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Showing papers on "Protoplast published in 1997"


Journal ArticleDOI
01 Jan 1997-Planta
TL;DR: In this article, the conditions for the induction of mature zygotic embryos were established in both culture systems and the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones).
Abstract: Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryo-genesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed.

94 citations


Journal ArticleDOI
TL;DR: The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.
Abstract: We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20–35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

92 citations


Journal ArticleDOI
TL;DR: Somatic Hybrids between Sinapis alba and rapid-cycling Brassica oleracea were generated for transferring of resistance to Alternaria brassicae to B. Oleracea, showing intermediate morphology with partially divided leaves and some trichomes on stems and leaves.
Abstract: Somatic Hybrids between Sinapis alba and rapid-cycling Brassica oleracea were generated for transferring of resistance to Alternaria brassicae to B. oleracea. A. brassicae causes the significant disease black spot in cruciferous crops. A total of 27 plants were regenerated from protoplast fusion using 0, 5, 10, 20 and 30 krad γ-irradiation of the resistance donor and iodoacetate treatment of B. oleracea. All plants showed intermediate morphology with partially divided leaves and some trichomes on stems and leaves. Flow cytometry and banding patterns of the enzymes leucine amino peptidase (LAP) and phosphoglucose isomerase (PGI) confirmed the hybrid status of the regenerated plants. Some of the plants obtained from cuttings from the somatic hybrids showed a resistance to A. brassicae that was similar to that found in S. alba. The flowers of the somatic hybrids had reduced anthers with little pollen production.

91 citations


Journal ArticleDOI
TL;DR: The authors' procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures.
Abstract: Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures.

77 citations


Journal ArticleDOI
TL;DR: A comparative analysis has been carried out with purified membranes of L-form cells, of parent vegetative hyphal cells, and of protoplasts derived from the latter, finding the phospholipid composition of the protoplast membrane differs quantitatively from that of the N form and the L form.
Abstract: The cells of an L-form strain of Streptomyces hygroscopicus have been grown for 20 years without a cell wall. Their cytoplasmic membranes have high stability and an unusual structural polymorphism. To clarify the importance of the lipid components for these membrane properties, a comparative analysis has been carried out with purified membranes of L-form cells, of parent vegetative hyphal cells (N-form cells), and of protoplasts derived from the latter. The phospholipid classes and fatty acids were determined by thin-layer chromatography (TLC), two-dimensional TLC, high-performance liquid chromatography, gas chromatography, and mass spectrometry. The qualitative compositions of cardiolipin (CL), lyso-cardiolipin (LCL), phosphatidylethanolamine (PE1 and PE2), lyso-phosphatidylethanolamine (LPE), phosphatidylinositolmannoside (PIM), phosphatidic acid (PA), dilyso-cardiolipin-phosphatidylinositol (DLCL-PI), and the 13 main fatty acids were the same in the three membrane types. However, significant quantitative differences were observed in the L-form membrane. They consist of a three- to fourfold-higher content of total, extractable lipids, 20% more phospholipids, an increased content of CL and PIM, and a reduced amount of the component DLCL-PI. Furthermore, the L-form membrane is characterized by a higher content of branched anteiso 15:0 and anteiso 17:0 fatty acids compared to that of the membranes of the walled vegetative cells. These fatty acids have lower melting points than their straight and iso-branched counterparts and make the membrane more fluid. The phospholipid composition of the protoplast membrane differs quantitatively from that of the N form and the L form. Whereas the phospholipid classes are mostly similar to that of the N form, the fatty acid pattern tends to be closer to that of the L-form membrane. The membranes of both the L-form cells and the protoplasts need to be more fluid because of their spherical cell shape and higher degree of curvature compared with N-form membranes.

