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Han, Jong-Eun, Han-Sol Lee, Hyoshin Lee, Hyunwoo Cho, and So-Young Park. "Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture." International Journal of Molecular Sciences 23, no. 19 (September 30, 2022): 11556. http://dx.doi.org/10.3390/ijms231911556.
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Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.
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Perera, Srini C., and Peggy Ozias-Akins. "Regeneration from Sweetpotato Protoplasts and Assessment of Growth Conditions for Flow-sorting of Fusion Mixtures." Journal of the American Society for Horticultural Science 116, no. 5 (September 1991): 917–22. http://dx.doi.org/10.21273/jashs.116.5.917.
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Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).
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Struck, C., R. Rohringer, and R. Heitefuss. "Isolation and lectin-binding properties of barley epidermal and mesophyll protoplasts." Canadian Journal of Botany 72, no. 11 (November 1, 1994): 1688–91. http://dx.doi.org/10.1139/b94-207.
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Protoplasts from primary leaves of barley (Hordeum vulgare L.) were obtained by enzymatic digestion and fractionated by discontinuous density gradient centrifugation to yield highly enriched fractions of mesophyll and epidermal protoplasts. A characterization of both protoplast types resulted in a clear differentiation of the outer protoplast surfaces. The protoplasts were examined for affinity to various lectins by agglutination tests and by labeling with lectin – fluorescein isothiocyanate conjugates. Both types of protoplasts agglutinated with soybean lectin. Fluorescein isothiocyanate-labeled soybean lectin was uniformly distributed on the protoplast surface. Mesophyll protoplasts, but not protoplasts from the epidermis, were agglutinated by Concanavalin A. Both types of protoplasts exhibited fluorescence labeling with Concanavalin A – fluorescein isothiocyanate conjugate. This label often showed a patchy distribution on the protoplast surface. Tetragonolobus lectin and β-D-glucosyl Yariv artificial antigen agglutinated mesophyll but not epidermal protoplasts. One of three tested monoclonal antibodies with specificity for arabinogalactans had affinity to the surface of mesophyll and epidermal protoplasts. Key words: agglutination, arabinogalactan protein, cell surface, epidermal protoplasts, fluorescence labeling, lectin.
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Sinha, Anupam, Andrew C. Wetten, and P. D. S. Caligari. "Effect of biotic factors on the isolation of Lupinus albus protoplasts." Australian Journal of Botany 51, no. 1 (2003): 103. http://dx.doi.org/10.1071/bt01104.
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Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.
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Qiu, Quan-Sheng, Ze-Zhou Wang, Nang Zhang, Qi-Gui Cai, and Rong-Xi Jiang. "Aquaporins in the plasma membrane of leaf callus protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward." Functional Plant Biology 27, no. 1 (2000): 71. http://dx.doi.org/10.1071/pp99033.
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The water transport activity of Actinidia deliciosa protoplasts was determined using a cell imaging system. Results showed that the protoplast volume increased swiftly when placed in a hypoton-ic medium, and also increased with an increase in medium osmotic gradients. The osmotic water permeability coefficient (Pf) values were 0.118 × 10–3, 0.121 × 10–3, and 0.133 × 10–3 cm s–1 when the osmotic gradients were 75, 100, and 125 mosmol, respectively. The water transport activity of protoplas-ts could be inhibited by HgCl2 and stimulated by amphotericin B. Moreover, ZnCl2 and ZnSO4 had a significant inhibitory effect on the water transport activity of the protoplasts. Our results indicate that the Actinidia deliciosa protoplasts had properties typical of aquaporins, suggesting that aquaporins were present at the plasma membrane.
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Ishikawa, Francine Hiromi, Quélen de Lima Barcelos, Elaine Aparecida de Souza, and Eustáquio Souza Dias. "Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum." Ciência e Agrotecnologia 34, no. 1 (February 2010): 74–79. http://dx.doi.org/10.1590/s1413-70542010000100009.
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The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.
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Leinhos, Volker, and Rodney Arthur Savidge. "Isolation of protoplasts from developing xylem of Pinusbanksiana and Pinusstrobus." Canadian Journal of Forest Research 23, no. 3 (March 1, 1993): 343–48. http://dx.doi.org/10.1139/x93-050.
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Protoplasts were isolated from developing xylem of Pinusbanksiana Lamb, and Pinusstrobus L. by incubating freshly harvested tissue in a cellulose–pectinase mixture having mannitol as osmoticum. Protoplasts were then purified using a discontinuous sucrose–mannitol gradient. More than 70% of the isolated protoplasts were of small diameter (12–27 μm) and had dense cytoplasm and many small vacuoles, suggesting that they originated from ray cells. Larger protoplasts constituted about 25% of the protoplast population; these contained single large vacuoles and only parietal cytoplasm, suggesting that they originated from fusiform cells. Using combined gas chromatography–mass spectrometry, coniferin was confirmed to be present in protoplast preparations. By high-performance liquid chromatography (258 nm UV detection), coniferin was readily detected in protoplasts and in extracts of developing xylem from both species. On a fresh-weight basis, coniferin occurred at 1.0–1.6 mM in protoplasts. In late June, coniferin in developing xylem could be accounted for totally by protoplast coniferin content. In late July, protoplasts contained 93 and 61% of the coniferin content in developing xylem of P. strobus and P. banksiana, respectively.
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Tusa, N., J. W. Grosser, and F. G. Gmitter. "Plant Regeneration of `Valencia' Sweet Orange, `Femminello' Lemon, and the Interspecific Somatic Hybrid following Protoplasm Fusion." Journal of the American Society for Horticultural Science 115, no. 6 (November 1990): 1043–46. http://dx.doi.org/10.21273/jashs.115.6.1043.
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Protoplasm culture following the chemical fusion of `Valencia' sweet orange [Citrus sinensis (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon [Citrus limon (L.) Burro. f.] leaf protoplasts resulted in the regeneration of an interspecific allotetraploid somatic hybrid plant, two autotetraploid lemon plants, and diploid plants from both parents. The regeneration of plants from lemon leaf protoplasts is an example of protoplast-to-plant regeneration from non-nucellus-derived tissue for Citrus. Regenerated plants were classified according to leaf morphology, chromosome number, and analyses of phosphohexose isomerase (PHI), peroxidase (PER), and 6-phosphoglucose dehydrogenase (PGD) zymograms. The somatic hybrid plant was vigorous, with leaves morphologically intermediate to the parents. The tetraploid lemon plants were similar to diploids, although less vigorous and with thicker leaves. The tetraploid lemon and somatic hybrid plants, if fertile, could be used in interploid sexual crosses to breed triploid seedless lemon cultivars with tolerance of mal secco disease from sweet orange. Further investigation of plant regeneration from leaf protoplasts could increase the number of totipotent Citrus clones amenable to somatic hybridization and genetic transformation experiments.
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Saxena, Praveen K., and John King. "Optimal conditions for the isolation and culture of protoplasts from a cell suspension culture of an isoleucine–valine-requiring auxotroph of Datura innoxia." Canadian Journal of Botany 65, no. 8 (August 1, 1987): 1736–40. http://dx.doi.org/10.1139/b87-237.
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Conditions have been standardized for obtaining high yields of viable protoplasts from cell suspension cultures of an isoleucine–valine-requiring auxotroph (IV-1) of Datura innoxia P. Mill. Isolation of protoplasts critically required the use of a salt solution, containing sodium chloride and potassium chloride (0.125 M each), as osmotic stabilizers. The yield, viability, and divisional activity of the protoplasts isolated with mannitol or sucrose were poor. Protoplast-releasing enzymes used to isolate IV-1 protoplasts could be used twice without any loss in the yield or viability of the protoplasts. Cultured protoplasts developed cell walls and underwent sustained divisions in a modified Murashige and Skoog medium enriched with organic acids. Pretreatment of isolated protoplasts with glycine (0.1 M) and calcium chloride (0.05 M) prior to culture increased the frequency of cell colony formation. Protoplast-derived cells developed calli on transfer to agar-solidified medium.
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Liu, Donglong, and Nancy A. Reichert. "PROTOPLAST ISOLATION AND CULTURE OF KENAF (HIBISCUS CANNABINUS L.)." HortScience 29, no. 7 (July 1994): 729e—729. http://dx.doi.org/10.21273/hortsci.29.7.729e.
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Protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars [Everglades 41 (E41), E71, Guatemala 4 (G4), G45, G51, and Tainung 1]. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin (1% w/v; Calbiochem) plus macerase (0.5% w/v; Calbiochem), with a 24 hour digestion at 30°C in the dark. Yields reached 7.2 (10)6 protoplasts/g leaf tissue. Protoplast viabilities ranged from 65% to 96%. Minor cultivar differences were observed related to protoplast yield, but all viability estimates were in an acceptable range. Greatest cell division frequencies and plating efficiencies were obtained when protoplasts were initially cultured in liquid medium at a density of 1.0 (10)5 protoplasts/ml. Electrofusion protocols were developed for kenaf protoplasts testing the range from 1200 to 3000 V/cm. A fusion voltage of 2000 V/cm yielded the highest fusion frequency and retained viability above 80%.
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Riyadi, Imron. "Isolasi Protoplas Tanaman Kacang Panjang secara Enzimatis." Buletin Plasma Nutfah 12, no. 2 (October 6, 2016): 62. http://dx.doi.org/10.21082/blpn.v12n2.2006.p62-68.
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<p>The technique, kind and concentration of enzyme that were appro-priate and optimum affected the isolation process and rendement result of plant protoplasts. A research was conducted to enhance the protoplast rendements of long bean (Vigna sinensis, L.) that was isolated by enzyme Cellulase RS and Macerozyme R-10 as single and combination in a solution. Concentrations of enzyme were used as much as 2.0-3.0% w/v for Cellulase RS and 0.4-0.6% w/v for Macerozyme R-10. Those solutions contain mannitol 25 mM as osmotycum. Isolation process was done on shaker with 50 rpm (rotation per minute) speed in dark room for 3 hours. Results show that C3 treatment (concentration of Cellulase RS enzyme as much as 3.0% w/v) yielded protoplasts density 17.40 x 105 protoplasts/ g fresh weight of mesophyl and M2 treatment (concentration of Macerozyme R-10 enzyme as much as 0.5% w/v) resulted 17.46 x 105 protoplasts/g. As a whole, the best treat-ment was achieved by C2M2 (combination between Cellulase RS as much as 2.5% and Macerozyme R-10 enzyme as much as 0.5% w/v) which resulted protoplasts density 32.67 x 105 protoplasts/g fresh weight of mesophyl</p><p> </p><p><strong>Abstrak</strong></p><p>Teknik, jenis, dan konsentrasi enzim yang tepat dan optimum berpengaruh dalam proses isolasi dan hasil rendemen protoplas tanaman. Penelitian ini bertujuan untuk meningkatkan rendemen protoplas kacang panjang (Vigna sinensis L.) yang diisolasi dengan enzim Cellulase RS dan Macerozyme R-10 secara individu dan penggabungan dua enzim dalam satu larutan. Konsentrasi enzim yang digunakan adalah 2,0-3,0% b/v untuk Cellulase RS dan 0,4-0,6% b/v untuk Macerozyme R-10. Zat osmotikum yang digunakan adalah mannitol 25 mM. Proses isolasi dilakukan di atas gyotoric shaker dengan kecepatan 50 ppm (putaran per menit) dalam kondisi gelap selama 3 jam. Hasil penelitian menunjukkan bahwa perlakuan C3 (konsentrasi enzim Cellulase RS 3,0% b/v) menghasilkan densitas 17,40 x 105 protoplas/g dan perlakuan M2 (konsentrasi enzim Macerozyme R-10 0,5% b/v) menghasilkan densitas 17,46 x 105 protoplas/g berat segar mesofil daun. Secara keseluruhan, perlakuan terbaik dicapai oleh C2M2 (konsentrasi enzim Cellulase RS 2,5% dan enzim Macerozyme R-10 0,5% b/v) yang menghasilkan densitas 32,67 x 105 protoplas/g berat segar mesofil daun.</p>
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Attree, S. M., D. I. Dunstan, and L. C. Fowke. "Initiation of embryogenic callus and suspension cultures, and improved embryo regeneration from protoplasts, of white spruce (Picea glauca)." Canadian Journal of Botany 67, no. 6 (June 1, 1989): 1790–95. http://dx.doi.org/10.1139/b89-227.
