I. Negrutiu

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes)

SummaryTwo S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.

Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

SummaryProtoplast-derived colonies of haploid N. plumbaginifolia leaves were used to select for resistance to NaCl, KCl and polyethylene glycol 6000 (PEG). Salt-and PEG-tolerant cell lines were isolated on the basis of growth in a culture medium containing inhibitory concentrations of either NaCl or KCl (200 mM) or PEG (25%). The frequency of resistant lines ranged from 10-5 to 10-6. One resistant line from each treatment was regenerated into plants. All resistant lines produced 10–25 times more proline than the wild type when grown on a non-selective medium. Similar values were also observed in the leaves of resistant progeny plants. In each mutant line, salt or PEG resistance was transmitted as a single dominant nuclear gene as shown by segregation ratios in progenies of crosses between resistant and wild-type plants. The latter observation demonstrates clearly the existence of a genetic basis for increased salt tolerance.

Regeneration of fully nitrate reductase - deficient mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani).

SummaryProtoplast-derived colonies of haploid N. plumbaginifolia were selected for by chlorate resistance in media supplemented with casamino acids. Eighty resistant lines were confirmed by a second passage on a higher concentration of chlorate. Frequency of spontaneous mutation ranged from 10−5 to 10−6. Fifty of the resistant lines could be regenerated into plants, and 30 were characterized biochemically. Ninety percent were fully deficient for nitrate reductase activity. The lines were further tested for xanthine dehydrogenase activity and subsequently classified as defective in the apoenzyme (nia type, 26 lines) or the cofactor (cnx type, 4 lines). Two groups had been identified up until now within the cnx type by growth tests on high concentrations of molybdate supplied to the medium. Nitrate reductase deficiency was stably and continously expressed in both variant cell cultures and regenerants. Genetic analysis demonstrated that nitrate reductase deficiency was inherited as a single recessive nuclear gene.

Advances in somatic cell genetics of higher plants — the protoplast approach in basic studies on mutagenesis and isolation of biochemical mutants

SummarySelection strategies developed in microbial genetics were successfully extrapolated to in vitro cell culture systems of higher plants and are having a major impact in the elucidation of regulatory mechanisms of basic cellular processes in eukaryotes. Although an increasing number and wide spectrum of biochemical variants have been isolated in such cell culture systems, their routine selection, characterization, and manipulation have not yet been achieved. Methodological limitations are considered to be one of the major reasons. Suspension or callus cultures, so extensively employed during the last decade in mutation-selection experiments and so useful in demonstrating the potentialities of in vitro screening techniques in obtaining various biochemical markers, have inherent drawbacks which limit in our opinion their further contribution in this field. Protoplast cultures represent an ideal tool for mutation and selection experiments. It is the purpose of this review to show how, due to recent methodological advances in the manipulation of some model protoplast culture systems, essential aspects of mutagenesis and selection of biochemical mutants can be reconsidered. These systems are simple and efficient, and lend themselves to statistical interpretation. Genetic analysis of selected variants should help us to understand and define better the new set of problems and concepts revealed by the somatic cell genetics of higher plants; combined with biochemical analyses it should elucidate the basic relationship between control of biological processes at cellular and whole organism level.

High threonine producer mutant ofNicotiana sylvestris (Spegg. and Comes)

SummaryMutagenesis and the subsequent selection of mesophyll diploid protoplasts ofNicotiana sylvestris on growth inhibitory concentrations of lysine plus threonine has led to the isolation of an LT-resistant mutant. Regeneration of this line (RLT 70) and analysis of its descendants demonstrated the dominant monogenic nuclear character of the resistance gene, further namedak-LT1. When the inhibition properties of aspartate kinase were examined in the homozygous mutant, lysine-sensitive activity could no longer be detected. In comparison, 70%–80% of the wild-type enzyme activity was usually inhibited by lysine, and the rest by threonine. Evidence for the existence of at least two AK isoenzymes was obtained by ion-exchange chromatography, where two peaks of activity could be detected: the first one to be eluted is lysine sensitive, and the second one threonine sensitive. One consequence of the altered regulation of AK in the mutant was the enhanced production of soluble threonine. Threonine accumulation was observed to occur throughout the life cycle of the mutant plant as well as in its different organs. In particular, leaves exhibited a 45-fold increment of soluble threonine, which corresponds to a 13-fold increase in total threonine: almost one-third of the total amino acids was free and proteinbound threonine. In RLT 70 seeds, 20% of the free amino acid pool was in the form of threonine (70-fold accumulation compared to the wild type), and total threonine content was increased five fold. As a general rule, the other amino acids were also more abundant in RLT 70 seeds, such that the total of amino acids present was between two to four times higher, but in contrast with the situation encountered in leaves, this was also due to a higher protein-bound amino acid content.