72 citations


Journal ArticleDOI
TL;DR: New types of cytoplasmic male sterility (CMS) in Brassica oleracea would be useful for F1 hybrid seed production and Mitochondrial segregation in the cybrids was slightly biased towards `Anand' mitochondria, and the presence of ` Anand' mtDNA fragments was strongly associated withmale sterility.
Abstract: New types of cytoplasmic male sterility (CMS) in Brassica oleracea would be useful for F1 hybrid seed production. The `Anand' cytoplasm derives from the wild species B. tournefortii. Rapid cycling stocks of B. rapa and B. oleracea were used in cybridization experiments as donor and recipient of `Anand' (=`tour') CMS, respectively. Prior to fusion with PEG, donor protoplasts were inactivated with 30 krad γ-rays and recipient ones with 3 mM iodoacetate, respectively. No calli were obtained from the pre-treated protoplasts. The frequency of shoot regeneration was 21–43% in untreated B. oleracea controls, but only 0–0.5% in `Anand' B. rapa. Putative cybrids were regenerated from about 3% of the calli from fused protoplasts. Regenerated plants were analyzed for nuclear DNA content, plant and flower morphology, pollen production, female fertility, cold tolerance, and organelle composition. Eighty-one percent of the regenerated controls and 63% of fusion-derived plants were diploid. The rest showed DNA contents corresponding to 2x–4x, 4x, or higher ploidy levels, presumably due to somatic doubling in vitro and/or fusions in which the donor nucleus was not completely eliminated. Sixty-four percent of the cybrids had stamens and petals varying in size and shape and were male-sterile, with indehiscent anthers. Their phenotype was otherwise similar to that of B. oleracea. The remaining plants had normal flowers and were male-fertile. Data from crosses with fertile pollinators indicated good female fertility in some of the sterile lines, both after hand and insect pollinations in cages. Mitochondrial (mt) segregation in the cybrids was slightly biased towards `Anand' mitochondria, and the presence of `Anand' mtDNA fragments was strongly associated with male sterility. Evidence of mtDNA rearrangements was obtained in some cybrids. Segregation of chloroplasts was slightly biased towards B. oleracea. The presence of `Anand' chloroplasts with a B. oleracea nucleus did not result in cold temperature chlorosis, as seen in `Ogura' CMS plants.

67 citations


Journal ArticleDOI
TL;DR: A prerequisite for the development of a successful transformation system is the availability of efficient regeneration systems, and up to 1995 the only available regeneration system in cassava was an organized type of somatic embryogenesis.
Abstract: A prerequisite for the development of a successful transformation system is the availability of efficient regeneration systems Up to 1995 the only available regeneration system in cassava was an organized type of somatic embryogenesis Transformation of these organized somatic embryogenic cultures with particle bombardment or Agrobacterium tumefaciens resulted in chimeric transformed embryos However, the transformed sector was lost after repeated cycles of secondary somatic embryogenesis After 1995 a less organized system of somatic embryogenesis was developed, so called friable embryogenic callus (FEC) and a system of adventitious shoot regeneration The FEC regeneration system was combined successfully with particle bombardment Selection of transgenic plants was based on either luciferase activity, or resistance to the aminoglycoside paromomycin or the herbicide phosphinothricin Furthermore, protoplasts of FEC are able to regenerate into plants and can be transformed by electroporation The adventitious shoot regeneration system was combined successfully with Agrobacterium tumefaciens For this mature somatic embryos were cocultivated with Agrobacterium and cultured for adventitious shoot development After selection based on the aminoglycoside geneticin or on hygromycin transgenic plants were formed

55 citations


Journal ArticleDOI
TL;DR: Observations of the cultures developed on media containing maltose indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration, and Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety.
Abstract: The effects were studied of various carbohydrates and osmotic stress, created by high agarose or carbohydrate concentrations, on the regeneration of fertile plants from protoplast-derived colonies of several indies (IR43, Jaya, Pusa Basmati 1) and japonica (Taipei 309) rice varieties. Observations of the cultures developed on media containing one of these carbohydrates (cellobiose, fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose), each at 88 mM, indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration. Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety, compared to shoot regeneration from 4-32% of the tissues in sucrose-supplemented medium. Media containing 288 mM maltose or an equimolar combination of 88 mM maltose and 200 mM mannitol, caused water loss from calli and promoted the growth of embryogenic calli. These calli formed shoots with greater frequencies when subsequently transferred to shoot regeneration medium with 88 mM maltose. A medium containing 88 mM maltose and semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarose had a similar beneficial effect on the growth of embryogenic calli and simultaneously supported high-frequency (48-55%) shoot formation. The optimum shoot regeneration frequencies (60-78%) were obtained when protoplast-derived colonies were serially cultured on to shoot regeneration medium containing 1.0% (w/v) agarose for 4 weeks, followed by a 2-week culture period on the same medium with 0.5% (w/v) agarose. Plants regenerated on medium containing maltose and/or 1.0% (w/v) agarose were phenotypically normal and fertile.