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Rapid and high frequency somatic embryo regeneration from protoplasts isolated from 10 embryogenic cell lines of white spruce (Picea glauca) is reported. Embryogenic callus was initiated from immature zygotic embryos as source material for protoplast isolation. Individual cell lines exhibited different capabilities for sustained growth. Protoplast plating efficiencies depended on the concentrations of macroelements included in the medium. Using a medium with reduced salts, individual protoplasts developed directly into embryos with no disorganized growth period. Protoplasts from newly established suspension cultures regenerated to recognizable somatic embryos within 8 days of culture. This embryo development was faster than that from protoplasts isolated from longer term suspension cultures. However, the latter suspensions yielded more protoplasts, displayed higher plating efficiencies, and differed in their response to media.
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Moon, Ki-Beom, Ji-Sun Park, Su-Jin Park, Hyo-Jun Lee, Hye-Sun Cho, Sung-Ran Min, Youn-Il Park, Jae-Heung Jeon, and Hyun-Soon Kim. "A More Accessible, Time-Saving, and Efficient Method for In Vitro Plant Regeneration from Potato Protoplasts." Plants 10, no. 4 (April 16, 2021): 781. http://dx.doi.org/10.3390/plants10040781.
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Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of protoplast extraction, which was 6.36 ± 0.51 × 106 protoplasts/g fresh weight (FW), was approximately 3.7 times higher than that previously reported for potato protoplasts. To obtain data, wounded leaves were used by partially cutting both sides of the midrib, and isolated protoplasts were purified by the sucrose cushion method, with a sucrose concentration of 20%. We confirmed a significant effect on the extraction efficiency by measuring enzymolysis during a 6 h period, with three times more washing buffer than the amount normally used. Protoplasts fixed in alginate lenses with appropriate space were successfully recovered and developed into microcalli 2 weeks after culture. In addition, to induce high efficiency regeneration from protoplasts, calli in which greening occurred for 6 weeks were induced to develop shoots in regeneration medium solidified by Gelrite, and they presented a high regeneration efficiency of 86.24 ± 11.76%.
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Stajič, Ester. "Improvements in Protoplast Isolation Protocol and Regeneration of Different Cabbage (Brassica oleracea var. capitata L.) Cultivars." Plants 12, no. 17 (August 27, 2023): 3074. http://dx.doi.org/10.3390/plants12173074.
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Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation protocol in cabbage (Brassica oleracea var. capitata L.), different enzyme solutions were tested for the isolation of leaf mesophyll protoplasts. In our experiments, the combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 showed the best result. The optimized protocol proved suitable for the isolation of protoplasts from five different cabbage cultivars with yields ranging from 2.38 to 4.63 × 106 protoplasts/g fresh weight (fw) and a viability of 93% or more. After three weeks in culture, protoplasts from all of the tested cultivars formed micro-calli, but further callus growth and shoot regeneration depended strongly on the genotype and regeneration protocol used. For shoot formation, 1 mg/L BAP in combination with auxin 0.2 mg/L NAA showed the best results with a regeneration of 23.5%. The results obtained will contribute to the development of different applications of cabbage protoplasts and facilitate the breeding process of this important horticultural crop.
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Tran, Huong Thanh, and Cuong Quoc Vo. "Protoplast isolation and culture from different explants of Musa spp. Cau man." Science and Technology Development Journal - Natural Sciences 1, no. 6 (December 7, 2018): 106–16. http://dx.doi.org/10.32508/stdjns.v1i6.621.
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In this paper, the roles of type and concentration of enzymes on protoplast isolation from in vitro leaves, multi-scalps (highly proliferating meristem culture), and young male flower of banana cv. cau man were studied. Respiration rate and content of plant hormones of these materials were analysed. Different techniques were used to culture these protoplasts. The development of protoplasts was observed under fluorescence microscope. The highest yield of protoplast (69.5 x 106 protoplasts / g fresh weight) was obtained from young male flowers after 16 hours treatment with 1.5 % cellulase, 0.25 % pectinase and 0.25 % hemicellulase. The combination of hanging drop cell technique (in 6 days), and carrot feeder layer cells in N6PKM medium supplemented with 0.2 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D), 1 mg/L α- naphthalene acetic acid (NAA), and 0.5 mg/L zeatin are suitable for protoplast development. Protoplasts that were isolated from multi-scalps and young male flowers created the wall and divided when cultured by this method. The development of protoplasts from young male flower began with cell walls creation after 4 days, the cell division was after 6 days, and small colonies formation was after 28 days of culture. The differentiation and physiological activity of cells play an important role on quantity and quality of protoplasts, as on the well as protoplast development.
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Li, Cai Hong, De Feng Xu, Ri Ying Ye, Ya Ling Wang, and Li Jun Sun. "An Efficient Method for Monitoring and Improving the Regeneration Efficiency of Protoplasts from Aspergillus Oryzae HN3042." Advanced Materials Research 781-784 (September 2013): 808–11. http://dx.doi.org/10.4028/www.scientific.net/amr.781-784.808.
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Protoplast technique is a powerful tool for strain improvement and the preparation of protoplasts with high regeneration efficiency is the basis of subsequent manipulations. In this study, the effects of enzymatic hydrolysis time on release and regeneration of protoplasts from Aspergillus oryzae HN3042 were investigated by analyzing the time-series image. The results showed that there was a significant correlationship between regeneration efficiency and morphological size of protoplasts (P < 0.01). An efficient method for monitoring and improving the regeneration efficiency of protoplast was thus established by controlling the enzymatic hydrolysis process and the results can be referred to other species during the preparation and regeneration of protoplasts.
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Pauls, K. P., and P. V. Chuong. "Flow cytometric identification of Brassica napus protoplast fusion products." Canadian Journal of Botany 65, no. 5 (May 1, 1987): 834–38. http://dx.doi.org/10.1139/b87-113.
