Showing papers on "Protoplast published in 1990"
287 citations
TL;DR: An efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309 is established and southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration.
Abstract: We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L) cultivars Nipponbare and Taipei 309 Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse Two Taipei 309 and 35 Nipponbare plants set viable seeds Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed
169 citations
TL;DR: The regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus produced healthy plants that were successfully transferred to soil and grown in the greenhouse.
Abstract: We describe the regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus. Proto-colonies were also obtained from a non-regenerable cell line established from friable callus that arose spontaneously on the aged callus, but did not give rise to any plants. Plating efficiencies well in excess of twenty percent were routinely obtained from protoplasts isolated from the regenerable as well as the non-regenerable cell lines in Kao and Michyaluk, as well as Murashige and Skoog based, nutrient media. Four to five week old protocolonies, derived from regenerable suspension culture protoplasts, formed distinct somatic embryos and shoots upon transfer to various regeneration media. Shoots were rooted on transfer to rooting media. Successive transfer of the plantlets to MS or half-strength MS medium, with or without activated charcoal, produced healthy plants that were successfully transferred to soil and grown in the greenhouse.
164 citations
TL;DR: The analysis of RNAs transiently expressed in transfected plant protoplasts appears to be at present the most suitable approach for addressing the problems of plant mRNA processing.
Abstract: Publisher Summary The analysis of RNAs transiently expressed in transfected plant protoplasts appears to be at present the most suitable approach for addressing the problems of plant mRNA processing. Transient expression experiments have been reported with protoplasts originating from a number of different tissues or suspension cultures from both dicotyledonous and monocotyledonous plants. In most instances the expression of introduced plasmids has been determined by assaying the activity of enzymes encoded by reporter genes. When protoplasts are to be transfected by electroporation, the filtrate is distributed into 12-ml polystyrene round-bottomed Falcon tubes (10 ml/tube), and the protoplast suspension in each tube is overlayed with 1 ml of EP solution. After centrifugation at 100 g for 10 min the floating protoplasts are collected, washed 2 times with EP, resuspended in EP, and used immediately for electroporation. Two alternative procedures can be used for transfecting protoplasts to study transient gene expression. Electroporation involves the use of an electric field to stimulate the uptake of exogenous DNA by the protoplasts.
149 citations
TL;DR: Regenerants from a 30-month-old haploid and a 10- month-old diploid tissue culture were cross-pollinated to generate a synthetic genotype (HE/89) with improved competence for maintenance of totipotency in various cultured expiants to regulate the maintenance of in vitro embryogenesis.
Abstract: Regenerants from a 30-month-old haploid and a 10-month-old diploid tissue culture were cross-pollinated to generate a synthetic genotype (HE/89) with improved competence for maintenance of totipotency in various cultured expiants. The HE/89 zygotic embryos developed friable, embryogenic cultures in the commonly used MS-and N6-based media without the addition of L-proline. By optimalization and changing the culture conditions, we were able to regulate the maintenance of the earlier, more synchronous (Type II) and the later, asynchronous (Type I) in vitro embryogenesis, as well as the shift between different ontogenic stages. Within 70 days after the inoculation of immature embryos a relatively homogeneous, early-embryogenic suspension culture usable for protoplast isolation was established from the initially surface-grown cultures. Using modified solutions for protoplast isolation and culture, viable protoplasts were reproducibly obtained from which plants were regenerated via defined ontogenic steps. Despite the long in vitro history of the parental genotypes, 60–70% of the more than 500 plants derived from the HE/89 protoplasts set seeds following self or sib-pollination.
127 citations
TL;DR: The transfer of kanamycin resistance of bacterial origin into wheat via pipetting Agrobacterium suspension into wheat spikelets via pollen-mediated gene transfer represents an alternative to protoplast-based transformation and leaf disk techniques by avoiding regeneration problems and somaclonal variation, introducing fertile plants.
109 citations
TL;DR: The stage of zygotic embryo development for optimal somatic embryogenesis was found to be the early stage of polyembryony and the appearance and nutritional requirements of the established protoplast-derived cell suspension were similar to those of the original cell suspension.
103 citations
Patent•
27 Feb 1990TL;DR: In this paper, an improved process for transforming plant protoplasts using simple, purely chemical, process steps is described, and the plant material obtainable by this method is obtained.
Abstract: The present invention relates to an improved process for transforming plant protoplasts using simple, purely chemical, process steps, and to the plant material obtainable by this method. The transfer of the genetic material into the plant cell is carried out directly, without the use of a natural plant-infective system such as a plant bacterium or plant virus, and without transfer by insects or phytopathogenic fungi, by joint incubation in a suitable incubation medium of the DNA to be transformed and plant protoplasts. In this manner, desired genes can very simply and efficiently be transferred to plant material, resulting in plants with improved properties.
