Showing papers on "Protoplast published in 1998"

Somaclonal Variation: Mechanism and Applications in Crop Improvement

Abstract: Somaclonal variation refers to the variation arising in cell cultures, regenerated plants and their progenies, and this general term was given by Larkin and Scowcroft (1981). However, other types of variation arise by specific culture of cells or tissues, which include culture of: protoplasts (protoclonal); anthers and microspores (gametoclonal); callus (calliclonal); apical meristem (mericlonal); leaf, stem, root or other somatic tissues (somaclonal). There are different approaches to create somaclonal variation, which include: (1) growth of callus or cell suspension cultures for several cycles; (2) regeneration of a large number of plants from such long-term cultures; (3) screening for desirable traits in the regenerated plants and their progenies, e.g. in-vitro selection to select agronomically desirable somaclones for tolerance to various biotic and abiotic stresses using toxic levels of pathotoxins, herbicides, salts, etc.; (4) testing of selected variants in subsequent generations for desired traits; and (5) multiplication of stable variants to develop new breeding lines.

PEG-Mediated Protoplast Transformation with Naked DNA

Abstract: 1. Introduction Direct introduction of DNA into plant protoplasts facilitates a rapid analysis of transient gene expression, as well as the generation of stably transformed transgenic plants. Transient gene expression assays performed after DNA transformation permit a comparative analysis of cisacting regulatory sequences and their function in transcriptional control of plant genes by signaling pathways mediating cellular responses to different environmental and hormonal stimuli (I). There are a number of methods for introducing DNA into plant protoplasts, but the most commonly used technique is the polyethylene glycol (PEGjmediated DNA uptake. The PEG-mediated transformation is simple and efficient, allowing a simultaneous processing of many samples, and yields a transformed cell population with high survival and division rates (2). The method utilizes inexpensive supplies and equipments, and helps to overcome a hurdle of host range limitations of Agrobacterium-mediated transformation. The PEG-mediated DNA transfer can be readily adapted to a wide range of plant species and tissue sources. In Arabidopsis thaliana, several methods of direct gene transfer to leaf mesophyll(3-5) and root-derived protoplasts (6) have been reported. They are all derived from a PEG-mediated direct gene transfer technique established originally for tobacco protoplasts by Negrutiu et al. (7). This chapter describes a method for PEG-mediated transformation of protoplasts derived from leaves, roots, and cell suspensions of A. thaliana. Leaf mesophyll protoplasts are able to regenerate after embedding into alginate, but their yield is relatively low. In comparison, cell suspensions provide an unlimited source of rapidly dividing protoplasts that can be obtained within 2-3 h and show a transient expression

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI).

Abstract: We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ∼3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.

Insertional mutagenesis of Aspergillus fumigatus

Abstract: We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme

Abstract: The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion.

Abstract: An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents.

Intertribal somatic hybridization between rapid cycling Brassica oleracea L. and Camelina sativa (L.) Crantz

Abstract: Black spot, caused by Alternaria brassicae and A. brassicicola, is an important disease in all Brassica oleracea vegetables. Sufficient resistance to the pathogen is not found within the species, nor in species that readily cross to B. oleracea. Camelina sativa (false flax) is highly resistant to Alternaria spp. and has, in addition, other desirable characters for the improvement of B. oleracea. Protoplast fusions were performed between rapid cycling B. oleracea (tribe Brassiceae), which has good regenerability, and C. sativa (tribe Sisymbrieae) by polyethylene glycol (PEG) treatment. The B. oleracea fusion partner was inactivated by treatment with iodoacetate. C. sativa has poor regenerability; hence, no pretreatment was needed for this species. The protoplasts were cultured using a feeder layer system. A total of 2903 calli were isolated from the fusions. Fourteen of these initiated shoots, i.e., 0.5% regeneration frequency. Approximately 110 shoots were excised from 6 of these calli and transferred to rooting medium. Rooted plantlets grew vigorously in vitro and flowering was frequently observed. However, establishment of rooted shoots in soil was unsuccessful. Hybrid identity was confirmed by intermediate shoot morphology, RAPD marker analysis, and flow cytometric estimation of nuclear DNA content.

Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts : shoot formation in less than two weeks

Abstract: Regeneration of plants from protoplasts is regarded a difficult and lengthy procedure which requires well developed skills on the side of the experimenter. Therefore, where alternative procedures for genetic engineering of plants are available, protoplast-based techniques are frequently avoided. Here, we demonstrate, that by our newly developed thin-alginate-layer technique it is possible to regenerate shoots from leaf protoplasts ofNicotiana tabacum L. at very high efficiency and very rapidly, with the first shoots appearing within less than two weeks. Root formation is induced on a third medium with first roots being found after only 10 more days of culture.