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A flow cytometric method to identify Brassica somatic hybrids has been developed. The procedure is based on the use of fluorescein isothiocyanate stained hypocotyl protoplasts and unstained mesophyll protoplasts as partners for polyethylene glycol induced protoplast fusion. The fluorescein isothiocyanate stained hypocotyl protoplasts and unstained mesophyll protoplasts were passed through the flow cytometer – cell sorter separately to maximize the sensitivity of the red and green detectors to chlorophyll and fluorescein isothiocyanate fluorescence, respectively, and to ensure that there was no spillover of chlorophyll fluorescence into the green detector or fluorescein isothiocyanate fluorescence into the red detector. When adjusted properly, suspensions of a single protoplast type gave populations that fell on either the green or red axis of a two-dimensional green versus red fluorescence plot. With the same machine settings three populations of protoplasts could be identified in fusion mixtures of the two types of protoplasts: namely, the parental protoplasts that fell on the green or red axis as well as a third that had significant green and red fluorescence.
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Kurita-Tashiro, Asami, Noriko Hayashi, Tomoya Oyanagi, and Hamako Sasamoto. "New Factors for Protoplast-Callose-Fiber Formation in Salt-Tolerant Mangrove Plants, Avicennia alba and Bruguiera sexangula and Analysis of Fiber Substructures." Journal of Plant Studies 9, no. 2 (May 2, 2020): 1. http://dx.doi.org/10.5539/jps.v9n2p1.
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Elongated and spiral &beta;-1,3-glucan (callose) fibers were obtained by new factors from protoplasts cultured in liquid medium from suspension cultured cells of two salt-tolerant mangrove species; Avicennia alba and Bruguiera sexangula. Differences in salt factor for protoplast-fiber formation were compared with those of the callose fibers developed from protoplasts of non-mangrove tree plants, Larix leptolepis and Betula platyphylla, which high concentrations of divalent cations, Mg2+ (50 mM) or Ca2+ (100 mM), were stimulatory. In the halophilic A. alba protoplasts, whose cell division was stimulated by up to 400 mM NaCl, addition of Mg2+, Ca2+, K+ ions inhibited protoplast-fiber formation. In B. sexangula, protoplast-fibers were rapidly and efficiently formed only by another new factor, electric cell fusion treatment of protoplasts. Spiral fibers developed from mangrove protoplasts were detected under an inverted microscope, and their specific blue-green color for callose after staining with Aniline Blue dye was detected under a fluorescence microscope. Enzymatic certification of callose was further performed with laminarinase, specific for callose, in comparison with cellulase CBH1, specific for cellulose. Differences in sub-structures, fibrils and sub-fibrils of two mangrove protoplast-fibers were analyzed using laser confocal scanning microscopy, atomic force microscopy and image J analysis. Tube-like fine structure was observed using transmission electron microscopy in single protoplast-fiber of B. sexangula selected with a micromanipulator.
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Kantharajah, AS, and WA Dodd. "Factors That Influence the Yield and Viability of Cucumber (Cucumis sativus L) Cotyledon Protoplasts." Australian Journal of Botany 38, no. 2 (1990): 169. http://dx.doi.org/10.1071/bt9900169.
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Protoplasts isolated from cotyledons of aseptically germinated cucumber seedlings were divided into three size classes. The relationships between tissue age, isolation procedure, yield and protoplast size were investigated. During germination and up to an age of 13 days, the percentage of protoplasts in each size class underwent considerable change with a big reduction in percentage of the largest protoplasts in older cotyledons. Protoplast size and yield could also be manipulated by varying the isolation technique. In this context, temperature, incubation time and shaker speed were significant. By selecting tissue of appropriate age and using a carefully selected isolation procedure the percentage of viable cucumber protoplasts with the ability to form a cell wall and divide can be increased considerably.
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Sasamoto, Hamako, Tsukasa Iwashina, Sakae Suzuki, Yoshitaka Azumi, and Yoshiharu Fujii. "Evaluation of an Anthocyanin, Cyanidin 3,5-di-O-glucoside, as an Allelochemical in Red Callus of a Mangrove Sonneratia ovata, Using Protoplast Co-Culture Bioassay Method with Digital Image Analysis." Journal of Plant Studies 7, no. 2 (March 28, 2018): 1. http://dx.doi.org/10.5539/jps.v7n2p1.
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protoplasts to examine the allelopathic activities. Protoplasts were isolated with Cellulase R10 and Driselase 20 in 0.6 M mannitol solution and purified by density gradient centrifugation on 0.6 M sucrose. Protoplasts were co-cultured in 50 μL of liquid Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine and 0.6 M mannitol solution in a 96-well culture plate. Protoplast density ranged from 5 × 103/mL to 105/mL. Cell division of lettuce protoplasts was strongly inhibited by addition of S. ovata protoplasts, and non-spherical cell enlargement was slightly inhibited. By contrast, digital image analysis of scanned 96-well culture platesrevealed no inhibition in accumulation of yellow color in lettuce protoplasts. An anthocyanin, cyanidin 3,5-di-O-glucoside (cyanin), was identified and its content in the red callus was ca. 1 mM of fresh weight. The effects of cyanin on the growth of lettuce protoplasts at three stages were similar to those of red S. ovata protoplasts. From these results, cyanin was most likely the allelochemical contained in red callus of S. ovata. The allelopathic activity of cyanin was compared with that of other putative allelochemicals in several plant materials, using the protoplast co-culture method with digital image analysis.
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Al-Nema, Qutaiba, and Mozahim AL-Mallah. "Electrofusion of mesophyll protoplasts from two varieties of sugar beet, (Beta vulgaris L.)." Journal of Life and Bio Sciences Research 1, no. 1 (April 23, 2020): 22–25. http://dx.doi.org/10.38094/jlbsr117.
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Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.
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Meng, Ruirui, Chenchen Wang, Lihua Wang, Yanlong Liu, Qiuwen Zhan, Jiacheng Zheng, and Jieqin Li. "An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9." PeerJ 8 (October 13, 2020): e10077. http://dx.doi.org/10.7717/peerj.10077.
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Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.
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Inoue, Aya, Daisuke Mori, Reiko Minagawa, Yoshiharu Fujii, and Hamako Sasamoto. "Allelopathy in a Leguminous Mangrove Plant, Derris indica: Protoplast Co-culture Bioassay and Rotenone Effect." Natural Product Communications 10, no. 5 (May 2015): 1934578X1501000. http://dx.doi.org/10.1177/1934578x1501000512.
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To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the ‘Protoplasts Co-culture Method’ for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 × 103 - 105 / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.