92 citations
TL;DR: An overview of tissue culture techniques and their applications in plant propagation and genetic improvement of plants is presented in this paper, which includes embyro culture, meristem culture, micropropagation, in vitro selection, anther culture and protoplast culture.
Abstract: Plant tissue culture involves the culture of all types of plant cells, tissues and organs under aseptic conditions. This definition also extends to the culture of excised embryos and to protoplast culture. An overview of tissue culture techniques and their applications in plant propagation and genetic improvement of plants is presented. The areas under review include: (1) embyro culture, (2) meristem culture, (3) micropropagation, (4) somatic embryogenesis, (5) somaclonal variation, (6) in vitro selection, (7) anther culture and (8) protoplast culture. Problems and limitations of each of the techniques are also discussed. Examples are given of work that has been undertaken or that is currently in progress on the application of these techniques to the improvement of Queensland's subtropical horticultural industries. Key examples are: (1) embryo culture to facilitate incorporation of genes conferring disease-resistance from wild Cucurbita species into cultivated varieties, (2) meristem culture for virus elimination in strawberries (Fragaria × ananassa) and sweet potato (Ipomoea batatas), (3) micropropagation for rapid increase in new varieties of ginger (Zingiber officinale) and pineapple (Ananas comosus) to enable more rapid field evaluation and early release, (4) micropropagation of disease-free, genetically uniform planting material of superior female papaya (Carica papaya) selections and banana (Musa spp.) selections and (5) the use of somaclonal variation and gamma-irradiation for the genetic improvement of banana. Finally, future opportunities for the utilisation of tissue culture in plant propagation and improvement in Queensland's horticultural industries are summarised.
84 citations
TL;DR: The antagonistic abilities of the prototrophic strains were found to vary with each pathogenic fungus, and the protOTrophic strain A2 overgrew all the pathogenic fungi more rapidly than the parental strains.
Abstract: Protoplasts from two auxotrophic mutants of Trichoderma harzianum Rifai (ATCC 32173), obtained from young thalli following cell wall digestion by NovoZym 234, were fused in 33% PEG suspended in 10 ...
66 citations
TL;DR: Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplast of Brassica napus c v Westar to show hybridization patterns similar to B. napus.
Abstract: Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers All of the regenerated plants had morphology identical to B napus and 10 of them possessed the diploid chromosome number of B napus The remaining plants had chimeric or aneuploid chromosome numbers The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B napus were examined by Southern hybridization analysis Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns Of these 4 plants, 1 was male sterile, and 3 were male fertile The remaining plants showed mitochondrial DNA patterns identical to B napus and were male fertile
TL;DR: This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.
Abstract: Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.
TL;DR: Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.
Abstract: To determine whether regeneration of rice plants from protoplast culture induces DNA polymorphisms, progeny plants from direct regenerants of such cultures were examined for restriction fragment length polymorphisms (RFLP analysis). Significantly increased levels of DNA polymorphism were found compared with those in non-tissue culture control plants. Analysis with gene sequences representative of different functional domains, revealed that such polymorphisms are apparently widespread and not associated with any particular region. Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.
TL;DR: Protoplast fusion was investigated as a means of obtaining somatic hybrids between alfalfa, Medicago sativa L., and two sexually incompatible annual species, M. intertexta and M. scutellata, to confirm that the selected calli were nuclear hybrids.
TL;DR: A marine aerobic bacterium identified as Pseudomonas alginovora was isolated from decaying fronds of Laminaria digitata and secreted an endo- acting alginate lyase of the guluronate-specific type that was effective in producing protoplasts from Laminationaria species.
TL;DR: 2D NEPHGE of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related proteins are used, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts, and it is suggested that wound healing callus and in vitro callus correspond to the same differentiation state.
Abstract: We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells. The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.
TL;DR: The myrosinase activity in protoplast and tissue cultures as well as in regenerated shoots and plants have been examined and compared to the activity in intacts plants.
Abstract: A detailed investigation of myrosinases in Brassica napus at different developmental stages and organs is reported. In addition, the myrosinase activity in protoplast and tissue cultures as well as in regenerated shoots and plants have been examined and compared to the activity in intacts plants
TL;DR: The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt- DNA were involved in the rearrangements following protoplast fusion, and all different chloroplast types could be established within each combination.
Abstract: Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.
TL;DR: Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated and are useful for studies in maize requiring large numbers of stably transformed or co- transformed cell lines.
Abstract: Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 1∶1 ratio to 65% at a 100∶1 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.
TL;DR: Mitochondrial DNA probes, capable of discriminating different cytoplasms ofBeta spp.
