Showing papers on "Protoplast published in 2012"

Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

Abstract: Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

Biotechnology in Forage and Turf Grass Improvement

Abstract: 1 Introduction.- 1.1 Agronomic Importance of the Festuca-Lolium Complex..- 1.1.1 Major Festuca Species.- 1.1.2 Major Lolium Species.- 1.2 Distribution of Fescues and Ryegrasses.- 1.3 Biotechnology in Festuca-Lolium Improvement: General Considerations.- References.- 2 Meristem Culture.- 2.1 Introduction.- 2.2 Culture of Vegetative Meristems in Festuca and Lolium.- 2.3 Culture of Floral Meristems in Festuca and Lolium.- 2.4 Meristem Culture in Other Grasses.- 2.5 Summary and Conclusions.- References.- 3 Callus Cultures and Somaclonal Variation.- 3.1 Introduction.- 3.2 Regeneration from Callus Cultures in Festuca and Lolium.- 3.3 Somaclonal Variation in Festuca and Lolium.- 3.4 Regeneration from Callus Cultures in Other Grasses.- 3.5 Summary and Conclusions.- References.- 4 Anther Culture and Production of Haploids.- 4.1 Introduction.- 4.2 Anther Culture and Haploids in Festuca.- 4.3 Anther Culture and Haploids in Lolium.- 4.4 Anther Culture in Other Grasses.- 4.5 Summary and Conclusions.- References.- 5 Plant Regeneration from Suspension Cells and Protoplasts.- 5.1 Introduction.- 5.2 Cell Suspension and Protoplast Cultures in Festuca.- 5.2.1 Plant Regeneration from Embryogenic Cell Suspensions in Festuca.- 5.2.2 Plant Regeneration from Protoplasts in Festuca.- 5.3 Cell Suspension and Protoplast Cultures in Lolium.- 5.3.1 Plant Regeneration from Embryogenic Cell Suspensions in Lolium.- 5.3.2 Plant Regeneration from Protoplasts in Lolium.- 5.4 Suspension and Protoplast Cultures in Other Grasses.- 5.5 Summary and Conclusions.- References.- 6 Somatic Hybridization.- 6.1 Introduction.- 6.2 Somatic Hybridization in Festuca and Lolium.- 6.3 Cybridization in Festuca and Lolium.- 6.4 Somatic Hybridization in Other Grasses.- 6.5 Summary and Conclusions.- References.- 7 Transgenic Plants from Protoplasts.- 7.1 Introduction.- 7.2 Direct Gene Transfer to Protoplasts in Festuca.- 7.3 Direct Gene Transfer to Protoplasts in Lolium.- 7.4 Direct Gene Transfer to Protoplasts in Other Grasses.- 7.5 Summary and Conclusions.- References.- 8 Protoplast-Independent Production of Transgenic Plants.- 8.1 Introduction.- 8.2 Protoplast-Independent Transformation in Festuca.- 8.3 Protoplast-Independent Transformation in Lolium.- 8.4 Protoplast-Independent Transformation in Other Grasses.- 8.5 Summary and Conclusions.- References.- 9 Molecular Markers.- 9.1 Introduction.- 9.2 Molecular Markers in Festuca and Lolium.- 9.2.1 Isozyme Markers.- 9.2.2 Species-Specific Repetitive DNA Sequences.- 9.2.3 RFLP Markers.- 9.2.4 RAPD Markers.- 9.3 Molecular Markers in Other Grasses.- 9.4 Summary and Conclusions.- References.- 10 Perspectives.- 10.1 Introduction.- 10.2 Forage Quality.- 10.2.1 Manipulation of Lignin Biosynthesis.- 10.2.2 Manipulation of Fructan Metabolism.- 10.2.3 Transgenic Expression of "Rumen By-pass" Proteins.- 10.3 Disease and Pest Resistance.- 10.3.1 Fungal Pathogens.- 10.3.2 Viruses.- 10.3.3 Pests.- 10.4 Growth and Development.- 10.4.1 Manipulation of Pollen Allergens.- 10.4.2 Manipulation of Flowering Time and Senescence.- 10.4.3 Manipulation of Apomixis.- 10.4.4 Manipulation of Self-Incompatibility and Cytoplasmic Male Sterility.- 10.4.5 Grasses as Bioreactors.- 10.5 Summary and Conclusions.- References.