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Wang, Lun, Jiaojiao Zhang, and Xiaoyong Xu. "A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)." Plants 13, no. 20 (October 15, 2024): 2878. http://dx.doi.org/10.3390/plants13202878.
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The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.
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Sun, M., H. Kieft, and AAM van Lammeren. "Cotyledon-derived diploid and haploid protoplast culture and diploid plant regeneration in Brassica napus cv. ' Topas '." Canadian Journal of Botany 76, no. 3 (March 1, 1998): 530–41. http://dx.doi.org/10.1139/b98-022.
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The present paper describes a simple and reliable protocol for the successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L. cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32°C instead of 25°C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.Key words: cotyledon protoplast culture, haploid culture, plant regeneration.
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Jenes, Barnabas, Matti Puolimatka, Pedro Bittencourt, and Seppo Pulli. "Time saving method for protoplast isolation, transformation and transient gene expression assay in barley." Agricultural and Food Science 3, no. 2 (March 1, 1994): 199–205. http://dx.doi.org/10.23986/afsci.72695.
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This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutellum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene glycol) method. Plasmid pAct1-F containing the rice Act1 promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transformation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligible time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture.
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Wang, Peilin, Yuanchun Pu, Muhammad Ali Abid, Linglin Kang, Yulu Ye, Man Zhang, Chengzhen Liang, Yunxiao Wei, Rui Zhang, and Zhigang Meng. "A Rapid and Efficient Method for Isolation and Transformation of Cotton Callus Protoplast." International Journal of Molecular Sciences 23, no. 15 (July 28, 2022): 8368. http://dx.doi.org/10.3390/ijms23158368.
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Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.
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Schulz, Margot, and Gottfried Weissenböck. "Isolation and Separation of Epidermal and Mesophyll Protoplasts from Rye Primary Leaves — Tissue-Specific Characteristics of Secondary Phenolic Product Accumulation." Zeitschrift für Naturforschung C 41, no. 1-2 (February 1, 1986): 22–27. http://dx.doi.org/10.1515/znc-1986-1-205.
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Abstract We have develop ed a technique for the large-scale isolation of epidermal and mesophyll protoplasts, as well as the vascular strands, of rye primary leaf blades. Separation of the two types of protoplasts has been successful only from leaves harvested at the end of a 13-h light period, when chloroplasts were enriched in starch. The occurrence of different flavonoid compounds, and amounts, in epidermal and mesophyll protoplasts can be used as criteria for protoplast purity and viability since C-glucosylflavone O-glycosides are characteristic of epidermal protoplasts whereas flavone O-glucuronides and anthocyanins are typical of mesophyll protoplasts. Several non-flavonoid phenolic compounds are found only in the epidermal protoplast. These patterns of secondary product accumulation reflect the high tissue specificity of the rye leaf.
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Mieth, Hannelore, Volker Speth, and Jürgen Ebel. "Phytoalexin Production by Isolated Soybean Protoplasts." Zeitschrift für Naturforschung C 41, no. 1-2 (February 1, 1986): 193–201. http://dx.doi.org/10.1515/znc-1986-1-229.
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Abstract Protoplasts isolated enzymatically from suspension-cultured cells of soybean (Glycine max L. Merr., cv. Harosoy 63) were used to study the production of the isoflavonoid-derived phytoalexin, glyceollin. A large enhancement in the in vivo rates of synthesis and catalytic activities of two of the enzymes associated with glyceollin biosynthesis, phenylalanine ammonia-lyase and chalcone synthase, preceded phytoalexin accumulation during early stages of culture of isolated protoplasts while cell wall regeneration occurred. A glucan elicitor from cell walls of the fungus Phytophthora megasperm a f. sp. glycinea, an effective inducer of the phytoalexin response in cultured cells, was not capable of enhancing phytoalexin formation in protoplasts. Lack of responsiveness of the protoplasts to the glucan elicitor could either be associated with their stressed metabolic state in which the response system is already saturated or with the removal from cultured cells of an essential factor of the glucan elicitor-mediated phytoalexin induction during protoplast isolation. At least two components of the protoplast isolation medium, the osmoticum and the fungal endopolygalacturonase, have the potential to initiate the observed phytoalexin synthesis during protoplast isolation. Our results indicate that under the methods employed isolated soybean protoplasts display a stress response which other types of soybean cells show following microbial attack or treatment with elicitor.
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Sasamoto, Hamako, and Shinso Yokota. "In vitro Bioassay of Allelopathic Activities of a Mangrove Tree, Kandelia obovata, and Fast-growing Trees, Betula platyphylla and Populus alba, Using Protoplast Co-culture Method." Journal of Plant Studies 10, no. 2 (July 17, 2021): 8. http://dx.doi.org/10.5539/jps.v10n2p8.
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Allelopathic activities of a salt-tolerant and low-temperature tolerant mangrove tree, Kandelia obovata, which grows in brackish water regions of sub-tropical areas, and two fast-growing trees, Betula platyphylla and Populus alba, which grow in the temperate area, were examined by two in vitro bioassay methods, the sandwich method using dried leaves and the protoplast co-culture method using leaf protoplasts. Lettuce root growth examined by the sandwich method, was inhibited 50% by 50 mg dried mature leaves of K. obovata. In the protoplast co-culture method, inhibition rates of cell division of lettuce protoplasts were 31% and 69% by leaf protoplasts of K. obovata at densities of 1 &times; 104 mL-1 and 5 &times; 104 mL-1, respectively. These results were compared with the inverse relationship between allelopathic activities and salt tolerance of mangrove plants of different families. B. platyphylla showed 37% inhibition by the sandwich method using dried young leaves, but only 10% inhibition at 5 &times; 104 mL-1 by the protoplast co-culture method using leaf protoplasts of B. platyphylla. Dried young leaves of P. alba showed 66% inhibition, but the leaf protoplasts at the density of 5 &times; 104 mL-1 showed highly stimulatory activity. Abscisic acid, of which contents in leaf protoplasts of three tree species varies from high to low in relation to salt tolerance and recalcitrance of tissue culture, was discussed as a putative allelochemical.
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Echeverria, Ed. "Preparation and Characterization of Protoplasts from Citris Juice Vesicles." Journal of the American Society for Horticultural Science 112, no. 2 (March 1987): 393–96. http://dx.doi.org/10.21273/jashs.112.2.393.