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts.

Abstract: A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×106 were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 105 protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.

Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus.

Abstract: Substantial somaclonal variation in growth rate, morphology, and alkaloid production of Hyoscyamus muticus L. hairy root clones obtained by transformation with four Agrobacterium strains was shown. The hyoscyamine content of the root clones (n = 100) obtained from the same origin varied from 0.03 to 0.59% of dry weight. The clones produced 25-320 times less scopolamine than hyoscyamine. The best producing root clone was used as a starting material for protoplast isolation. The hyoscyamine content of protoplast-derived hairy root clones (n = 171) ranged from 0.04 to 1.45 % of dry weight. Most clones showed improved alkaloid production in relation to the parent clone, but the mean hyoscyamine content of the clones was the same as that of the parent clone. All the studied hairy root clones showed relatively stable alkaloid production during long-term cultivation. No correlation was found between alkaloid production and growth rate or morphology of the clones.

The relationship between the regeneration system and genetic variability in the cucumber (Cucumis sativus L.)

Abstract: Somaclonal variation in the Borszczagowski line of Cucumis sativus L was determined for five regeneration systems: micropropagation (MP), direct leaf callus regeneration (DLR), leaf callus regeneration (LCR), recurrent leaf callus regeneration (RLCR), and direct protoplast regeneration (DPR) The frequency at which new phenotypes appeared in R1 lines and the stability of the rDNA region analysed using of five probes were investigated MP was not subject to change, while DLR caused only infrequent changes The highest frequency of change arose through DPR (90% of lines) and RLCR (428%), as opposed to 59% with LCR Tetraploids were produced only in the case of LCR (47%) and RLCR (28%)

Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.)

Abstract: Results are reported on the production and characterization of somatic hybrids between Allium ampeloprasum and A. cepa. Both symmetric and asymmetric protoplast fusions were carried out using a polyethylene-based mass fusion protocol. Asymmetric fusions were performed using gamma ray-treated donor protoplasts of A. cepa and iodoacetamide-treated A. ampeloprasum protoplasts. However, the use of gamma irradiation to eliminate or inactivate the donor DNA of A. cepa proved to be detrimental to the development of fusion calli, and thus it was not possible to obtain hybrids from asymmetric fusions. The symmetric fusions yielded a high number of hybrid calli and regenerated plants. The analysis of the nuclear DNA composition using interspecific variation of rDNA revealed that most of the regenerated plants were hybrids. Flow cytometric analysis of nuclear DNA showed that these hybrid plants contained a lower DNA content than the sum of the DNA amounts of the parental species, suggesting that they were aneuploid. A shortage of chromosomes in the hybrids was confirmed by genomic in situ hybridization. Chromosome counts in metaphase cells of six hybrids revealed that these plants lacked 2–7 leek chromosomes. One hybrid showed also the loss of onion chromosomes. The hybrids had an intermediate phenotype in leaf morphology. The application of these somatic hybrids in breeding is discussed.

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Abstract: Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×106 protoplasts/g fresh weight). Protoplasts plated at a density of 105–106/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N6-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP.

Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species

Abstract: The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3–4 months and set seeds, of which 70–80% germinated.

Plant parasitic nematodes: Digesting a page from the microbe book

Abstract: It is somewhat ironic that an important finding concerned with cellulose biosynthesis in higher plants (1) appeared just before the paper in this issue of the Proceedings by Smant et al. (2) describing genes from a plant parasitic nematode that encode proteins that degrade cellulose. Cellulose is the most abundant polymer on the planet and is an essential component of the plant cell wall. Although the cell wall or extracellular matrix, as it is sometimes called, is complex (3), crystalline cellulose is the structural linchpin. Given this situation, it is not surprising that hungry pathogens commonly attack components of the plant cell wall, including cellulose. Genetic evidence has established that cellulases that depolymerize cellulose often are required for high virulence (the quantitative degree of pathogenicity) by microbial pathogens (e.g., refs. 4 and 5). These entrepreneurs damage the plant cell wall to facilitate their movement through plant tissue and to obtain nutrients from the digested cell wall components or the contents of unclothed plant protoplasts. Curiosities abound, including questions such as: Where and when did cell wall degrading genes originate? How were such genes recruited by pathogens? What do plants do about this mode of attack? How can human activity intervene and prevent such plant diseases without secondary downsides? Answers are not abundant, but we are improving our understanding of plant pathogenesis and what to do about it so that humans reap more of the harvest. First, pathogens are turning out to be copycats. For example, microbial pathogens of plants and animals share clusters of highly homologous genes involved in pathogenicity and virulence. These have come to be …

