ABSTRACT
Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (Solanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotiana tabacum) and the wild tomato species Lycopersicon peruvianum is reported. The microprotoplasts contained one or a few chromosomes. Monosomic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of potato using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting enzyme mixture. The microprotoplasts were isolated from the micronucleated protoplasts by high-speed centrifugation. By using sequential filtration, small microprotoplasts containing one or few chromosomes were separated from the bigger subdiploid microprotoplasts. These small microprotoplasts were fused with recipient protoplasts of tobacco or tomato using polyethylene glycol. The selectable marker kanamycin resistance (KanR) and the reporter gene β-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion products and the isolation of hybrid calli. Several monosomic addition plants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus and one or two copies of the neomycin phosphotransferase (nptII) gene conferring KanR, and the complete set of chromosomes of tobacco or tomato, as revealed by genomic in situ hybridization and Southern blot hybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions, and some introgression plants showing integration of gus and nptII in the tomato genome, were recovered in the first backcross progeny. The potential value of MMCT for the transfer of economically important traits, genome analysis, and gene expression is discussed. Key words : chromosome transfer, microprotoplast fusion, monosomic-disomic additions, sexual transmission, DNA integration, alien gene expression.
ABSTRACT
Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan(R)) and ß-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan(R) calli. A high frequency of plants regenerated from Kan(R) calli expressed both Kan(R) and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.
ABSTRACT
Data on isolation, purification and transfer of mitochondria from a cytoplasmic male sterile line of the "Ogura" type of Raphanus sativus to a male fertile line of Brassica napus are reported. Microinjection has been used for the transfer of the donor mitochondria to the recipient protoplasts. The injected protoplasts were identified and followed individually throughout their development using a computerized microscope stage which greatly enhanced the number of injections (five-fold). The transferred donor mitochondria were stably maintained during several successive cell divisions, revealing that they were viable and functional. Several calluses were obtained from injected protoplasts without using any selection pressure. Restriction fragment length analysis of seventeen calluses, using mitochondrial DNA probes, indicated that three contained the donor "Ogura" type mitochondria. No recombinant types of mitochondria have been observed. Flow cytometric and karyotype analyses of the calluses revealed the presence of similar amount of DNA and chromosome number as those of the recipient plants of B.napus. The application of microinjection for the manipulation of cytoplasmic composition is discussed.
ABSTRACT
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.
ABSTRACT
The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant "Doba" line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.
ABSTRACT
Flow cytometry and karyological analysis were used to study polysomaty and polyploidization during the first 15 days of callus formation in leaf segments from shoot cultures and greenhouse-grown plants of various lines and genotypes of Solanum tuberosum and S. phureja. The greenhouse-grown plants showed a higher degree of polysomaty (77% and 49% of polyploidized nuclei) than the shoot cultures (< 3%). During the in vitro culture period, polyploidization occurred through endoreduplication. Segments of the five shoot cultures showed up to 87%, 53%, 59%, 45% and 56% polyploidization, respectively; the DNA content of corresponding interphase nuclei amounted to 8C, 16C, 16C, 16C and 8C, and the chromosome numbers to 96. Segments from the two greenhouse-grown genotypes showed up to 87% and 84% polyploidization; the DNA content amounted to 32C and 16C, and the chromosome numbers to 192 and 96. The number of reduplication cycles was species-dependent; the degree of polyploidization was dependent on the initial ploidy level of the genotypes. Cell proliferation did not take place at a constant rate. The maximum frequencies of metaphases (52-171 per segment) occurred after 1 week of culture and were correlated with the ploidy level of the genotypes. Cells were triggered to mitosis rather than to endoreduplication. Cell cycles with normal monochromosomes could be shorter than 1 day, and those with diplochromosomes lasted at least 1 day. Polysomaty, degree of polyploidization and abnormal nuclear processes are discussed in relation to the origin of genetic instability early in culture.
ABSTRACT
Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli ß-glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the ß-glucuronidase gene are expressed conferring resp. kanamycin resistance and ß-glucuronidase activity to the plants.
ABSTRACT
Amiprophos-methyl (APM) is a potential herbicide which acts at the level of microtubules. By exposure of suspension cells of Nicotiana plumbaginifolia to this agent, a high degree of metaphase arrest was observed and single as well as groups of chromosomes were scattered throughout the cell, offering good prospects for application in cytology and chromosome isolation. After prolonged exposure to the drug, the chromosomes decondensed and micronuclei were formed. Based on their DNA content, the micronuclei were sorted by flow cytometry. Prospects for application of isolated micronuclei for partial genome transfer and gene mapping are discussed.
ABSTRACT
The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.
ABSTRACT
Attempts were made to obtain plant regeneration in vitro from leaf tissue and stem internodes of Haplopappus gracilis. The morphogenetic effects of several combinations of auxin/kinetin and light with three types of basal media were investigated. A modified White's medium supplemented with IAA gave optimum callus growth, and permitted root and shoot differentiation.
ABSTRACT
The transmission of variation from protoplast-derived plants of tetraploid potato cultivar 'Bintje' to tuber progeny was examined. The morphological alterations of a majority of the variant protoclones were transmitted to corresponding tuber progeny. Some of the normal and variant protoclones gave new phenotypes, or segregated into parental and new phenotypes after vegetative propagation. The ploidy levels of almost all these clones remained unchanged after propagation. It was concluded that the occurrence of variation after vegetative propagation was due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part of the regenerated plant. The origin of variation is discussed in the light of these results.
ABSTRACT
A wide range of phenotypic variation occurred among protoplast - derived plants of tetraploid potato cultivar 'Bintje'. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.
ABSTRACT
The self-incompatibility system of Oenothera organensis is used to analyze the frequency of S locus mutations induced by low doses (2.5-20 rad) of fast neutrons and x-rays. Th principles and methods of the screening system for detection of low dose effects have been presented. The results show that low doses induced significantly higher frequencies of seeds and seedlings than those produced spontaneously. The analyses of part of the progenies derived from the control and treated series suggested that they were due to revertible mutations. A modified in vitro method of culturing pollinated cut styles has been developed for rapid screening of compatible pollen tubes.
Subject(s)
Mutation , Plants/genetics , Culture Techniques , Fast Neutrons , Fertility , Plants/radiation effects , Pollen , Radiation DosageABSTRACT
Attempts were made, through mentor pollen techniques, to overcome self-incompatibility in species belonging to the genera Nicotiana and Oenothera and in a hybrid of Lycopersicum, which are characterized by a gametophytic system of incompatibility. While radiation-killed incompatible pollen did not generate mentor effects in any of the material tested, radiation-killed compatible pollen was found to promote a high level of illegitimate fertilizations by incompatible pollen in N. alata. No evidence was obtained that radiation-killed compatible pollen could induce mentor effects in strictly self-incompatible clones of O. organensis and of the interspecific hybrid L. esculentum x L. peruvianum.