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Abstract A method for the preparation of protoplasts from juice vesicles of citrus fruits is described. Protoplasts were obtained after incubating juice vesicles for 16 hr at room temperature in a medium containing cellulase and pectinase. The solution containing the protoplast was filtered through a 200-μm nylon mesh and the filtrate layered on a Percoll density gradient of 4%, 3.5%, and 3%. After 1 to 2 hr at 4°C, protoplasts were collected from the 4% Percoll layer. Protoplast viability was assessed by different methods and estimated to be ≈ 85%. The use of several stains and observations with light and electron microscopy provided information on some anatomical and physiological characteristics.
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Bekkaoui, Faouzi, and David I. Dunstan. "Permeabilization of Piceaglauca protoplasts to macromolecules." Canadian Journal of Forest Research 19, no. 10 (October 1, 1989): 1316–21. http://dx.doi.org/10.1139/x89-202.
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Chemical permeabilization (polyethylene glycol, molecular weight 3350) and electropermeabilization (electroporation) treatments were applied to white spruce protoplasts to determine their effectiveness for uptake of membrane impermeable macromolecules. The two techniques have been compared using the membrane impermeable fluorescent dye calcein (molecular weight 622). The effects of varying the polyethylene glycol concentration, or the capacitance and voltage, were tested. In both techniques, the viability of protoplasts decreased after treatment compared with the controls. However, electroporation (capacitance 25 μF; voltage 300 V, 750 V•cm−1) gave better-permeabilization results (55% protoplast viability with 96% of these being fluorescent protoplasts) than the best treatment with polyethylene glycol (20%) (30% protoplast viability with 15% being fluorescent protoplasts). An investigation was made with the dye fluorescein isothiocyanate dextrans at different average molecular weights: 4000, 70 000, and 150 000. The degree of internalization by electroporation of each of these molecules did not substantially differ, though they were all low compared with calcein, which is suggestive of a limitation in permeability. The protoplasts subjected to either polyethylene glycol or electroporation treatments gave rise to callus and proembryos.
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Shao, Yingying, Detian Mu, Limei Pan, Iain W. Wilson, Yajie Zheng, Lina Zhu, Zhiguo Lu, et al. "Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis." International Journal of Molecular Sciences 24, no. 4 (February 11, 2023): 3633. http://dx.doi.org/10.3390/ijms24043633.
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Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.
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Krasova, Yuliya V., Vladimir V. Fadeev, Yelizaveta М. Moiseeva, Yury S. Gusev, and Mikhail I. Chumakov. "Optimization of the technique for maize protoplast isolation and their nativity after electroporation." Izvestiya of Saratov University. Chemistry. Biology. Ecology 22, no. 4 (December 15, 2022): 445–54. http://dx.doi.org/10.18500/1816-9775-2022-22-4-445-454.
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We optimizes the composition and concentration of the enzymes, the time for enzymatic treatment and the volume of the enzyme mixture. We also optimized the concentration osmotic of agent, the centrifugation mode, and filter pore size for protoplasts isolating from epidermal cells of maize roots (Zea mays L.) of the Brown Marker (BM) line. It was found that 150 minutes is the optimal time for 150 mg root tissue maceration. The yield of intact protoplasts was ~ 4.4 ± 0.2 × 105 cells/mL at the following concentrations of enzymes and osmotic stabilizer: cellulase – 17,4, pectolase – 1.2, hemicellulase – 0.07, D-mannitol – 9.3%. The the concentration of protoplasts was to 23 times higher (p < 0.05) in 800 μl, compared with 200 μl of the enzyme mixture with equal concentrations of enzymes and osmotic stabilizer. It was found that filtration of 800 μl protoplast suspension through a filter with a pore size of 15 × 15 microns increases the yield of protoplasts up to 3.3 times, compared with a filter with a pore size of 15×39. Fractional centrifugation without preliminary filtration of the solution and the flotation method did not produce an increase in the yield of protoplasts. The residual number and protoplast wholeness after ~ 20 hours at +3 °C incubation was evaluated. The protoplast number decreased up to 2 times (p < 0.05) after electroporation.
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Balasubramanian, N., G. Annie Juliet, P. Srikalaivani, and D. Lalithakumari. "Release and regeneration of protoplasts from the fungus Trichothecium roseum." Canadian Journal of Microbiology 49, no. 4 (March 1, 2003): 263–68. http://dx.doi.org/10.1139/w03-034.
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A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 × 104 protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.Key words: Trichothecium roseum, protoplast, isolation, regeneration, lytic enzymes, osmotic stabilizers.
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Li, Xiaobin, Ying Qin, Yufei Kong, Samantha Chandranath Karunarathna, Yunjiang Liang, and Jize Xu. "Optimization of Protoplast Preparation Conditions in Lyophyllum decastes and Transcriptomic Analysis Throughout the Process." Journal of Fungi 10, no. 12 (December 21, 2024): 886. https://doi.org/10.3390/jof10120886.
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Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in Lyophyllum decastes. The results indicated an optimal protoplast yield of 5.475 × 106 cells/mL under conditions of fungal age at 10 days, digestion time of 2.25 h, enzyme concentration of 2%, and digestion temperature of 28 °C. The Z5 medium supplemented with L. decastes mycelial extract achieved a high regeneration rate of 2.86. RNA-seq analysis revealed 2432 differentially expressed genes (DEGs) during protoplast formation and 5825 DEGs during regeneration. Casein kinase I, cytochrome P450 (CYP52), and redox-regulated input receptor (PEX5) were significantly upregulated during the protoplast stage, while β-1,3-glucan synthase (SKN1), chitin synthase (CHS2), hydrophobin-1, and hydrophobin-2 showed significant upregulation during the protoplast regeneration phase. These findings provide a reference for the efficient preparation and regeneration of protoplasts and offer new insights into the molecular mechanisms of protoplast formation and cell wall regeneration in fungi.
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Sun, Yuan, Zheng, Liang, Jiang, Wang, Chen, et al. "An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts." Plants 8, no. 10 (September 28, 2019): 385. http://dx.doi.org/10.3390/plants8100385.
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In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.
