Protoplast

About: Protoplast is a research topic. Over the lifetime, 5474 publications have been published within this topic receiving 122468 citations.

Expression of foreign genes in regenerated plants and in their progeny.

Abstract: Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km and Cm). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.

Experiments in plant tissue culture

Abstract: Part I. Background Information: 1. Culture of plant cells, tissues, and organs 2. Laboratory facilities 3. Aseptic techniques 4. Media composition and preparation Part II. Experimental: Callus and Callus-Derived Systems: 5. Initiation and maintenance of callus 6. Organogenesis 7. Cell suspensions 8. Somatic embryogenesis Part III. Experimental: Culture of Organs and Organized Systems: 9. Isolated roots 10. Micropropagation by bud proliferation 11. Anther and pollen cultures Part IV. Experimental: Isolated Cells: 12. Transdifferentiation of parenchyma cells to tracheary elements 13. Isolation and culture of protoplasts 14. Protoplast fusion and somatic hybridization Part V. Supplementary Topics: 15. Cryopreservation of germplasm 16. Production of secondary metabolites 17. Quantitation of procedures 18. Formulations of tissue culture media 19. Commercial sources of supplies Author index Subject index.

Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion

Abstract: We have previously regenerated tobacco plants from fused protoplasts isolated from two varieties of Nicotiana tabacum. The parent cells had distinct morphological nuclear and cytoplasmic markers, enabling us to recognise, among the whole regenerated plants, those with a single parental nucleus and a hybrid cytoplasm produced by the mixing of the two parental cytoplasms1. Genetic analysis of their progeny has confirmed that the phenotypes of these cytoplasmic hybrids (or cybrids) are stable and maternally inherited. Analysis with restriction endonuclease has shown that only one or the other parental chloroplast DNA is present in the progeny of the cybrids2. We report here, however, that the mitochondrial (mt) DNAs of cybrids are different from those of the parents and from the mixture of the two. The new DNAs result from mitochondrial recombination.

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids

Abstract: Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.

Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

Abstract: Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca2+ ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants.