Showing papers on "Protoplast published in 2006"

A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.

Abstract: SUMMARY The transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stem- and sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate large-scale functional analysis of defence-related genes in rice.

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

Abstract: Background: Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. Results: We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7–14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60–70% transformation efficiency. In contrast, using 50 µg of a 12 kb plasmid we obtained a maximum of 25–30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. Conclusion: We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

A Comparison of the Phenotypic and Genetic Stability of Recombinant Trichoderma spp. Generated by Protoplast- and Agrobacterium-Mediated Transformation

Abstract: Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion

Abstract: The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T.␣atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 $$\upmu$$ g/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in␣vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 $$\upmu$$ g/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T.␣atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

Abstract: In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.

Macrotubule-dependent protoplast volume regulation in plasmolysed root-tip cells of Triticum turgidum: involvement of phospholipase D.

Abstract: The probable involvement of phospholipase D (PLD)/phosphatidic acid (PA) signalling in the hyperosmotic stress response of Triticum turgidum root cells was investigated by examining the effects of butanol-1, butanol-2, phosphatidylbutanol (PtdBut), N-acylethanolamine (NAE) and PA on the hyperosmotic response, the organization of the tubulin cytoskeleton and the accumulation of a phosphorylated p38-like mitogen-activated protein (MAP) kinase (phospho-p46) in plasmolysed root cells. The effects of all the treatments were assessed by differential interference contrast (DIC) microscopy of living cells, tubulin immunofluorescence, conventional transmission electron microscopy (TEM), tubulin immunogold localization, protoplast volume measurements and western blot analysis. Butanol-1 and NAE compromised the viability of plasmolysed cells, induced a marked reduction in the plasmolysed protoplast volume, and inhibited hyperosmotically induced tubulin macrotubule formation and the accumulation of phospho-p46. Exogenous PA reinforced the hyperosmotic response of T. turgidum root cells and positively affected tubulin macrotubule formation. Additionally, PA reduced the effects of butanol-1 in plasmolysed cells. Taken together, the data suggest that PLD-mediated PA synthesis occurs upstream of the accumulation of phospho-p46 to regulate hyperosmotically induced macrotubule formation in plasmolysed T. turgidum root cells.

Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases.

Abstract: Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e. they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18,2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam

Abstract: Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l−1 IAA and 1.0–3.0 mg l−1 BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l−1 BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.

Preparation of recombinant polysaccharide-degrading enzymes from the marine bacterium, Pseudomonas sp. ND137 for the production of protoplasts of Porphyra yezoensis

Abstract: We previously reported that a marine bacterium, strain ND137, produced the enzymes to make protoplasts from the thalli of a marine red alga Porphyra yezoensis. In this study, the bacterium was classified as Pseudomonas sp. based on the sequence of the gene encoding 16S rRNA and the G + C content of the DNA. The β-1,4-xylanase-, cellulase-, β-1,3-xylanase-, porphyranase-, and β-1,4-mannanase-encoding genes were cloned from the genomic library of Pseudomonas sp. ND137 and expressed in Escherichia coli. The protoplasts from Porphyra yezoensis were produced by treating the thalli with several combinations of these recombinant crude enzymes. Treatment of the Porphyra thalli with any of these enzymes on their own failed to produce protoplasts. However, a mixture of β-1,3-xylanase, porphyranase, and β-1,4-mannanase produced protoplasts, although β-1,4-xylanase and cellulase were not observed to have any effect on protoplast production. These results indicate that among the five recombinant crude enzymes, β-1,3-x...

An Efficient Protocol for Ulmus minor Mill. Protoplast Isolation and Culture in Agarose Droplets

Abstract: We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 107 protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 105 and 2 × 105 protoplasts ml−1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 105 protoplasts ml−1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.