Somatic hybridization in mint: identification and characterization of Mentha piperita (+) M. spicata hybrid plants

Abstract: Twenty eight somatic hybrid plants were identified following protoplast fusions between peppermint (Mentha piperita L. cv Black Mitcham), producing high-quality oil, and spearmint (Mentha spicata L. cv Native Spearmint), likewise producing high-quality oil and also possessing resistance to verticillium wilt. Prior to fusion, peppermint protoplasts were subjected to iodoacetic acid to inhibit cell division. Protoplasts of peppermint and spearmint were fused using polyethylene glycol plus DMSO. Fusion products were cultured according to an efficient protoplast-to-plant-cycle protocol developed for peppermint. Using this protocol, iodoacetic acid-treated peppermint protoplasts were not able to divide, whereas untreated spearmint protoplasts had the ability to produce callus but not shoots. Therefore, selection of somatic hybrid calli was based on the presumed capability of hybrid cells to form calli and shoots. Shoots in vitro were initially identified as hybrids using RAPD profiles. Subsequently, observations on morphology, chromosome counts, and Southern-hybridization patterns confirmed their hybrid status. The results of verticillium tests revealed that 18 somatic hybrids were more susceptible than Native Spearmint, while hybrid II-14 had a level of susceptibility intermediate between that of the fusion parents. Oil-analysis of hybrid plants indicated that they all have a GC-profile typical of spearmint oil.

Protoplast ion fluxes: their measurement and variation with time, position and osmoticum

Abstract: The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating cell wall. In this study net H+ and Ca2+ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a non-invasive microelectrode technique for ion flux measurements. For Ca2+, the flux distribution was almost symmetrical, ranging ±30 nmol · m−2 · s−1 around zero. For H+ it was skewed towards efflux ranging from −100 to +10 nmol · m−2 · s−1. The distribution of H+ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H+ and Ca2+ fluxes increased, which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange.

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts.

Abstract: Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl2, 0.92 mM NaH2PO4 and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS–8P (2.5 ml). MS-8P medium consisted of Murashige and Skoog salts without NH4NO3, 1 mg l–1 thiamine HCl, 100 mg l–1 myo-inositol, 3.1 g l–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×106 protoplasts g–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×105 ml–1 in 0.4 M MS–8P for 2–3 weeks, followed by subculture in 0.15 M MS–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed.

Comparison of UV light, X-ray and restriction enzyme treatment as tools in production of asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana

Abstract: Asymmetric somatic hybrids between Brassica napus (receptor) and Arabidopsis thaliana (donor) have been produced by three different methods supposed to induce asymmetry. The donor protoplasts were either UV- or X-irradiated, or the mixture of protoplasts was treated with the restriction enzyme PvuII immediately before fusion. The genome composition of the hybrids was analysed with Southern blot hybridisations using 15 different mapped A. thaliana RFLP markers as probes. Both UV- and X-irradiation were found to be efficient treatments for induction of asymmetry in somatic hybrids in a dose-dependent manner. The addition of a restriction enzyme to the protoplast mixture did not have any effect on the frequency of asymmetric hybrids or on the degree of asymmetry in the hybrids produced. UV- and X-irradiation resulted in higher fertility in the hybrids, while PvuII treatment did not have any effect on seed set. A significant positive correlation between degree of asymmetry in different plants and seed set after selfing was detected.

Asymmetric protoplast fusions between wild species and breeding lines of potato - effect of recipients and genome stability

Abstract: The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation.

Combination of resistance to potato late blight in foliage and tubers by intraspecific dihaploid protoplast fusion

Abstract: Five dihaploid Solanum tuberosum genotypes, encompassing different levels of polygenically inherited resistance to potato late blight disease (Phytophthora infestans (Mont.) de Bary) in foliage and tubers, were used in four intraspecific protoplast fusion combinations. Vigorous growing putative hybrid calli were selected four weeks after electrofusion. Intact plants were regenerated from 6% of the selected calli. Verification of hybridity was accomplished by use of RAPD analysis which revealed that 53% of the regenerated plants were true somatic hybrids. The score of true somatic hybrids in the different fusion combinations ranged within 21% – 100%. The hybrid plants were analysed for resistance to foliage blight in a field trial and assessed for tuber blight resistance by use of a laboratory test. Resistance to late blight in foliage and tubers varied between the hybrids. Very high levels of resistance to both foliage and tuber blight were obtained in some hybrids. However, loss of resistance in some hybrids as compared to the parental plants were also observed. Possible reasons for the phenotypic disappearance of resistance to either foliage or tuber blight are suggested.

