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1.
Yasuyuki Yamada Kuniaki Kiso Jiro Sekiya Takeshi Yasuda 《Bioscience, biotechnology, and biochemistry》2013,77(6):1055-1059
Protoplasts were isolated from cultured tobacco cells by removing the cell wall enzymatically. We examined the time courses of treatment with some different concentrations of enzymes (cellulase and macerozyme) and with some different kinds and concentrations of sugars (sucrose, sorbitol and mannitol) which produce good conditions for protoplast isolation. The best conditions for protoplast preparation from tobacco cells cultured in vitro were: use of actively growing cells and isolation of protoplasts with 5% cellulase and 0.1% macerozyme in 0.5% sorbitol. 相似文献
2.
Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on
the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion
and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were
approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 107 protoplasts g-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 106 cells mL-1 in culture medium and developed into microthalli of a line of two to six cells. 相似文献
3.
Summary
Beauveria bassiana protoplast formation from blastospores, conidia and mycelia was studied. The method of protoplast formation involves preincubation of the fungal cells with dithiothreitol and subsequent treatment with an enzyme mixture consisting of: cellulase, chitinase, -glucuronidase and lysozyme. Using this procedure protoplasts were formed from blastospores and mycelia but not conidia. Formation of protoplasts from 24 hour old mycelia was 100% efficient using the above conditions. A number of ionic and osmotic protoplast stabilizing agents were tested. Ammonium sulfate was shown to be the stabilizer of choice. Protoplasts were stable when stored at 4° C with a loss of only 17% in 6 days. We suggest that this procedure of protoplast production will allow a gentler method for the extraction and isolation of intact high molecular weight DNA from B. bassiana. 相似文献
4.
5.
Isolation of protoplasts from Fucus serratus and F. vesiculosus(Fucales, Phaeophyceae): factors affecting protoplast yield 总被引:1,自引:0,他引:1
Protoplasts were isolated enzymatically from meristematic tissues of the brown algae, Fucus serratus, using a combination of 2% cellulase R-10 Onozuka, 0.5% macerozyme and 1% crude extract of gland gut of Aplysia
vaccaria. The main factors affecting protoplast yield were identified. Protoplasts were produced in large quantities from apical region of thallus and from plantlets compared to mature explants. Yields were greatly improved by the addition of sodium citrate and bovine serum albumin in the enzymatic solution and could reach 5.8 × 106 protoplasts per gram of fresh wt. The applicability of these optimal parameters to other species Fucus vesiculosus was shown. 相似文献
6.
J. Jásik I. N. Smolenskaya S. E. Zorinyants A. V. Nosov O. I. Baulina J. KriŠtín 《Biologia Plantarum》1992,34(3-4):193-201
Protoplasts were obtained from tetraploid wheat (Triticum timopheevi Zhuk.) suspension culture by incubation in solution of 1 % pectinase 500, 1 % driselase and 1 % cellulase and cultivated in Schenk and Hildebrandt medium. Freshly isolated protoplasts contained dense cytoplasm and constricted organellae exhibited negative contrast of their membranes. Together with normal protoplasts huge multinucleate protoplasts were present in the population. 3 h after plating, the cytoplasm showed normal appearance, the negative contrast of membranes was not evident any longer. Cisternae of endoplasmic reticulum and Golgi apparatus were numerous. There were some vesicles and fibres on the protoplast surface. 8 d after plating, many dividing cells were found out and cell clumps arosen in this way were present in the culture. Some of the protoplasts particularly those originally multinucleate ones were upset. 相似文献
7.
Genotypes of Solanum tuberosum, assessed for their differential response to a protoplast regeneration protocol, were identified. This enabled comparisons to be made in relation to their abilities to regenerate into dividing cells and colonies. Protoplasts from genotypes with contrasting regeneration responses were plated as single or mixed genotype cultures at various plating densities in replicated, randomised experiments, and the effect on the regeneration of particular cell types observed. Protoplasts from single genotype cultures showed intergenotypic variation in the extent of cell regeneration and there were significant effects of genotype mixing and density × genotype mixing interactions. Overall, the effect of mixing was beneficial to a regenerating culture but significant density genotype mixing interactions showed only a positive benefit at the lowest plating density. The protoplast mixing phenomenon was not correlated with the behaviour of the same genotypes at the plantlet level in experiments with in vitro meristem cultures. 相似文献
8.