Calcium phosphate nanoparticle mediated genetic transformation in plants

Abstract: We have produced ultra-small sized (20–50 nm diameter) calcium phosphate (CaP) nanoparticles encapsulating a reporter gene, pCambia 1301 The material in the nanoparticles is of crystalline nature having a hydroxyapatite structure as revealed from the XRD pattern The maximum loading of pCambia 1301 in the nanoparticles and the pH-dependent dissolution of CaP nanoparticles were studied using gel electrophoresis DNA is highly protected in the cell from cellular nucleases when it is encapsulated into the CaP nanoparticles The transformation efficiency was found to be about 807% compared to 544% by Agrobacterium tumefaciens and only 8% using naked DNA Our results indicate that CaP nanoparticles could be used as a better transforming vector in plants as compared to the Agrobacterium tumefaciens mediated genetic transformation technique In our experiment we presume that the plasmid DNA released from CaP nanoparticles in the cell has, perhaps, been able to enter into the nucleus Transgenic GUS (β-glucuronidase) integrates into the genomic DNA by non-homologous recombination as in the case of Agrobacterium tumefaciens infection Because of high transformation frequency, the method seems to be an attractive option for delivering a transgene into plant cells and protoplast

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

Abstract: An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 106 and 106 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.

Production of lipase from genetically improved Streptomyces exfoliates LP10 isolated from oil-contaminated soil

Abstract: Lipases (triacyl glycerol acyl hydrolase) catalyze hydrolysis and syntheses of ester formed from glycerol and long-chain fatty acids. They have many industrial applications, especially in food and detergent industries. Out of 33 bacterial isolates, a group of 20 bacterial isolates produced lipase enzyme on tributyrin agar and Tween 80 agar media. In liquid medium, the lipase activity was ranged from 1.5 to 6.9 IU/ml. Among the evaluated bacteria, the isolate LP10 that was isolated from soil collected from fuel station. It was the most active isolate in lipase production (6.9 IU/ml). Using morphological, physiological and biochemical studies, it was identified as an isolate belonging to the genus Streptomycesand identified as Streptomyces exfoliates LP10. Identification was confirmed using 16S rDNA analysis. Growth of the selected bacterium in medium containing tributyrin and Tween 60 at initial pH 6 in addition to incubation at 37°C for three days yielded the maximum lipase production. The molecular weight of the purified enzyme was 60 kDa, determined using gel electrophoresis. Improvement of lipase production was carried out between Streptomyces exfoliates LP10 and Streptomyces niveus using protoplast fusion. Five fusants were obtained. Fusant LP3 was the best lipase producer (3 times higher) compared to its parents. Key words: Lipase, Streptomyces exfoliates, tributyrin, protoplast fusion, 16S rDNA.

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Abstract: Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 106 ml−1) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 106 ml−1). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

Abstract: Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus

Abstract: A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets. Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9 % in C. intybus genotypes and efficiencies of up to 0.7 % in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5 mg l−1 indole-3-acetic acid-enriched medium (up to 87.5 % of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

Abstract: The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv Marketer Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density In contrast, cell density was significantly decreased following the addition of catalase to the medium Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation

Characteristics of fertile somatic hybrids of G. hirsutum L. and G. trilobum generated via protoplast fusion.

Abstract: Fertile somatic hybrids between tetraploid upland cotton G. hirsutum L. cv. Coker 312 and wild cotton G. trilobum were generated by symmetric electrofusion. Comparisons of morphology, combined with flow cytometric, RAPD, SRAP and AFLP analyses confirmed the hybrid nature of the regenerated plants. The hybrids differed morphologically from the parent plants. Flow cytometric analysis showed that the hybrids had DNA similar in amount to the total combined DNA content of the two parents, and the use of molecular markers revealed that the hybrids contained genomic fragments from both fusion parents, further indicating the hybrid nature of the regenerated plants. The stability of the morphological features of the hybrids was examined in following generations. The hexaploid fusion plants showed strong photosynthesis and a high expression level of some photosystem-related genes. Our results suggest that novel traits may be incorporated in cotton breeding programs through the production of somatic hybrids and the backcrossing of these plants with elite cultivars.

Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii.

Abstract: The production of a low-temperature alkalophilic lipase from Acinetobacter johnsonii was improved using genome shuffling. The starting populations, obtained by UV irradiation and diethyl sulfate mutagenesis, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 0.15 mg lysozyme/ml for 45 min at 37°C. The protoplasts were inactivated under UV for 20 min or heated at 60°C for 60 min and a fusant probability of ~98% was observed. The positive colonies were created by fusing the inactivated protoplasts. After two rounds of genome shuffling, one strain, F22, with a lipase activity of 7 U/ml was obtained.