52 citations


Journal ArticleDOI
Kirimura Kotaro1, Toshiyuki Sato1, N. Nakanishi1, M. Terada, Shoji Usami1 
TL;DR: Interspecific protoplast fusion between Aspergillus terreus and A.usamii was done to breed new koji molds producing itaconic acid from starch and indicated that the fusants obtained were haploids like the parental strains.
Abstract: Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10−5−10−4. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml).

50 citations


Journal ArticleDOI
TL;DR: Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular.
Abstract: The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular.

49 citations


Journal ArticleDOI
TL;DR: With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.
Abstract: A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

Journal ArticleDOI
TL;DR: The results suggest that alfalfa mosaic virus may move from cell to cell via tubules, and the AlMV MP is solely required for the induction of the tubular structures.
Abstract: The alfalfa mosaic virus (AlMV) movement protein (MP) was fused with the green fluorescence protein (GFP). The MP-GFP fusion was transiently expressed in protoplasts prepared from Nicotiana tabacum, resulting in the production of long, extending, tubular structures protruding from the protoplast surface. Deletions of MP amino acids 1 to 77, 84 to 142, or 226 to 300 all affected tubule formation. Hence, the AlMV MP is solely required for the induction of the tubular structures, and the results suggest that AlMV may move from cell to cell via tubules.

Journal ArticleDOI
TL;DR: Laccase could be the only effective polymerizing enzyme during the first day of protoplast culture and could contribute in the first steps of healing in wounded leaves, substituting for POX activity in cell wall reconstitution when hydrogen peroxide is not yet available.

Journal ArticleDOI
TL;DR: Brachycytes are thick-walled, drought-tolerant brood cells or parts of brood bodies that develop in protonemata of Funaria (and other mosses) that can be induced by ABA in the chloronema of normally growing protonema, either after spore germination, or after protoplast regeneration or in the auxin-deficient mutant 87.25.

Journal ArticleDOI
TL;DR: It was demonstrated that the incubation of both protoplasts and chloroplasts with TRIA resulted in a rise of the excimer/monomer (IE/IM ) ratio of pyrene (Py) fluorescence, thus indicating remarkable fluidization and/or disordering of the lipid matrix of their membranes.
Abstract: The effects of a long chain aliphatic alcohol 1-triacontanol (TRIA) on the photosynthesis and membrane properties of mesophyll protoplasts and chloroplasts isolated from pea leaves were studied. In vitro treatments of isolated protoplasts caused a large enhancement (166% ) of the CO2-fixation rate after 60 min of TRIA (10-6 M) application as compared to the control. An enhanced photosynthetic response was observed in in vitro treated leaf pieces. Application of octacosanol (OCTA) under the same experimental conditions did not result in any stimulating effects. In vivo treatments of pea seedlings also resulted in a significant increase of the net CO2 uptake to 109% and 119% in 10-8 M and 10-6 M TRIA-treated plants respectively. It was demonstrated that the incubation of both protoplasts and chloroplasts with TRIA resulted in a rise of the excimer/monomer (IE/IM ) ratio of pyrene (Py) fluorescence, thus indicating remarkable fluidization and/or disordering of the lipid matrix of their membranes. This effect depended on the incubation time and became evident at very low concentrations of TRIA (0.3 μM). The increase of membrane fluidity was accompanied by TRIA-induced alterations in the dielectric environment in the membrane regions where Py molecules are situated. The results are discussed in terms of specific concentration dependent TRIA-induced alterations of the dynamic properties of protoplast and chloroplast membranes and their possible involvement in the initiation of the integral physiological response to exogenous application of TRIA.

Journal ArticleDOI
TL;DR: Results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred.
Abstract: In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the cells. The morphology 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 had about 24 chromosomes, fewer than the sum of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred.

Journal ArticleDOI
TL;DR: The results demonstrate the high efficiency of the direct gene transformation in Lolium species, and the integration of foreign DNA into the genomes of the transgenic plants was confirmed by Southern hybridization.

