Showing papers on "Protoplast published in 1994"

Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs

Abstract: A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.

Plant cell biology: a practical approach

Abstract: 1.: An introcuction to optical microscopy for plant cell biology. 2.: The use of fluorescent probes for studies of living plant cells. 3.: General and enzyme histochemistry. 4.: Electron microscopy. 5.: In situ hybridization of RNA. 6.: DNA-DNA in situ hybridization - methods for light microscopy. 7.: Immunochemistry for light and electron microscopy. 8.: Plant protoplast techniques. 9.: Chemical analysis of the components of the primary cell wall. 10.: Immunofluorescent techniques for analysis of the cytoskeletan. 11.: Isolation of endo- and plasma membranes. 12.: Protein transport into intact chloroplasts and isolated thylakoids. 13.: Ion-selective micro-electrodes. 14.: Microsampling and measurements of solutes in single cells

Perception of Gibberellin and Abscisic Acid at the External Face of the Plasma Membrane of Barley (Hordeum vulgare L.) Aleurone Protoplasts.

Abstract: The response of protoplasts isolated from aleurone layers of barley (Hordeum vulgare L. cv Himalaya) to internally and externally applied hormone was analyzed to localize the site of perception of the hormonal signal. Protoplasts responded to externally applied gibberellic acid (GA3) with increased synthesis and secretion of [alpha]-amylase, transient expression of the glucuronidase reporter gene fused to the hormone-responsive elements of the [alpha]-amylase promoter, and the vacuolation typical of GA3-treated aleurone cells. When up to 250 [mu]M GA3 was microinjected into the protoplast cytoplasm, none of these responses were observed. This did not reflect damage to the protoplasts during the microinjection procedure, since microinjected protoplasts remained responsive to externally applied hormone. Nor did it reflect loss of microinjected GA3 from the protoplast, since 50% of microinjected [3H]GA20 was retained by protoplasts for at least 24 h. Externally applied abscisic acid (ABA) could reverse the stimulation of [alpha]-amylase synthesis and secretion, whereas microinjecting up to 250 [mu]M ABA was ineffective at antagonizing the stimulatory effect of GA3. These results suggest that the site of perception of GA3 and ABA in the barley aleurone protoplast is on the external face of the plasma membrane.

Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells.

Abstract: During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Abstract: Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH

Abstract: We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400–430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

Genetic Transformation of Rice

Abstract: Rice was the first major monocot crop species to be transformed and regenerated. Initially, rice transformation was limited to japonica cultivars. Subsequently, a number of indica and javanica cultivars have also been transformed and regenerated into fertile transgenic plants. Most transformation studies in rice have used direct DNA uptake into protoplasts, induced by polyethylene glycol treatment or electroporation. Recently, other transformation methods have been developed that are less genotype dependent, such as microprojectile bombardment of cell suspensions and immature embryos. This review summarizes progress in both protoplast-based and other transformation methods.

Intertribal somatic hybrids between Brassica napus and Thlaspi perfoliatum with high content of the T. perfoliatum-specific nervonic acid.

Abstract: Protoplast fusions were performed between hypocotyl protoplasts of Brassica napus and mesophyll protoplasts of Thlaspi perfoliatum. The two species are members of the Lepidieae and Brassiceae tribes, respectively, in the family of Brassicaceae. Seeds of T. perfoliatum are rich in the fatty acid C24∶1 (nervonic acid), an oil valuable for technical purposes. In the search for renewable oils to replace the mineral oils, plant breeders have been trying to develop oil crops with a high content of long-chain fatty acids. After fusion of B. napus protoplasts with non-irradiated as well as irradiated protoplasts of T. perfoliatum selection was carried out by flow cytometry and cell sorting. Of the shoots regenerated from different calli 27 were verified as hybrids or partial hybrids using the isoenzyme phosphoglucose isomerase (PGI) as a marker. Another 6 plants were identified as partial hybrids using a T. perfoliatum-specific repetitive DNA sequence. Slot blot experiments were performed to estimate the copy number of the repetitive DNA sequence in the parental species and in the hybrids. In T. perfoliatum there were approximately 105 copies per haploid genome, and the range in the hybrids was 1–37% of the value in T. perfoliatum. When the nuclear DNA content of the regenerated shoots was analysed we found partial as well as symmetric hybrids. Even though the rooting and establishment of hybrid shoots in the greenhouse were difficult, resulting in the death of many plants, 19 plants were cultured to full maturity. Seeds obtained from 15 plants were analysed to determine whether they contained nervonic acid, and 5 of the hybrids were found to contain significantly greater amounts of nervonic acid than B. napus.

