Ioan Negrutiu

Intergeneric asymmetric hybrids between Nicotiana plumbaginifolia and Atropa belladonna obtained by "gamma-fusion".

SummaryAsymmetric nuclear hybrids have been obtained by fusion of cells from a nitrate-reductase deficient mutant of Nicotiana plumbaginifolia (cnx20) and gamma irradiated protoplasts of Atropa belladonna (irradiation doses tested were 10, 30, 50 and 100 krad). The hybrid formation frequency following selection for genotypic complementation in the NR function was in the range of 0.7%–3.7%. Cytogenetic studies demonstrated that all hybrid plants tested possessed multiple (generally tetra- or hexaploid) sets of N. plumbaginifolia (n = 10) chromosomes along with 6–29 Atropa chromosomes (n = 36), some of which were greatly deleted. Besides the cnxA gene (the selection marker), additional material of the irradiated partner was expressed in some of the lines, as shown by analyses of multiple molecular forms of enzymes. Surprisingly, rDNA genes of both parental species were present and amplified in the majority of the hybrids. Whenever studied, the chloroplast DNA in the hybrids was derived from the Nicotiana parent. Regenerants from some lines flowered and were partially fertile. It is concluded that irradiation of cells of the donor parent before fusion can be used to produce highly asymmetric nuclear hybrid plants, although within the dose range tested, the treatment determined the direction of the elimination but not the degree of elimination of the irradiated genome.

Arabidopsis thaliana as a model system in somatic cell genetics I. Cell and tissue culture

Abstract Induction and growth of callus from seeds, stems and pieces of leaf of Arabidopsis thaliana were successful on a Gamborg B 5 medium and on a modified B 5 medium (PG 2 ). Actively growing calluses have been obtained on agar and in liquid cultures. Cell suspension cultures were establiched on the same culture media, with cell densities up to 1.6·10 6 cells/ml. A culture system is described which enables us to use large cell aggregates as a continuous source of single cells and small cell aggregates (containing up to 10 cells) for mutagenesis, plating and protoplast production. Arabidopsis plants were regenerated from young calluses grown in solid or liquid cultures, by a sequential transfer from a medium (PG 3 ), where shoot initiation takes place, to a PG 4 medium which induces root formation. The morphogenetic potential appeared to depend on the age of the callus in culture. A wide range of heteroploidy has been observed in tissue cultures. The influence of factors such as the age of the callus, the type of organ used to induce calluses, the hormone ratio and the composition of the medium was determined. A procedure for the isolation of protoplasts from leaves and cell cultures is described.

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

SummaryExtremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt60,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via “gamma”-fusion.

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. InNicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F1 monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the “discordant” segregation values observed between corresponding R1 and F1 progenies in up to 9% of the evaluated transformants. In addition, 5% of the transformants showed a phenotypic loss of resistance. In the F3 generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F4 generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved.N. plumbaginifolia exhibited more instability thanN. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.

Some Factors Controlling in vitro Morphogenesis of Arabidopsis thaliana

Summary In vitro morphogenesis of Arabidopsis callus cultures is shown to be under the control of defined hormonal ratios and concentrations. Low kinetin concentrations (10 -7 M) are desirable during callus induction to obtain subsequent organ differentiation. Leaf formation was optimal with a ratio 10 -6 M kinetin/10 -7 M IAA. Root formation from the callus occured on a wide range of cytokinin/auxin ratios. The importance of a defined sequence of culture media to establish an optimal hormonal balance for leaf formation is discussed. Ageing of the callus in culture is accompanied by a progressive decline of the morphogenetic response and by an increase of the degree of ploidy.

Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids.

SummaryWe report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR+, NR+, KmR, and NR-KmR). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred.

Protoplast-Derived Plants in Nicotiana plumbaginifolia Viviani: Improving the Regeneration Response of Wild Type and Mutant Cultures

Summary Nicotiana plumbaginifolia is considered a good plant for studies of somatic cell genetics and development of genetic manipulation procedures. Substantial improvements of the plant regeneration capacity in this species which is a good source of protoplasts was obtained by (1) developing a general scheme of protoplast processing that suits the requirements of both advanced cell technology (selection of mutants, fusion of protoplasts, genetic transformation) and totipotency, and (2) modifying the regeneration medium with respect to sugar, hormone, and major mineral content. An optimal sequence of culture steps was established which allows us to produce fertile plants from mutant cell lines, revertants, fusion products among metabolically deficient mutants, and DNA-treated cultures within three months of culturing.

Factors which Enhance in vitro Morphogenesis of Arabidopsis thaliana

Summary The regeneration efficiency of Arabidopsis callus cultures can be influenced by several factors as the culture medium (solid or liquid medium, method of sterilization, nitrogen source), the inoculum size, the light and low temperature treatment, and the interval between subcultures. These factors, alone or in combination, were shown to enhance at various degrees the morphogenetic response of callus cultures. Subculture intervals of 6 to 8 weeks in continuous low light or darkness, 4 °C cold treatment for 3 days prior to regeneration, and the use of a low inoculum size in liquid, filter-sterilized regeneration medium exert a positive effect on the regeneration of Arabidopsis calluses.

In vitro Morphogenesis of Arabidopsis thaliana: The Origin of the Explant

Summary Callus cultures derived from seeds, anthers, leaf and stem segments can be induced to regenerate into whole plants. In function of the origin of the explant, the appropriate hormonal balance for callus proliferation and regeneration was found to be different. Callus cultures derived from anthers exhibit the highest morphogenetic response, which is maintained for longer than 18 months in culture. Morphologically distinct callus lines, isolated from callus cultures of common origin, respond very differently to the morphogenetic induction conditions. Lines with high, low or no morphogenetic response were selected and represent a valuable tool for studies on in vitro morphogenetic processes.

Leaf Formation and Peroxidases from Arabidopsis Callus

Summary A progressive decline of leaf formation capacity was observed in long-term cultures of Arabidopsis callus on 2,4-D. It corresponded to a gradual loss of activity of total peroxidase and of specific isoperoxidases. A passage on media without auxin was required to restore the morphogenetic capacity. It was accompanied by a general increase of activity for most of the isoperoxidases. Leaf formation by passing on a third medium with low IAA level and high kinetin content paralleled a continuous increased enzyme activity as mentioned before by different authors on other materials. The results suggested that morphogenetic potential is dependent upon an endogenous hormonal balance controlled by the exogenous regulators.