Efficient transformation of rice protoplasts mediated by a synthetic polycationic amino polymer

Abstract: A very simple and efficient transformation system for rice was established using a synthetic polycationic amino polymer (polycation). Improvements in the culture conditions, especially filtration of the suspension cells before and after protoplast culture, greatly contributed to a large yield of high-quality protoplasts and an increased ability of the cells to regenerate. Transformation parameters, such as the ratio of DNA and polycation concentrations, preincubation of the DNA and polycation prior to DNA transfer, and precentrifugation and resuspension of protoplasts before DNA transfer, were analyzed. Fertile transgenic plants containing the bar gene were selected and shown to demonstrate resistance against high concentrations of bialaphos. Southern blot analysis showed four to nine bands representing the bar gene in polycation-mediated transgenic rice plants compared with two to three bands in electroporation-mediated transgenic rice plants. The regeneration efficiency of the polycation-mediated method was compared to that of the electroporation-mediated method; while the polycation-mediated method tended to show a relatively lower regeneration rate, regenerants showed a normal phenotype.

Rapid production of fertile transgenic barley (Hordeum vulgare L.) by direct gene transfer to primary callus-derived protoplasts

Abstract: Protoplasts were isolated from primary calli of barley (Hordeum vulgare L.), and an antibiotic (G418) resistance gene was introduced into these protoplasts using a polyethylene glycol (PEG) DNA uptake method. Sixty-four G418 resistant calli were obtained in nine experiments, and two plants were regenerated from these calli. NPTII ELISA and Southern analysis indicated that the G418 resistance gene was introduced and expressed in two T0 plants. These plants set seed and the introduced gene was transmitted to T1 plants. These results suggest that our transformation system using primary callus-derived protoplasts is a useful method for the generation of transgenic barley.

Plant regeneration from leaf protoplasts of evening primrose (Oenothera hookeri)

Abstract: A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture in B5 medium containing 3 mg l–1 α-naphthaleneacetic acid (NAA) and 1 mg l-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4) plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l–1 NAA and 1 mg l–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency of the procedure for protoplast to cell line formation is high (about 80%).

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Abstract: Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120.

Protoplast fusion between Monostroma nitidum and Porphyra yezoensis and subsequent growth of hybrid plants

Abstract: Protoplasts from Monostroma nitidum (Chlorophyta) and Porphyra yezoensis (Rhodophyta) were fused utilizing the PEG method. The heterofusants were easily identified by the color of the hybrid cells. The frequency of fusion between the different protoplasts was 1.4%. The heterofusants were transferred into ES medium and cultured. The survival rate of heterofusants after 30 days cultivation was 2–7%. Two plants exhibited hybridizable DNA fragments as compared to parental DNA fragments. Additionally, these two plus a third hybrid plant had characteristic fatty acid components of both the Chlorophyta and Rhodophyta. The results indicate that genetic recombination is possible between these two genera. The field for breeding by protoplasts fusion should be extended across the divisions.

Protoplast fusion between Lycopersicon esculentum and L. peruvianum-complex: somatic embryogenesis, plant regeneration and morphology

Abstract: Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid.

An optimising protocol for protoplast regeneration of three peppermint cultivars (Mentha x piperita)

Abstract: An efficient protocol for plant regeneration from protoplasts of peppermint ‘Mitcham Digne 38’, ‘Mitcham Ribecourt 19’ and ‘Todd's#x2019; was developed by stepwise optimization of first cell division, microcalli formation and shoot differentiation. The rate of first cell divisions was strongly dependent on the addition of 2,4-D to callus induction medium. Best results were obtained with 1 μM 2,4-D in combination with NAA (2.5 μM) and BA (4 μM). Although liquid medium was more efficient to support first protoplast divisions, solid medium was clearly more suitable to sustain subsequent cell divisions leading to the formation of microcalli. Shoot organogenesis was induced from protoplast-derived calli by using reduced auxin concentration (0.5 μM NAA) and high concentration of cytokinins. Addition of 2.3 μM thidiazuron increased bud formation, allowing a regeneration frequency of more than 50% from calli of ‘Mitcham Digne 38’ and ‘Todd's’. Genotypic differences were noticed for regeneration capability and the pathway of shoot regeneration.