Regeneration of interspecific somatic hybrids between Helianthus annuus L. and Helianthus maximiliani (Schrader) via protoplast electrofusion

Abstract: Helianthus maximiliani is one of the wild Helianthus species with the genes for resistance to many pathogens including Sclerotinia sclerotiorum. Unfortunately, a transfer of disease resistance genes from this species into the cultivated sunflower is limited by its poor crossability with the cultivated sunflower and sterility of interspecific hybrids. To overcome this problem, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clone of H. maximiliani were electrically fused with etiolated hypocotyl protoplasts of the cultivated sunflower inbred line PH-BC1-91A. Fusion products were embedded in agarose droplets and subjected to different regeneration protocols. Developed microcalluses were released from the agarose and transferred into solid media. Shoot regeneration was achieved by culture of calluses on regeneration medium containing 2.2 mg l−1 BAP and 0.01 mg l−1 NAA after the treatment with a high concentration of 2,4 D for a limited period of time. A morphological and RAPD analysis confirmed a hybrid nature of the regenerated plants.

Sugar effects on membrane damage during desiccation of pea embryo protoplasts

Abstract: Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.

Protoplast electrofusion between common wheat (triticum aestivum l.) and italian ryegrass (lolium multiflorum lam.) and regeneration of mature cybrids

Abstract: This study describes the development of electrofusion techniques using the ‘donor-recipient’ model for the production of cybrids between common cultivated winter wheat (Triticum aestivum L.) cv. Jinghua No. 1 and a phylogenetically remote, sexually incompatible grass species, Italian ryegrass (Lolium multiflorum Lam.), which belong to two different subtribes: Triticinae and Loliinae. Wheat protoplasts were metabolically inactivated by iodoacetamide before fusion, while protoplasts of Italian ryegrass were X-ray irradiated before protoplast isolation. The suspension cells were directly used to optizmize the inactivation parameters. By exploring the minimum irreversible membrane breakdown strength, the electrofusion parameters were optimized just a few minutes before electrofusion began. A total of 108 green plantlets were obtained, and about half of the green plants uncontrollably necrotized. Among all green plants, 14 were rooted normally and transplanted in growth chamber or field and developed to maturity. All these transplanted plants were male sterile with smaller and off-white anthers. Seeds were obtained by crossing with Jinghua No. 1. Three transplanted regenerants possessed the characteristics of glume facing the rachis, which was the taxonomic characteristic distinguishing the two subtribes of Triticinae and Lolliinae. Although Southern blot hybridization analysis of 33 randomly selected regenerants using a wheat ribosomal DNA probe (pHA71) did not find any differences to wheat, analysis using two mitochondrial probes B342 (cox l), 490 (Pro II) and one chloroplastidic probe pHve H5 revealed that 31 plants were ‘true cybrids’ by showing ryegrass-specific band(s) or new band(s). It also showed that the mitochondria and chloroplasts were not coexistent as the restriction fragment length polymorphism band of Italian ryegrass was not detected by the mitochondrial probes 7 (26s), B342 (cox I), pHJ2-7-1 (cox II), B30 (atp9), and the chloroplast probe pHvc P5. To regenerate the cybrids, the regeneration capacity of the recipient (wheat) was crucial in this study.

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts ( Musa spp.)

Abstract: One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma2 and PCM, significantly affected the number of microcalli produced, which was 90 × 103 per Petri dish on Ma2 with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 103 per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma2 and the frequency of embryogenic cell aggregates was higher among microcalli on Ma2-medium. However, no cell division was observed in protoplasts cultured on Ma2 in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

Abstract: A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 105 protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.8 mg l−1 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.

Physiological characterization of the sex pheromone protoplast‐release‐inducing protein from the Closterium peracerosum‐strigosum‐littorale complex (Charophyta)

Abstract: SUMMARY In the unicellular charophycean alga Closterium peracerosum-strigosum-littorale complex, the protoplast-release-inducing protein (PR-IP), a sex pheromone responsible for gametic protoplast release from mating-type minus (mt–) cells, was found to stimulate secretion of mucilage from the cells. Induction of sexual cell division by PR-IP was also confirmed. Bioassays were used to determine the minimum doses required to induce these functions, revealing that 5 · 10−16 M of PR-IP stimulated mucilage secretion, and that 5 · 10−10 M of PR-IP were required for protoplast release. Exposure of the cells to 5 · 10−11 M of PR-IP resulted in the induction of sexual cell division as well as mucilage secretion. These results strongly suggest that PR-IP is a multifunctional pheromone that independently promotes multiple steps in conjugation at the appropriate times through different induction mechanisms.

