Donor-recipient Protoplast Fusion Research Articles

NEW CYBRIDS RESULTING FROM ASEXUAL PATHWAY: A PROMISE OF CYBRIDIZATION FOR CREATING NEW CITRUS ROOTSTOCKS AND CULTIVARS

Cybridization should be a way to induce phenotypical variations in highly heterozygous secondary citrus species or genotypes, without breaking their very complex multilocus structure by sexual recombination. It should have particular interest to manipulate the nucleo-cytoplasmic male sterility, to transfer tolerance traits with cytoplasmic determinisms and eventually to improve tolerance to oxidative stress. The present work is focused on the creation of new cybrids through three different approaches of protoplast fusion: (i) symmetric hybridization, (ii) donor-recipient protoplast fusion by UV irradiation of donor protoplasts, and (iii) cytoplasts + protoplast fusion. Sixteen combinations of symmetric protoplast fusion have produced cybrids. No cybrid was obtained after UV irradiation of donor protoplasts while three combinations of cytoplasts + protoplast fusion produced cybrids. Therefore, cytoplast fusion appears much more promising than the UV approach for targeted cybridization. The innovative plant material is currently propagated for further cultivar and rootstock selection and for basic researches on the impact of nucleo-cytoplasmic interaction on desirable traits. (Resume d'auteur)

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line.

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.

A Review of Plant Embryo Culture

Departme Tusa, N., J.W. Grosser, and F.G. Gmitter, Jr. 1990. Plant regeneration of ‘Valencia’ sweet orange, ‘Femminello’ lemon, and the interspecific somatic hybrid following protoplast fusion. J. Amer. Soc. Hort. Sci. 115:1043– 1046. Vardi, A., P. Arzee-Gonen, A. Frydman-Shani, and S. Bleichman. 1989. Protoplast fusion mediated transfer of organelles from Microcitrus and Citrus and regeneration of novel alloplasmic trees. Theor. Applied Genet. 78:741–747. Vardi, A., A. Breiman, and E. Galun. 1987. Citrus cybrids: Production by donor–recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles. Theor. Applied Genet. 75:51–58. Vardi, A. and E. Galun. 1988. Recent advances in protoplast culture of horticultural crops: Citrus. Scientia Hort. 37:217–230. Vardi, A., P. Spiegel-Roy, and E. Galun. 1982. Plant regeneration from Citrus protoplasts: Variability in methodological requirements among cultivars and species. Theor. Applied Genet. 62:171–176.

Effect of gamma-irradiation on protoplast viability and chloroplast DNA damage in Lycopersicon peruvianum with respect to donor-recipient protoplast fusion

Irradiation can be used in donor-recipient protoplast fusions to achieve inter/intraspecific chloroplast transfer. The effects of gamma-rays on the viability, cell division frequency and chloroplast DNA integrity of Lycopersicon peruvianum protoplasts were studied to determine the critical radiation dose, defined as that dose which inactivates the protoplasts through damage to the nuclear DNA but which does not inhibit functioning of chloroplasts DNA. No effect on the viability of the treated protoplasts was visible 5 hr after irradiation of L. peruvianum protoplasts with gamma-ray doses from 50 to 1000 Gy. However, after a dose of 2500 Gy, protoplast viability decreased to 64%. After 120 hr, protoplast viability decreased in all treatments with increased dose. Some cells were able to divide after the 50 Gy dose. However, sustained divisions were not observed. Cell division did not take place after any of the other doses. The effects of 50, 300 and 1000 Gy on chloroplast DNA of irradiated protoplasts and isolated chloroplasts were investigated using conventional and pulsed-field gel electrophoresis. No dramatic chloroplasts DNA fragmentation was observed. Results showed that the proportion of linear chloroplast DNA increased with increasing dose. Indications of chloroplasts DNA fragmentation were found only after irradiation with 1000 Gy. On the basis of these results a gamma-ray dose of 300 Gy was selected for chloroplast transfer in asymmetric hybridization experiments. At this dose, cell division is prevented but chloroplast DNA integrity is not significantly disrupted.

Nuclear-organelle interaction in Solanum: Interspecific cybridizations and their correlation with a plastome dendrogram

Alloplasmic compatibility, namely the functional interaction between the nuclear genome of a given species with plastomes and chondriomes of alien species, is of considerable relevance in plant biology. The genus Solanum encompasses a wide spectrum of species and is therefore suitable for a study of this compatibility. We thus chose the nuclear genome of Solanum tuberosum (potato) and organelles (chloroplast and mitochondria) from 14 other Solanum species to initiate an investigation of intrageneric nucleus/organelle interactions. An assessment of the diversity of the chloroplast DNAs from these 15 species resulted in the construction of a plastome dendrogram (phylogenetic tree). In parallel we extended a previous study and performed ten additional fusion combinations by the "donor-recipient protoplast fusion" procedure, using potato protoplasts as recipients and protoplasts from any of ten other Solanum species as donors. We found that two fusion combinations did not yield cybrids and that the chloroplasts of S. polyadenium and the mitochondria (or mitochondrial components) from S. tarijense could not be transferred to cybrids bearing potato nuclei. In general, there is a correlation, albeit not perfect, between the cybridization data and the plastome dendrogram. These results furnish valuable information toward future transfer of plasmone-encoded breeding traits from wild Solanum species into potato. This information should also be useful for the planning of asymmetric protoplast fusion between potato and wild accessions for the improvement of pathogen and stress resistance of potato cultivars.

Transfer of the petaloid-type CMS in carrot by donor-recipient protoplast fusion.