Asymmetric hybridization in Nicotiana by "gamma fusion" and progeny analysis of self-fertile hybrids.

SummaryMesophyll protoplasts of the nitrate-reductase (NR)-deficient Nicotiana plumbaginifolia mutant, “Nia26”, were fused with γ-irradiated mesophyll protoplasts of Nicotiana sylvestris, V-42. Hybrid selection was based on complementation of NR deficiency by transfer of the donor NR gene to N. plumbaginifolia. Regenerated hybrids had different numbers of donor chromosomes in a tetraploid background of N. plumbaginifolia. The transfer and expression of different isozymes from the donor were also observed. Six self-fertile regenerants were obtained from 21 independently isolated cell colonies. Progeny analyses revealed: (1) the linkage of NR and shikimate dehydrogenase (ShDh); (2) a stabilization of the transmission rate of NR; and (3) the obtainment of mono- and disomic addition lines in the first and second progeny of the original regenerants. Southern hybridization analyses demonstrated unequivocally the presence of the NR gene from the donor partner in progeny plants.

Complementation analysis by somatic hybridisation and genetic crosses of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

SummaryFusion complementation experiments between nitrate reductase (NR) deficient lines CNX 20, 27, 82 and 103 of Nicotiana plumbaginifolia were performed with the already characterized N. plumbaginifolia mutants nx 1, 24 and 21, belonging respectively to the complementation groups cnx A, B and C. CNX 20 and 82 were identified as belonging to the group of cnx A. CNX 27 complemented with NX 1 and NX 21 but not with NX 24 indicating another B type. The fourth line, CNX 103 showed complementation with CNX 20, NX 21 and NX 24, revealing a fourth cnx complementation group, cnx D, that until now has not been described in higher plants. Genetic crosses inside respectively the NIA and the CNX group, and between NIA and CNX confirmed the fusion complementation results, and showed allelism for the nia mutants

Localization of the T-DNA on marker chromosomes in transformed tobacco cells by in situ hybridization

SummaryChromosome and molecular analyses were conducted on tobacco cells which had been transformed by the T-DNA of the Ti-plasmid. These analyses showed that there were specific chromosome rearrangements in the transformed cells (marker chromosomes). There was a positive correlation between the number of marker chromosomes per cell and the oncogenic potential of the transformed cells. However, we show, using the Southern hybridization method, that the TL fragment of T-DNA, but not the TR, clearly hybridizes with nuclear DNA. In situ hybridization was used to locate the insertion site of T-DNA: the hybridization signal was found on a small metacentric chromosome. This chromosome may occur single or translocated onto other chromosomes, to make marker chromosomes. Thus, by locating the T-DNA, we have confirmed the correlation between the marker chromosomes and the oncogenic potential.

Symmetric Versus Asymmetric Fusion Combinations in Higher Plants

The terms symmetric and asymmetric hybrids refer to the nuclear constitution of fusion products. Symmetric hybrids are defined as consisting of complete sets of chromosomes from both the parents, while asymmetric ones result from a preferential or unidirectional loss of chromosomes belonging to one of the fusion partners. Since protoplasts can be isolated from almost any plant species and readily induced to fuse in all possible combinations, it was expected and even accepted that fusion technology was going to make important contributions in both basic and applied research. This matter was critically evaluted by Harms (1983 a), Burgess (1984) and reviewed in detail by Gleba and Sytnik (1984) and Horn et al. (1986).

Genetic analysis of revertants for the nitrate reductase function of Nicotiana plumbaginifolia

SummarySpontaneous revertants of nitrate reductase (NR)-less mutants were isolated by screening for nitrate utilization in diploid NR− protoplast cultures of Nicotiana plumbaginifolia. The revertants contained in vivo NR activity in the case of apoenzyme mutants (nia) as well as of a cofactor-deficient (cnx) mutant. Revertants of the NIA type proved to be tetraploid, and genetic analysis showed that only one out of the four NR structural genes had reverted to a functional allele.