Dr. M. C. Liechty S. G. Bradley K. L. Kasweck 《Journal of industrial microbiology & biotechnology》1987,2(2):71-77
Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses. 相似文献
9.
Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 104/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 %
Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others
(e.g., MgSO4) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract
medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance
usnic acid. 相似文献
10.
Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG
Polyethylene glycol
- DAPI
4,6-diaminido-2-phenylindole
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
11.
Summary Protoplasts were prepared from cells ofChlorella saccharophila by treatment with a mixture of pectinase and cellulase. The yield of protoplasts is dependent upon the culture conditions prior to cell wall digestion. In thin section chemically-fixed protoplasts were without wall remnants at the surface of the plasma membrane. Of particular interest is the relationship between the Golgi apparatus and a nuclear envelope-endoplasmic reticulum continuum. Protoplasts have a photosynthetic capacity lying between 70 and 80% of that of normal cells, but show the same response towards CO2 concentration and DCMU inhibition. Protoplasts also respond to changes in the osmolarity of the surrounding medium in accordance with the Boylevan't Hoff equation as if they are an osmometer. The nonosmotic volume (NOV) was calculated.Abbreviations GA
Golgi apparatus
- ER
endoplasmic reticulum
- NE
nuclear envelope
- PM
plasma membrane
- N
nucleus
- S
starch
- M
mitochondria
- V
vacuole 相似文献
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13.
Summary Protoplasts of the mossAnoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium. 相似文献
14.
Protoplasts were isolated from thalli of Dictyopteris prolifera using a mixture of crude enzymes from vicera of live oysters (Crassostrea gigas) and the following commercial enzymes: an abalone enzyme, cellulase, polygalacturonase and hemicellulase. The enzyme mixtures produced up to 3.3 × 107 cells per l g of tissue fresh weight. The conversion to protoplasts of the cells was about 100% using the oyster enzyme or the abalone enzyme alone. The optimum pH for protoplast isolation was 6.0 and 20 hours were required for conversion to protoplasts. 相似文献
15.
The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to
its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following
the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation
period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol.
The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher
yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white
M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell
division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained. 相似文献
16.
E. S. Dias E. F. Araújo W. V. Guimarães R. M. C. Muchovej 《World journal of microbiology & biotechnology》1996,12(6):625-628
Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×107/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings. 相似文献
17.
Summary Protoplasts of the desmidsMicrasterias angulosa, M. denticulata, M. thomasiana andCosmarium turpinii were obtained by incubating cells in Waris' liquid medium + 0.3 M mannitol + 2% Cellulysin for 1–3 hours. One osmotically fragile protoplast was formed at the isthmus from the joint contents of both semicells. The resultant protoplasts were bright green and remained so for more than 5 days in the osmotically protective medium. The protoplast yield was better than 80%. The empty cell walls were not digested by the Cellulysin or by autolytic enzymes. 相似文献
18.
Tomoaki Wakabayashi Toru Kuboi Takuya Tuboi Masayuki Kaji Masakazu Hara 《Marine biotechnology (New York, N.Y.)》1999,1(4):407-410
Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 μg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran
sulfate sodium salt, and 1 μl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 107 cells per gram of fresh weight alga.
Received January 26, 1998; accepted September 17, 1998. 相似文献
19.
Protoplasts were isolated from root cortical explants of thirteen cultivars of Pisum sativum. The roots were tested for starch in the cortical tissues. There was a considerable range in the average protoplast yields and the coefficient of variation of those yields from the thirteen cultivars. Cultivars with a higher incidence of starchy roots had lower average explam protoplast yields and greater variation in their protoplast yields. Significantly higher protoplast yields were observed from explants with no starch or few starchy cells. Increasing the germination time of the peas increased the starch content in the seedling roots and decreased the protoplast yields. 相似文献
20.
G. A. Birrer W. R. Chesbro R. M. Zsigray 《Journal of industrial microbiology & biotechnology》1989,4(4):325-331
Summary Protocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques. 相似文献