Journal ArticleDOI
TL;DR: It is found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protop last recalcitrance.
Abstract: Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.

Journal ArticleDOI
01 Oct 1997-Gene
TL;DR: The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm and was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation.

Journal ArticleDOI
TL;DR: A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa.
Abstract: Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa.

Journal ArticleDOI
TL;DR: Brassica napus somatic hybrids with low linolenic acid (18:3) content in their seed oil have been produced using fusion partners screened for low 18:3, and the low level proved stable in the R1 generation.
Abstract: Brassica napus somatic hybrids with low linolenic acid (18:3) content in their seed oil have been produced using fusion partners screened for low 18:3. One somatic hybrid contained only 3.5% 18:3, a level significantly below the mid-parental mean. The low level of 18:3 proved stable in the R1 generation. Oil content of the lowest 18:3 selection increased from the mid-parental mean (29.3%) in the R0 generation to 36% in a R1 field bulk. The R1 field population also showed some resistance to shattering.

Journal ArticleDOI
TL;DR: Driselase was found to produce higher protoplast yields than the other lytic enzymes tested (Glucanex, Novozyme, β-glucuronidase, Sigma lytic enzyme, or ICN lyticase), although yields differed for the various strains.

Patent
20 May 1997
TL;DR: In this article, the authors proposed a method for producing friable embryogenic callus from explants of cassave of closely related species and isolating protoplasts from such callus.
Abstract: Method for producing protoplasts of cassave or closely related species, which protoplasts are capable of regeneration into plants. The method comprises producing friable embryogenic callus from explants of cassave of closely related species and isolating protoplasts from said friable embryogenic callus. Protoplasts which are obtainable by such a method. Method for transforming such a protoplast of cassave or closely related species, and transformed protoplast obtainable thereby. Method for regenerating plants from these protoplasts, and cassave plant or closely related species obtainable thereby.

Journal ArticleDOI
TL;DR: An improved regeneration medium designated R1M was established by supplementing Polypepton and yeast extract which were shown to increase the S. griseus 2247 growth rate to R1, and the transformation efficiency for the plasmid pRES18 was increased to 106 transformants per μg DNA.

Journal ArticleDOI
TL;DR: Protoplast fusion induced by polyethylene glycol and Ca 2+ was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 as mentioned in this paper.
Abstract: Protoplast fusion induced by polyethylene glycol and Ca 2+ , was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade- and Aspergillus flavipes ATCC-16795 (F7) lys-. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1.0 x 10 -2 . The reversion frequency of the mutation in spores and protoplasts was low and ranged from 20 to 40 x 10 -7 . Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.

Journal ArticleDOI
TL;DR: Transgenic plants of Linum usitatissimum L. suffruticosum ssp.

Journal ArticleDOI
TL;DR: Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum and somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants.
Abstract: Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum (2n=16). (L.). The best media for induction of embryogenic cultures were based on Kao (1977) or Kao and Michayluk (1975). Somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants, somatic embryo protoplasts and cell suspension protoplasts, leaflets and various explants of immature zygotic embryos. The process of somatic embryogenesis was maintained for over two years on Murashige and Skoog's (1962) medium supplemented with 0.5 mg l-1 benzyladenine and 0.05 mg l-1 naphthaleneacetic acid. These long term cultures were capable of regenerating plants that were fertile and produced seeds. These results were compared with those from protoplast, tissue and organ culture of other species of the Trifolium genus.

Journal ArticleDOI
TL;DR: The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.
Abstract: Sunflower hypocotyl protoplasts (Helianthus annuus L. cv. Emil) divide symmetrically to form loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo-like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of β-linked glucan and the dynamics of microtubules during early phases of culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a β-glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30-40% of the protoplasts and was not related to either division type. No marked differences were observed in cortical arrays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well-defined basket around the nucleus; these microtubules were never observed in liquid-cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.


Journal ArticleDOI
TL;DR: Intact mycelium was not significantly induced by soluble carboxymethylated (alkaline) chitin in the production of chitinolytic enzymes, and enzymatically produced protoplasts, of M. anisopliae were highly inducible for these enzymes.