Abstract: For our program on the transfer of cytoplasmic male sterility (CMS) by cybridization inBeta vulgaris L. (sugar beet), we have developed a procedure for the isolation and culture of mesophyll protoplasts of sugar beet followed by shoot regeneration. A prerequisite proved to be the presence in the media of n-propylgallate (nPG), a lipoxygenase inhibitor. Sustained divisions were found in all accessions that were tested. Plating efficiencies and regeneration ability varied greatly from one experiment to the other and appeared to be accession-dependent. Shoots could be easily transferred to soil. A majority of the regenerants (72%) retained the diploid chromosome number. Somaclonar variation in phenotype was low (4.9%). Mitochondrial DNA probes, capable of discriminating different cytoplasms ofBeta spp. showed no rearrangements due to the protoplast and in vitro culture phase, indicating that these probes can be used to identify cybrids after asymmetric fusions. The data presented here open up possibilities for genetic engineering using protoplasts in one of the world's most important arable crops.
TL;DR: Results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion, and a greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.
Abstract: Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase β subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.
TL;DR: In this paper, plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation.
Abstract: Plasmids DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 104 transformants/μg DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformations efficiency.
TL;DR: The microspore system improves significantly on the protoplast system by virtually eliminating the large somaclonal variation associated with protoplas selection, by utilizing a true haploid cell system, and by resulting in a more synchronized embryo development that facilitates accurate mutation and selection methods.
Abstract: Pioneering research in Brassica microspore culture (1,2,3)rapidly led to the realization that microspores provide a powerful alternative to protoplast culture as a single-celled culture method in plants. These two single-celled systems are fundamentally different, both in tissue origin and in genetic variability. The microspore system improves significantly on the protoplast system by virtually eliminating the large somaclonal variation associated with protoplast selection, by utilizing a true haploid cell system, and by resulting in a more synchronized embryo development that facilitates accurate mutation and selection methods. Most critical, however, are the observations that plant regeneration frequencies in excess of 80% can be readily obtained and that the entire sequence from microspore isolation to plantlet development may take place in as little as 4 wk (4).
TL;DR: Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria, and most protoplasts were viable and regenerated a cell wall and divided.
Abstract: Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×108 protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.
TL;DR: It is suggested that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt− cells of thisClosterium complex.
Abstract: When mating-type minus (mt−) and plus (mt+) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt− cells also proceeded without pairing in a medium in which mt− and mt+ cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (Mr) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of Mrs 42000 and 19000, while the deglycosylated polypeptides had Mrs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·109 and the concentration required for 50% of the maximum response (ED50) as 4.1·10−9M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt− cells of thisClosterium complex.
TL;DR: An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented and two-thirds of transgenic plants grown to maturity in the greenhouse bore viable seeds.
Abstract: An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol seed calli from the varietiesNipponbare andTaipei 309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 × 106 protoplasts perg fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant transcriptional regulatory signals was used as a selectable marker gene. Hygromycin-resistant colonies were selected in the presence of 95 μM hygromycin B with apparent frequencies of 2×10−4 and 5×10−4 forNipponbare andTaipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.
TL;DR: Protoplasts were isolated from suspension cultured cells of a single genotype from tall fescue and cultured by the agarose bead method and some of the protoplasts from cultivar Nanryo grew into yellow compact calluses that differentiated into green plantlets.
TL;DR: Hypocotyl calli-derived protoplasts of two cultivars of Brassica juncea were fused with normal as well as γ-irradiated mesophyll protoplast of Eruca sativa, and resulted in somatic and partial somatic hybrids that could be regenerated from such fusion products.
Abstract: Hypocotyl calli-derived protoplasts of two cultivars of Brassica juncea (2n=36), a major oil-seed crop, were fused with normal as well as γ-irradiated mesophyll protoplasts of Eruca sativa (2n=22). The irradiation of the Eruca fusion partner increased the plating efficiency as well as the morphogenic potentiality of the fusion products over the normal fusion. Fertile plants could be regenerated from such fusion products. Analysis of 63 out of 181 plants regenerated showed that, indeed, 11 somatic hybrids (2n=58) and 10 partial somatic hybrids (chromosome number ranged between 50 and 56) had been obtained. Pollen viability (0%–82.9%) and seed set (0%–50%) of the hybrids indicated them to be useful for future studies.
TL;DR: Protoplasts from cauliflower and broccoli leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture and plants were recovered from protoplasts of previously recalcitrant Brassica genotypes.
Abstract: Protoplasts from cauliflower (Brassica oleracea ssp. botrytis) and broccoli (ssp. italica) leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture. Cells from rice, tomato and tobacco suspensions were not as effective as the B. campestris cells. Plants were recovered from protoplasts of previously recalcitrant Brassica genotypes. Protoplasts cultured in low numbers (10-100) on the feeder layer divided and formed colonies capable of plant regeneration, as did fused protoplasts.
TL;DR: 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.
Abstract: 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.