Abstract.Results on the enhancement of the frequency of protoplasts with micronuclei, and on the isolation and enrichment of smaller sub-diploid microprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reported. Suspension cells were treated with the spindle toxin amiprophos-methyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wall-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increased by a factor of 2-6. A shorter period (3 h) of incubation with a higher concentration of enzymes as well as a longer period (16 h) of incubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, synchronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, significantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono-and micronucleated) obtained after enzyme incubation was fractionated throuh a continuous iso-osmotic gradient of Percoll, using high-speed centrifugation. This resulted in one large and a few small bands, which contained a heterogeneous population of microprotoplasts, protoplasts and cytoplasts. In contrast to the large band, the small bands contained a relatively higher frequency of small sub-diploid microprotoplasts. To separate the small sub-diploid microprotoplasts from the large microprotoplasts and protoplasts of the bands, discontinuous Percoll gradients and sequential filtration through nylon sieves of decreasing pore size (48--20-15-10-5 [am) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (~80%) small subdiploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as revealed by miAbbreviations: APH =aphidicolin; APM = amiprophos-methyl; Au = arbitrary units; CB = cytochalasin B; DAPI = 4,6-diamidino-2-phenylindole; FDA = fluorescein diacetate; HU = hydroxy urea Correspondence to: K.S. Ramulu; FAX: 31 (8370) 18094 crodensitometric and flow-cytometric analyses. The application of this technique for partial genome and limited gene transfer is discussed.

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As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a ‘mutator’ phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for ‘mutator’ phenotypes in somatic hybrids between potato cvs. ‘Delikat’ and ‘Désirée’ and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with ‘mutator’ phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.

Results on efficient induction of micronuclei by Cremart in suspension cells and protoplasts of potato are reported. Cremart is a highly effective phosphoric amide herbicide, which acts on the mitotic spindle, and induces micronuclei through modification of mitosis. After treatment with Cremart, metaphase chromosomes changed directly into micronuclei without centromere division and chromatid separation. When suspension cells were treated with Cremart (3.7-15.0 μM) for 48h, and subsequently incubated in a mixture of cell wall-digesting enzymes in the presence of cytochalasin-B and Cremart for 18h, the frequency of micronucleation in the cell/protoplast mixture increased significantly, as compared to that obtained after treatment of suspension cells with Cremart (3.7-15.0 μM) for 48 h. Sieving after enzyme incubation resulted in the recovery of protoplasts, showing mass induction of micronuclei. Also synchronized suspension cells of Nicotiana plumbaginifolia responded with high frequency of micronucleation after Cremart (3.7 μM) treatment. The application of this procedure for partial genome transfer is discussed.

Cold treatment of seeds, obtained from crosses between cultivars of T. gesneriana L., affects the developmental stage of embryos, which in turn influences the frequency of callus induction and the development of different callus types. Cold-treated, mature embryos and basal segments of in vitro-derived bulblets, were suitable explants for the initiation of regenerative callus on medium with 2,4-dichlorophenoxyacetic acid. The bulblets were initiated on flower-stalk segments from cold-stored bulbs of T. gesneriana 'Christmas Marvel.' Histological analyses of regenerative callus revealed the regeneration of bulb-like structures. The influences of culture medium, culture conditions, growth regulators and acetylsalicylic acid, an inhibitor of ethylene, on the initiation and establishment of regenerative callus cultures are discussed.