Factors Affecting the Protoplast Isolation and Culture of Anubias nana Engler

Abstract: The effects of various factors on the isolation and culture of Anubias nana Engler protoplast were investigated. Several factors, such as kind and concentration of enzymes, mannitol concentration in digestion solution, incubation time, plant material age and sucrose concentration in purifying solution were extremely important to obtain highest number and viability of protoplasts. The highest yield (4.79±0.48x106 per gram fresh weight) and viability (82.90±4.31%) was obtained when protoplasts were digested from in vitro six-week-old leaves with 2% Cellulase Onozuka RS, 0.2% Pectolyase Y-23, 0.6 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM morpholinoethane sulfonic acid (MES), pH 5.6 for 4 h in the dark and purified with 18% sucrose gradient centrifugation. Purified protoplasts were cultured on KM8P medium supplemented with 0.2 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg L-1 α-naphthalene acetic acid (NAA), 0.5 mg L-1 zeatin by agarose-bead with thin layer liquid culture. The protoplasts regenerated cell walls within 24 h. First cell division was observed after culturing for 7-9 days and micro-colonies were formed within 30 days. The culture medium, culture method and plant growth regulator were significant factors influencing cell division and survival of protoplast.

Plant regeneration from mesophyll protoplasts of a medicinal plant, phellodendron amurense rupr.

Abstract: A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by the enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×105 protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×105–6×105 protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6 M mannitol, 2.0 μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when trans...

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast-protoplast fusion.

Abstract: Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.

Protoplast Isolation and Culture of Dendrobium Sonia "Bom 17"

Abstract: The optimization of Dendrobium Sonia “Bom 17” protoplast isolation and culture conditions were investigated. Protoplasts were successfully isolated from one-month-old leaves of in vitro-grown plantlets using an enzyme solution comprising 1% Cellulase Onozuka R-10, 0.2% Macerozyme, 0.3 M mannitol, 10 mM CaCl2.2H2O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Approximately 3.97±0.63 · 10 5 protoplasts per gram fresh weight were obtained when digested in enzyme solution for 4 h on a shaker under dark conditions, then washed with 0.3 M mannitol solution supplemented with 10 mM CaCl2.2H2O and 10 mM MES, and purified with 0.3 M sucrose solution. Freshly isolated protoplasts were then cultured at final density of 2 · 10 5 protoplasts/ml in Kao & Michayluk medium (KM8P) by agarose-bead cultures under dark conditions at 25°C. First cell division was observed after culturing for 2 days and multicellular colonies (15-20 cells) were formed after 2

Isolation, Regeneration and PEG-Induced Fusion of Protoplasts of Pleurotus pulmonarius and Pleurotus florida.

Abstract: Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulted in the maximum release of protoplasts from 3-day-old mycelia of P. florida (5.3~5.75 × 10(7) protoplasts/g) and P. pulmonarius (5.6~6 × 10(7) protoplasts/g). The isolated protoplasts of P. florida regenerated mycelium with 3.3% regeneration efficiency while P. pulmonarius showed 4.1% efficiency of regeneration. Polyethyleneglycol (PEG) - induced fusion of protoplasts of these two species resulted in 0.28% fusion frequency. The fusant produced fruiting bodies on paddy straw but required a lower temperature of crop running (24 ± 2℃) than its parents which could fruit at 28 ± 2℃. The stable fusant strain was selected by testing for the selected biochemical markers i.e. Carbendazim tolerance and utilization of the lignin degradation product, vanillin.

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Abstract: Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Abstract: Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N6BM and N6K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.

Callus Cultures of Arabidopsis

Abstract: Protoplasts are plant cells lacking cell walls. They can be generated from stationary callus cultures derived from Arabidopsis thaliana seedlings. After treatment of the callus with cellulase and pectinase, protoplasts are inoculated with viral RNAs using polyethylene glycol. After various times postinoculation, total RNA is extracted and subjected to electrophoresis on nondenaturing agarose gels for further analysis. Stationary callus cultures are simpler to maintain than more traditional suspension cultures and yield high-quality, uniform protoplasts. Protoplasts prepared in this fashion can also be used for uptake of DNA.