The CMS trait of petaloid-type flowers in carrot was tried to transfer to fertile varieties by donor-recipient protoplast fusion. X-irradiated protoplasts of the carrot CMS line, 31A, and iodoacetamide-treated protoplasts of 5 different fertile varleties were used as a cyioplasmic donor and recipients, respectively. Fifty-eight plants were regenerated from the fusion experiments and no plant with petaloid flowers was observed. A11 the regenerated plants were fertile or male sterile with brown anthers. To produce cybrlds with petaloid-type flowers, Z1, which is a regenerated plant obtained from protoplast fusion between 31A and K5, was used as a cytoplasmic recipient and fused with X-irradiated protoplasts of 31A. From this donor-recipient protoplast fusion, 41 fusion products were regenerated and bore flowers. Among them, 39 plants were CMS with petaloid stamens and 2 plants were the same male sterile with brown anthers as Z1. Chromosomal and mitochondrial DNA analyses of the fusion products revealed that all of the plants investigated had nuclei derived from Z1 and rearranged mtDNAs. After crossing with fertile varieties including K5, the petaloid male sterility of the fusion products was maternally inherited in the F1 and F1B1 generations. Other agronomic traits of the progenies were similar to those of the parental fertile line. The results indicate that the petaloid CMS trait can be transferred effectively to another carrot line by using two-step donor-recipient protoplast fusion.

DNA hybridization analysis of mitochondrial genomes of carrot cybrids produced by donor-recipient protoplast fusion

The mitochondrial genome composition of 17 carrot cybrids possessing petaloidy stamens and brown anthers that were produced by donor-recipient protoplast fusion between a petaloid CMS line, 31A and the brown anther type of a genic male sterile line, Z1, was analyzed by Southern hybridization with 12 heterologous mitochondrial gene probes. The hybridization analysis revealed that 6 out of the 17 cybrids had the same hybridization patterns as the CMS parent, 31A and the remaining 11 cybrids possessed rearranged mitochondrial genomes of the parental lines. Hybridization of maize cytochrome c oxidase subunit I ( coxI) to 4.2-kb and 2.8-kb fragment is characteristic of all the petaloid cybrids and the CMS parent, 31A: whereas hybridization to the 4.2-kb fragment is absent in the brown anther type cybrid.

Asymmetric hybridization between Nicotiana tabacum and N. repanda by donor recipient protoplast fusion: transfer of TMV resistance

Genetically asymmetric hybrids were recovered by fusion of Nicotiana tabacum protoplasts with irradiated protoplasts of kanamycin-resistant, nopalineproducing plants of N. repanda. Hybrid calli were selected by culture on media containing kanamycin and were regenerated. These plants were morphologically similar to N. tabacum but produced nopaline, indicating they retained genes from N. repanda. Esterase isozyme profiles also indicated that the plants are somatic hybrids, but are more similar to N. tabacum than N. repanda. Chromosome counts showed most of the hybrids had 55-62 chromosomes, which is consistent with extensive, although incomplete elimination of N. repanda chromosomes. The hybrids were largely male sterile, but about half of them set seed when crossed with N. tabacum. Chromosome numbers of the progeny and the pattern of inheritance of kanamycin resistance indicated the continued elimination of N. repanda genetic material in these backcrosses. The N. repanda parent used in these fusions gave a hypersensitive response to TMV, whereas the N. tabacum parent was TMV sensitive. When inoculated with TMV, plants from two hybrid clones gave a hypersensitive response. Plants from the other clones became systemically infected with the virus.

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, 'K5', were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, 'K5'. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.

Transfer of cytoplasmic organelles from an oligomycin-resistant Nicotiana cell suspension into tobacco protoplasts yielding oligomycin-resistant cybrid plants

Oligomycin-resistant lines were derived from a Nicotiana sylvestris cell suspension, after N-nitroso-N-methylurea mutagenesis followed by selection in the presence of 0.4 μg/ml oligomycin, a specific mitochondrial ATPase inhibitor. One of the lines, oliR38 was further analyzed to investigate the role of mitochondria in this resistance. The oliR38 line proved to be also highly resistant to venturicidin, another specific inhibitor of mitochondrial ATPase. By the donor-recipient protoplast-fusion procedure the cytoplasmic organelles of oliR38 were transferred to protoplasts of Line 92, a line of tobacco plants which contain the cytoplasmic organelles of N. undulata. Cell suspensions prepared from several cybrid plants, containing the cytoplasmic organelles of oliR38, exhibited the same level of oligomycin resistance as the oliR38 line.

Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles

Embryogenic nucellar-callus from three Citrus types was used for protoplast isolation. The protoplasts were fused by the ‘donor-recipient’ procedure by which the nuclear-division of the donor-protoplasts was arrested by gamma-irradiation and the metabolism of unfused recipient-protoplasts was transiently inhibited by iodoacetate. The following fusion combinations were performed: (1) Poorman x Poncirus trifoliata with Villafranca lemon; (2) Poorman x P. trifoliata with Sour orange; (3) Sour orange with Villafranca. Combinations 1, 2 and 3 resulted in 120 (125), 70 (5) and 19 (89) calli (regenerated plants), respectively. The mitochondrial-DNA (mtDNA) restriction profiles of Poorman x P. trifoliata obtained by fragmentation with Bcl I, Bam H1 or Sal I differed from the respective profiles of Villafranca and Sour orange but no differences in mtDNA restriction profiles were detected between Villafranca and Sour orange. When Southern blots of Villafranca and Sour orange mtDNAs were hybridized with radiolabelled heterologous mtDNA probes, the mtDNAs of these two Citrus types could be differentiated. The fusion-derived plants from all three donor-recipient combinations had the recipients morphological (nuclear-coded) features. MtDNA restriction profiles, with and without hybridization to heterologous probes, indicated that the analysed plants were cybrids.