Effect of silver nitrate on sugarcane cell suspension growth, protoplast isolation, ethylene production and shoot regeneration from cell suspension cultures

Abstract: Silver nitrate (AgNO), an inhibitor of the physiological action of ethylene, reduced cell growth, promoted ethylene production, increased the yield of protoplasts and reduced shoot regeneration from sugarcane heterogeneous cell suspension cultures. The increase in the rate of protoplast isolation from cultures treated with AgNO (0 to 59 μM) correlate with an increase in endogenous ethylene production by the cells. The addition to the culture medium of chemicals that either inhibited (aminoethoxyvinylglycine, AVG) or promoted (aminocyclopropane-1-carboxylic acid, ACC) ethylene biosynthesis did not alter the number of protoplasts isolated from these cultures. However, protoplasts were isolated with AVG in combination with AgNO even though ethylene production was inhibited. These results suggested that AgNO may be having another more direct effect on protoplast release. One such site may be the cell wall or on cell metabolism conditioning cells to release protoplasts after enzyme treatment.

Somatic Hybridization in Crop Improvement I

Abstract: Section I Protoplast Fusion, Somatic Hybrids, Asymmetric Hybrids, Cybrids - Transfer of Chloroplast Traits.- I.1 Somatic Hybridization - A Rich Source of Genetic Variability.- I.2 Fluorescence Microscope Study of Protoplast Fusion.- I.3 Somatic Hybridization by Microfusion of Protoplasts.- I.4 Asymmetric Somatic Hybrids.- I.5 Cybrids - Transfer of Chloroplast Traits Through Protoplast Fusion Between Sexually Incompatible Solanaceae Species.- Section II Somatic Hybridization in Cereals, Grasses, and Legumes.- II. 1 Somatic Hybridization in the Family Gramineae.- II.2 Somatic Hybridization Between Zea mays and Triticum sect, trititrigia.- II.3 Somatic Hybridization in Festuca and Lolium.- II.4 Somatic Hybridization Between Birdsfoot Trefoil (Lotus corniculatus L.) and Soybean (Glycine max L.).- II.5 Somatic Hybridization in the Genus Medicago.- Section III Somatic Hybridization in Potato, Tomato, Eggplant, and Lettuce.- III. 1 Cybridization in Potato.- III.2 Somatic Hybridization in Solanum Tuberosum x S. chacoense.- III.3 Somatic Hybridization Between Solanum tuberosum and Nicotiana plumbaginifolia.- III.4 Pomato: Potato Protoplast System and Somatic Hybridization Between Potato and a Wild Tomato.- III.5 Somatic Hybridization Between Lycopersicon esculentum Mill. and Lycopersicon peruvianum var. dentatum Dun.- III.6 Somatic Hybridization Between Tomato (Lycopersicon esculentum) and Pepino (Solanum muricatum).- III.7 Somatic Hybridization of Eggplant (Solanum melongena L.) with Its Close and Wild Relatives.- III.8 Somatic Hybridization in Lettuce (Lactuca Species).- Section IV Somatic Hybridization in Brassicaceae.- IV.1 Resynthesis of Brassica napus Through Protoplast Fusion Between B. oleracea and B. rapa.- IV.2 Analysis of Somatic Hybrids and Cybrids Obtained by Fusion of Brassica rapa and B. oleracea.- IV. 3 Somatic Hybridization Between Radish (Raphanus sativus) and Rapeseed (Brassica napus).- IV.4 Somatic Hybridization Between Brassica and Sinapi.- Section V Somatic Hybridization in Medicinal Plants - Including Tobacco.- V.I Somatic Hybridization of Medicinal Plants in the Family Solanaceae.- V.2 Somatic Hybridization in Datura.- V.3 Somatic Hybrids Between Nicotiana repanda and N. tabacum Show Resistance to Tobacco Mosaic Virus and Meloidogyne arenaria.- V.4 Somatic Hybridization Between Tobacco (Nicotiana tabacum L.) and Black Nightshade (Solanum nigrum L.), and the Selection of a New Strain, 694-L.- V.5 Transfer of Lincomycin Resistance Through Somatic and Sexual Cybridization in Nicotiana A. CSEPL? (With 4 Figures).- V.6 Somatic Hybridization in the Family Apocynaceae (Catharanthus, Rauwolfia, Rhazya, and Vinca Species).- Section VI Somatic Hybridization in Trees (Citrus, Poncinus, Prunus, Pyrus, and Populus Species).- VI. 1 Somatic Hybridization of Citrus with Sexually Incompatible Wild Relatives.- VI.2 Somatic Hybridization Between Citrus sinensis and Poncirus trifoliata.- VI.3 Somatic Hybridization Between Pyrus x Prunus Species.- VI.4 Somatic Hybridization in Populus Species (Poplars).- Section VII Somatic Hybridization in Algae, Bryophytes, and Ferns.- VII. 1 Somatic Hybridization in Algae.- VII.2 Somatic Hybridization in Bryophytes.- VII.3 Somatic Hybridization in Ferns.

Isolation and regeneration of haploid protoplasts from bangia atropurpurea (rhodophyta) with marine bacterial enzymes1

Abstract: Three kinds of enzymes, agarase, β-1,4-mannanase, and β-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY-234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa-pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β-1,4-mannanase, and β-1,3-xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 106 protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa.

