S. Hinnisdaels

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.

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.

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.

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P1, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.

Asymmetric Somatic Hybrids

In recent years, several methods for somatic gene transfer in plants have been developed (see Bajaj 1993). If the gene of interest has been cloned, advanced transformation systems based on viral or bacterial vectors provide an efficient way of introducing it into a desired recipient genome (Potrykus 1990). Unfortunately, the genetic and biochemical basis of many economically important traits such as disease resistance, stress tolerance, yield increase, etc. are largely unknown, and these traits might be subjected to complex regulatory mechanisms which will complicate the molecular biological approaches to elucidating their genetic and biochemical basis. Therefore, although very powerful cloning methodologies are available, the isolation and characterization of these traits should be anticipated on a relatively long-term basis. For the transfer of genes which are not available as cloned DNA sequences or for transferring “blocks” of genes of agronomic interest, somatic cell hybridization via protoplast fusion represents a good alternative method (Negrutiu et al. 1989a, b; Glimelius et al. 1991).

Analysis and Sorting of Plant Chromosomes by Flow Cytometry

The availability of purified individual chromosomes facilitates the study of molecular properties of eukaryotic genomes. This is based mainly on the reduced DNA amount compared to the total genomic content and the enrichment of markers or genes located on these chromosomes. Flow cytometry offers the possibility of isolating specific chromosomes in large quantities.

In situ hybridization to plant metaphase chromosomes using digoxigenin labeled nucleic acid sequences

In situ hybridization is the direct hybridization of specific nucleic acid sequences to morphologically preserved cytological preparations such as inter-phase nuclei, metaphase chromosomes, cells or tissue sections. The detection of specific nucleic acids in situ with radiolabeled probes was first introduced by Gall and Pardue [ 1]. At this time, autoradiography was the only means of detecting hybridized sequences and because molecular cloning was not possible in those days in situ hybridization was restricted to those sequences that could be purified by conventional biochemical methods. In spite of the high sensitivity and wide applicability of in situ hybridization it was not until the development of molecular cloning techniques and especially the introduction of a number of nonradioactive nucleic acid probe modification and detection procedures in the 1980s [2] that the technique came into general use. Indeed, nonradioactive labeled probes offer advantages over conventional isotope labeled probes, as they produce higher resolution of the hybridization sites and lower background interference. Moreover, signals can be detected in only a few hours compared to several days or even weeks of exposure time. Over the past few years, an overwhelming number of papers covering different applications of the technique has appeared [3]. Nonradioactive in situ hybridization and detection has made chromosomal localization of specific sequences on human chromosomes possible [4, 5], Moreover, nonradioactive in situ hybridization has also been used to identify chromosomes [6], to detect chromosomal abnormalities [7], to study the spatial organization of genes in interphase nuclei [8] and to investigate the mechanisms of gene amplification [9]. Because nucleic acid probe can be labeled with a variety of different nonradioactive haptens, each of which can be visualized with an independent detection system, it is now even possible to detect two [10, 11] or three [12, 13] DNA sequences simultaneously. Further optimization of this multicolored in situ hybridization in terms of compatible probe labels, fluorochromes and optical filters is needed. Unfortunately, such progress and practical applications have been restricted essentially to cytogenetic studies of human and other mammalian systems. The development of in situ hybridization strategies for gene mapping as well as for other applications in plants has lagged behind that for animal chromosome systems. The major factor contributing to the slow development of this technique in plant cytogenetics is the difficulty of obtaining high quality mitotic metaphase chromosomes free of cell wall material. Moreover, the highly condensed nature of plant chromosomes, as well as the lack of marker chromosomes and reproducible banding procedures, makes karyotyp-ing extremely difficult.

In situ hybridization to plant metaphase chromosomes: radioactive and non-radioactive detection of repetitive and low copy number genes

In situ hybridization is the direct hybridization of a specific nucleic acid probe to cytological preparations such as interphase nuclei, metaphase chromosomes or sectioned tissues. Since it was first established by Gall and Pardue [4], the technique has been improved continuously and considerable progress in the detection procedure has made chromosomal localization of specific sequences on human chromosomes possible [2, 19]. Moreover, in situ hybridization has also been used to identify chromosomes, to detect chromosomal abnormalities, to study the spatial organization of genes in interphase nuclei [9] and to investigate the mechanisms of gene amplification [18]. Unfortunately, such progress and practical applications have been restricted essentially to cytogenetic studies of human and other mammalian systems. The development of in situ hybridization strategies in plants still lags behind. The major attributes to the slow development of this technique in plant cytogenetics are: the presence of a cell wall which prevents proper accessibility of probe DNA to target DNA. the difficulty to produce high quality chromosome spreads. the lack of marker chromosomes and reproducible banding procedures makes karyotyping of plant chromosomes extremely difficult.