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Al-Ne'ma, Q. Sh, and M. K. Al-Mallah. "Protoplast isolation from leaf mesophyll of sugarbeet Beta vulgaris L. axenic seedlings." Journal of Biotechnology Research Center 7, no. 3 (December 1, 2013): 36–42. http://dx.doi.org/10.24126/jobrc.2013.7.3.280.
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Protoplasts were isolated from leaf mesophyll of sugarbeet (Beta vulgaris L.) axenic seedlings. Eight enzyme mixtures were tested for cell wall degrading ability. The efficient enzyme solution was mixture "II" that consist of 1.5% Cellulase RS, 2% Cellulase R10, 1% Macerozym R10 and 0.1% Pectolyase Y23. This mixture was efficient in releasing protoplasts and gave high yield of a density 7.3 × 104 protoplast / ml. These isolated protoplasts were viable 93%, their sizes ranged from 13 up to 52 µm, vacuolated and unvacuolated. This finding enable workers to focus on somatic hybridization through protoplast fusion to overcome some barriers facing gene transfer to improve plant species such as transfer of N2 fixation ability to non-legumes and producing resistant varieties to biotic stress and other traits.
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Martin, F. R., and R. A. Nolan. "American cockroach (Periplaneta americana) hemocyte response to Entomophaga aulicae protoplasts." Canadian Journal of Zoology 64, no. 6 (June 1, 1986): 1369–72. http://dx.doi.org/10.1139/z86-204.
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Contact through protoplasmic extensions from the protoplasts was infrequently observed and cell–cell contact involving large surface areas was not observed when adult Periplaneta americana hemocytes were incubated with Entomophaga aulicae protoplasts in in vitro culture for up to 30 min. Such contact did not appear to alter the subsequent morphology of either the protoplast or the hemocyte. Coagulocyte, plasmatocyte, and granulocyte aggregates along with individual coagulocytes and granulocytes produced a melaninlike material upon incubation with protoplasts. Incubation of the hemocytes in the presence of protoplasts did not alter the affinity of hemocytes for Sephadex beads. The results of this study indicate that there was no detectable cell-mediated immune reaction of cockroach hemocytes to E. aulicae protoplasts. The role of the melaninlike material is unknown.
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Du, Jiageng, Huitao Zhang, Weilong Li, Xiaoyan Li, Zhuo Wang, Ying Zhang, Aisheng Xiong, and Mengyao Li. "Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens." Agronomy 13, no. 8 (August 17, 2023): 2154. http://dx.doi.org/10.3390/agronomy13082154.
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Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function.
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KESKITALO, M. "Can protoplast production from in vitro cultured shoots of Tanacetum vary during the season?" Agricultural and Food Science 10, no. 3 (January 3, 2001): 145–51. http://dx.doi.org/10.23986/afsci.5688.
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Two different experiments were carried out to study the production of protoplasts and the variation of protoplast yield from in vitro cultured shoot tips of tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schiltz-Bip). In the first experiment, light had more pronouced effect for tansy than for pyrethrum. When the donor tissues of tansy were cultured under high light intensity the leaves contained anthocyanin and became brown during enzyme maceration. In contrast, donor tissues cultured under low light intensity produced leaves without anthocyanin. Depending on the light intensity of donor tissues, on average 5.8 - 6.8 x 106 and 3.4 - 4.3 x 106 protoplasts were isolated from one gram of mesophyll leaves of tansy and pyrethrum, respectively. In the second experiment, the production of protoplasts from tansy and pyrethrum varied seasonally. The most successful season for the production of protoplasts from in vitro cultured shoot tips was between December and April, when also the highest number of protoplasts could be isolated. It was not possible to state whether Tanacetum species have rhythms, which could cause physiological or chemical changes for the in vitro grown shoot tips. However, some external or internal, possible seasonal-dependent stimuli may have caused variation in the number of protoplasts isolated from tansy and pyrethrum and favoured protoplast production during winter and spring.
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Wei, Xiuyan, Xinyue Song, Dong Dong, Nemat O. Keyhani, Lindan Yao, Xiangyun Zang, Lili Dong, et al. "Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms." Canadian Journal of Microbiology 62, no. 7 (July 2016): 579–87. http://dx.doi.org/10.1139/cjm-2015-0770.
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The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box–Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R2 = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 107 cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl–Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·107 protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.
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Kropp, Bradley R., and J. A. Fortin. "Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Laccaria bicolor." Canadian Journal of Botany 64, no. 6 (June 1, 1986): 1224–26. http://dx.doi.org/10.1139/b86-167.
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Protoplasts were released from dikaryotic mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor using the lytic enzyme preparation NovoZyme 234. Protoplast release depended strongly on mycelium age, osmotic stabilizer, and temperature. The protoplasts could regenerate to form both monokaryotic and dikaryotic cultures capable of forming normal ectomycorrhizae with Pinus banksiana.
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Kiełkowska, Agnieszka, and Adela Adamus. "Embedding in Filter-Sterilized Alginate Enhances Brassica Oleracea L. Protoplast Culture." Acta Biologica Cracoviensia s. Botanica 56, no. 2 (March 1, 2015): 20–26. http://dx.doi.org/10.2478/abcsb-2014-0018.
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Abstract The influence of sodium alginate sterilization on the viability and mitotic activity of embedded protoplasts was studied in protoplasts of Brassica oleracea subsp. alba and rubra isolated from hypocotyl tissue and leaves of seedlings or plants grown in vitro. Both leaf and hypocotyl-derived protoplasts were more viable and divided more frequently when embedded in filtrated alginate. Division frequency was highest in cv. Reball F1 and the mitotic activity of its protoplasts was three times higher when embedded in filtrated alginate (36.1 ± 6.8%) than when cultured in autoclaved alginate (10.9 ± 5.0%). Protoplast-derived calli colonies were transferred to solid regeneration media and plants of all tested accessions were obtained.
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Jażdżewska, Elżbieta, Aleksandra Niklas, and Anna Majewska-Sawka. "Progress towards sugar beet improvement through somatic hybridization I. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts." Acta Societatis Botanicorum Poloniae 64, no. 4 (2014): 341–47. http://dx.doi.org/10.5586/asbp.1995.044.