Abstract: Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.

Fission yeast protein kinase C gene homologues are required for protoplast regeneration: a functional link between cell wall formation and cell shape control

Abstract: Two novel protein kinase C (n PKC) gene homologues, pck1+ and pck2+ were isolated from the fission yeast Schizosaccharomyces pombe (Toda et al. (1993) EMBO J. 12, 1987). We examined the functional differences of pck1+ and pck2+ in cell wall formation and actin organization of S. pombe. Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+ (delta pck1) and pck2+ (delta pck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization. Protoplasts of the wild-type strain and those of delta pck1 reverted to intact cells in osmotically stabilized liquid medium. A close spatial association between new cell wall formation and actin was observed in these two strains. In delta pck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed; (2) actin was not reorganized; and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts. These findings demonstrate that the pck2+ gene has a function essential for protoplast regeneration but the pck1+ gene does not. Involvement of n PKCs in cell wall formation and actin organization was also clarified. The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast.

Somatic hybridization as a tool for tomato breeding.

Abstract: Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Abstract: Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.

PEG-mediated direct gene transfer and electroporation

Abstract: For many years of genetic manipulation in plants, direct uptake of naked DNA by plant protoplasts has been the sole alternative to Agrobacterium tumefaciens-mediated gene transfer The first experiments demonstrating direct gene transfer included the delivery of isolated plasmid DNA to protoplasts of petunia and tobacco in the presence of poly-L-ornithine or polyethylene glycol (PEG) [1–4] During the following years, protoplast transformation mediated by PEG [5] or electroporation [6] was substantially simplified and their efficiency in model systems was increased by several orders of magnitude (reviewed by Paszkowski et al [7])

Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii

Abstract: Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.

Genetic transformation of plants by protoplast electroporation

Abstract: This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts.

Abstract: Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses ≤ 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.

Regeneration of plants from protoplasts of rapid cycling Brassica oleracea L.

Abstract: Rapid cycling Brassica species have great potential in plant genetic research because of their short life cycles and their minimal space requirements. Rapid cycling B. oleracea can be grown with up to six generations per year. Protoplast culture of this genotype can be applied for gene transfer by direct DNA uptake and by protoplast fusion. We here report on fast regeneration of flowering plants from protoplasts of rapid cycling B. oleracea. Regeneration frequencies of 27–65% were achieved with multiple shoots developing from individual calli. The regenerated plants were grown to maturity, and flowering and other morphological characteristics were monitored. The regenerants flowered within a similar time frame as plants grown from seeds. The ploidy level of regenerated and seed-grown plants was measured by flow cytometry. Many (20–45%) of the regenerants were tetraploid. Although only few seeds could be obtained from the tetraploids, large numbers of seeds with good germination were recovered from the diploid regenerants.

Factors influencing protoplast isolation from Coffea arabica cells

Abstract: Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K 3 salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×106 to 4.6×106 P g-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).

Significance of plasmolysis spaces as markers for periseptal annuli and adhesion sites.

Abstract: During hyperosmotic shock, the protoplast and stretched-out peptidoglycan layer first shrink together until the turgor pressure in the cell is relieved. Being non-compressible, the outer and inner membranes must fold their superfluous surfaces. While the protoplast contracts further, the inner membrane rearranges into plasmolysis spaces visible by phase-contrast microscopy. Two opposing theories predict a similar positioning of spaces in dividing cells and filaments: the 'periseptal annulus model', based on adhesion zones, involved in the predetermination of the division site; and a 'restricted, random model', based on physical properties of the protoplast. Strong osmotic shock causes retraction of the inner membrane over almost the entire surface forming the so-called 'Bayer bridges'. These tubular adhesion sites are preserved by chemical fixation, and can be destroyed by cryofixation and freeze-substitution of unfixed cells. Both the regular positioning of the plasmolysis spaces and the occurrence of tubular adhesion sites can be explained on the basis of physical properties of the membrane which necessitate rearrangements by membrane flow during shrinkage of the protoplast.

Synthesis of intraspecific somatic hybrids of Solanum tuberosum: assessments of morphological, biochemical and nematode (Globodera pallida) resistance characteristics

Abstract: In an attempt to produce novel agronomic traits, a number of intraspecific somatic hybrid plants have been produced following leaf mesophyll protoplast fusion between S tuberosum dihaploid clones PDH 40 (possessing good tuber shape and yield) and PDH 417 (possessing resistance to potato cyst nematode, G pallida) PDH 417 protoplast-derived calli failed to regenerate plants using the described culture conditions preventing this parental type amongst the mass of regenerated fusion products Initially, somatic hybrid plants were selected based on differential pigmentation in tuber sprouts and where possible on petal colour Differential mobility of patatin bands in electrophoresed tuber extracts further confirmed hybridity