance 532 Salt and Water Stress Tolerance 533 Disease Tolerance 534 SOMACLONAL VARIATION . ... . . . . . . .. . . . . . . . . . . .. .... . . . . . . . . ....... . . . . . . . 535 CONCLUSIONS AND REMARKS 536

Isolation and characterization of mutants in plant cell culture

Publisher Summary This chapter discusses several specific areas of plant tissue cultures, such as(1) rapid clonal propagation, (2) production of androgenetic (anther- or pollen-derived) haploid tissues and plants, and (3) culture and fusion of protoplasts and their use in attempts at genetic modification. Haploid callus tissues are mitotically unstable. By the use of suitable concentrations of p-fluorophenylalanine (PFP) in tissue cultures of tobacco, it is possible to maintain stable cultures of haploid cells and to select preferentially haploid cells from the mixed populations of cells of varying ploidy. Much of the work on germ plasm preservation involves the long-term storage of, for example, seeds and pollen grains. The plant tissue cultures also offer a useful method for the long-term storage of plant genetic resources, especially in those species where seeds cannot be easily obtained or stored for long periods of time, and particularly, in cultivars where vegetative propagation is essential for the maintenance of genetic stability. Two procedures developed for the long-term storage of plant tissue cultures are the storage and culture of excised shoot meristems and the freeze preservation of callus or suspension cultures.

Plant Tissue Cultures in Genetics and Plant Breeding

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.

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

If a double-mutant can be established with two markers that are dominant and counter-selectable, it can be serve as a universal hybridizer in selecting somatic hybrids exclusively after protoplast fusion with any other wild-type partner. Such a universal hybridizer was selected from the cell suspension of Sinapis turgida Del., members of the Brassicaceae. In a preliminary survey on tissue culture for about 50 species in Brassica and its wild allies, we found that S. turgida delivered cell suspension in a liquid culture (Toriyama et al 1987b). Following protoplast fusion between S. turgida and B. oleracea L., and between S. turgida and B. nigra (L.) Koch, somatic hybrids were selected and plantlets were obtained (Toriyama et al 1987a).

Selection of a Universal Hybridizer in Sinapis Turgida Del. and Regeneration of Plantlets from Somatic Hybrids with Brassica Species

Plants were regenerated from axenic plantlets by mesophyll protoplast culture, without mutagenic treatment. Two different lines of Nicotiana sylvestris were used: an original line, and a diploid androgenetic line derived from it. The regenerated plants were either diploid and phenotypically similar to their respective protoplast source line, or tetraploid. Genetic studies carried out on several diploid regenerated plants revealed genetic variability. Eight of 13 selfed progenies of plants regenerated from the original line, and 1 of 8 selfed progenies of plants regenerated from the androgenetic line, produced new mutant phenotypes never observed in the protoplast source lines. Two plants regenerated from the same protoplast-derived callus produced different mutations. Selfed progenies without a recognizable mutant phenotype were also different from their respective protoplast source line for quantitative characters; protoplast culture induced a depressive effect on the size of plants derived from protoplasts at younger and older stages of development. The origin of this depression and of the mutations is discussed.

Genetic variability induced in Nicotiana sylvestris by protoplast culture.

Nicotiana sylvestris cell lines resistant to the amino acid analogues S-2-aminoethyl-cysteine (AECR), or 5-methyl-tryptophan (5MTR), were isolated in suspension culture. Assuming these resistances to be dominant, we have attempted to determine if such variant cell lines can be used to select double resistant somatic cell hybrids. A total of 1.8 × 104 control calli from mixed AECR and 5MTR protoplasts, and AECR and 5MTR homokaryotic fusions were placed on double analogue selection, but none survived. Eight somatic hybrid calli (0.8%), able to grow without inhibition on the double analogue selection medium, were obtained after AECR + 5MTR protoplast fusion. These were further determined as hybrids on the basis of resistance level, chromosome number, and chlorophyll content, all characteristics differing in the parental cell lines.

D. W. R. White

Papers

Use of amino acid analogue-resistant cell lines for selection of Nicotiana sylvestris somatic cell hybrids.