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The isolation and culture of suspension-derived protoplasts from two sugar beet (<em>Beta vulgaris</em> L.) genotypes are described. Immobilization of protoplasts in agarose resulted in high frequency divisions and microcallus regeneration, with plating efficiency (PE) being clearly genotype-dependent. In further studies towards asymmetric fusion experiments, the effect of different doses of ultraviolet radiation (UV) and iodoacetic acid (IA) on protoplast physiology was assessed. Viability of both treated (UV, IA) and untreated protoplasts (control) was determined by FDA staining, and the biological effect was evaluated by testing the ability of protoplasts to divide and to form calli. The results are discussed in terms of the applicability of the methods for the production of asymmetric protoplasts suitable for somatic hybridization within the genus <em>Beta</em>.
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Nolan, Richard A. "Stage-specific changes in cytoplasmic protein synthesis in Entomophaga aulicae protoplasts." Canadian Journal of Microbiology 35, no. 3 (March 1, 1989): 373–78. http://dx.doi.org/10.1139/m89-057.
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The patterns of protein synthesis associated with three sequential stages in protoplast morphogenesis (spindle-shaped, early fusion sphere, and late fusion sphere protoplasts) of the fungus Entomophaga aulicae were studied using both one-dimensional gels with general protein staining and two-dimensional gels with [35S]methionine protein labelling and fluorography. A total of 332 proteins were observed with 63.5% (211) common to all three developmental stages. Of the individual totals, 3.3% (8 out of 245), 7.3% (22 out of 301), and 4.5% (13 out of 286) of the proteins were unique to the spindle-shaped, early fusion sphere, and late fusion sphere protoplasts, respectively. The molecular mass and pI distribution profiles for early fusion sphere protoplast proteins are discussed.Key words: protein synthesis, stage-specific proteins, fungal protoplasts, Entomophaga aulicae.
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Sihachakr, Darasinh, and Georges Ducreux. "Isolement et culture de protoplastes à partir de petioles et de tiges de deux variétés de patate douce (Ipomoea batatas)." Canadian Journal of Botany 65, no. 1 (January 1, 1987): 192–97. http://dx.doi.org/10.1139/b87-025.
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Protoplasts isolated from petioles and stems of two varieties of sweet potato showed high plating efficiency, and rhizogenic calli were obtained. The yield of protoplasts and the rate of survival of cell division were statistically analysed. Results are discussed in relation to the identification of different protoplast subpopulations and their histological origin.
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Stevenson, I. "Protoplast Formation from Submerged Mycelium and from Spore Germinants of Streptomyces coelicolor." Australian Journal of Biological Sciences 38, no. 1 (1985): 175. http://dx.doi.org/10.1071/bi9850175.
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Stages in the formation of protoplasts from S. coelicolor strain A3(2) have been studied by transmission electron microscopy. Protoplasts liberated from submerged mycelial growth were variable in size and were released when digestion of the cell wall by lysozyme had completely or almost completely taken place. Protoplasts did not fully adopt the typical rounded shape until after release. A single region of cytoplasm gave rise to more than one protoplast unit. Protoplasts released from spore germinants escaped from the tip of the germ tube, which was the region of the cell wall most susceptible to digestion. Protoplasts derived from spore germinants were more consistent in size and rounded up more rapidly. If a cross-wall had formed in a germinant then it gave rise to separate protoplasts from each cellular. compartment. Protoplasts of either type contained a single DNA region. These studies give an indication of the cellular organization of a streptomycete colony, which can be visualized as a multinucleated assemblage of cellular units in a common cytoplasm. The assembly of units separates into a number of protoplasts on digestion of the cell wall.
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Taylor, PWJ, HL Ko, and SW Adkins. "Factors Affecting Protoplast Isolation and the Regeneration of Shoot-Like Structures From Protoplast-Derived Callus of Sugarcane (Saccharum spp Hybrids)." Australian Journal of Botany 40, no. 6 (1992): 863. http://dx.doi.org/10.1071/bt9920863.
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High yields of protoplasts were isolated from non-regenerable, homogeneous cell suspension cultures of sugarcane, compared with regenerable, heterogeneous cell suspension cultures after incubation in an enzyme composition containing Cellulase RS, Pectinase, Macerozyme and Driselase. Higher yields of protoplasts were released from heterogeneous cell suspension cultures after the addition of 10 mg L-1 silver nitrate to the culture medium; however, ethylene production was not involved in protoplast isolation. Use of 0-05-2% Pectolyase Y23 pectinase rather than other pectinases resulted in higher yields of protoplasts from heterogeneous cell suspension cultures. These results suggest that there are differences in the cell walls between cells from heterogeneous and homogeneous cell suspension cultures which affect the isolation of protoplasts. Protoplasts isolated from heterogeneous cell suspension cultures failed to develop beyond the cell division stage. Protoplasts isolated from homogeneous cell suspension cultures and cultured in agarose droplets bathed in modified 8p medium, reformed cell walls, divided and developed into microcallus. Microcallus transferred to solid modified MS medium containing 1 mg -1 .2,4-D developed into callus. Protoplast-derived callus from one cultivar formed compact nodular callus when subcultured onto the same medium containing 1% activated charcoal. Incubation of this callus on MS medium containing BAP at 0.5 mg L-1, then a combination of BAP and fluridone each at 0.5 mg L-1, resulted in the regeneration of small, chlorophyll-containing shoot-like structures. As yet no intact plants have developed from these shoot-like structures.
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Chen, Chi-Chang, Feng-Ming Lee, and Yen-Yu Kao. "Endoreduplication in leaf protoplasts of haploid Nicotiana plumbaginifolia cultured in vitro." Genome 30, no. 5 (October 1, 1988): 615–20. http://dx.doi.org/10.1139/g88-104.
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When protoplasts isolated from the leaves of haploid plants of Nicotiana plumbaginifolia Viviani were examined cytologically during in vitro culture, a small fraction was found to contain diplochromosomes at the first mitosis. DNA measurements of freshly isolated protoplasts revealed that 92% contained G1 and 8% contained G2 haploid nuclei. The origin of the diplochromosomes was investigated by a simplified sister chromatid differential staining technique and autoradiography. Our results showed that nuclear division in the G1 protoplasts was normal, with each round of DNA replication being followed by mitosis. In the G2 protoplasts, however, one extra round of DNA replication often took place before the nucleus entered mitosis.Key words: Nicotiana plumbaginifolia, protoplast culture, endoreduplication, sister chromatid differentiation.