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

Abstract: The aim of this work was to improve plating efficiency of sugar beet mesophyll protoplast cultures. Preliminary experiments showed that cultures of good quality, viable protoplasts were obtained in rich media based on the Kao and Michayluk formulation and with the calcium alginate as an embedding matrix. Nevertheless, in these cultures cell divisions were either not observed or very seldom confirming earlier reported recalcitrance of sugar beet protoplasts. The recalcitrant status of these cultures was reversed upon application of exogenous phytosulfokine (PSK)—a peptidyl plant growth factor. The highest effectiveness of PSK was observed at 100 nM concentration. Plating efficiencies obtained in the presence of PSK reached approximately 20% of the total cultured cells. The stimulatory effect of phytosulfokine was observed for all tested breeding stocks of sugar beet. Our data indicate that PSK is a powerful agent able to overcome recalcitrance of plant protoplast cultures.

Protoplast fusion enhances antagonistic activity in Trichoderma spp.

Abstract: Genus Trichoderma is one of the most important filamentous fungi used as a biocontrol agent. Because of the absence of sexual reproduction in this fungus, other methods of genetic improvement have been developed such as proptoplast fusion to enhance its bicontrol potential. Therefore the objectives of this study were; 1) protoplast fusion and regeneration of two fungicide tolerant mutants of Trichoderma viride and T. harzianum 2) using ISSR for fingerprinting of inter-specific protoplast fusants and 3) assessment of antagonistic ability of Trichoderma fusants against three soil born diseases. Protoplast was isolated from two fungicide tolerant mutants PTv-V and PTz-F of Trichoderma,. The frequency of fusion tolerant to both pesticides was about 0.3 % and eight fusants were selected for further studies. In fusant stability experiments, only five of these fusants were the result of nuclear fusion of parental cells. Molecular characterization of two stable fusants using ISSR indicated the presence of novel fragments which may be due to recombination events between parents. In dual culture biocontrol experiments, five selected fusants showed growth inhibition against three pathogens namely; Rhizoctonia solani, Fusarium oxysporum and Pythium ultimum.. However, Fus 7 indicated the best ability of inhibition the growth of the three pathogens. In greenhouse experiments Fus 7 demonstrated a great ability to reduce tomato damping off of the three pathogens in the presence of the two fungicides understudy. It was concluded that, the protoplast fusion is a powerful tool to enhance the biocontrol ability of Trichoderma sp. against diseases. (A. I. Fahmi, A. D. Al-Talhi and M. M. Hassan Protoplast fusion enhances antagonistic activity in Trichoderma sp.) Nature and Science 2012; 10(5): 100-106). (ISSN: 1545-0740). http://www.sciencepub.net.

Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion.

Abstract: To increase thermotolerance and ethanol tolerance in Saccharomyces cerevisiae strain YZ1, the strategies of high-energy pulse electron beam (HEPE) and three rounds of protoplast fusion were explored. The YF31 strain had the characteristics of resistant to high-temperature, high-ethanol tolerance, rapid growth and high yield. The YF31 could grow on plate cultures up to 47 °C, containing 237.5 g L−1 of ethanol. In particular, the mutant strain YF31 generated 94.2 ± 4.8 g L−1 ethanol from 200 g glucose L−1 at 42 °C, which was 2.48 times the production of the wild strain YZ1. Results demonstrated that the variant phenotypes from the strains screening by HEPE irradiation could be used as parent stock for yeast regeneration and the protoplast fusion technology is sufficiently powerful in combining suitable characteristics in a single strain for ethanol fermentation.

Factors affecting protoplast isolation and cultivation of Cucumis spp.

Abstract: Protoplasts of Cucumis anguria, Cucumis melo (3 accessions), Cucumis metuliferus and Cucumis sativus were isolated from leaves, growing apices, hypocotyls and calluses. Plants were cultured on 2 concentrations of sucrose. The effect of plant culture medium, explant age and explant type on protoplast viability were investigated. The protoplasts were cultured in 3 types of culture medium and at two temperatures. Optimal age range for protoplast isolation was 1-5 weeks depending on explant type and genotype. Viabilities ranging between 50 % and 80 % were obtained from all explants and genotypes. Increased concentration of sucrose had negative impact on viability of protoplasts. The highest level of regeneration achieved was callus, regenerated from leaf protoplasts of C. melo cv. ‘Charentais’ and C. melo ‘MR-1’. The lowest regeneration capability was observed in hypocotyls. Liquid LCM medium (B5 macro and microelements (1 g · l -1 CaCl 2 ), B5 vitamins with 1g· l -1 myo-inositol, 2 mg· l -1 ascorbic acid, 0.8 mg· l -1 glycine, 20 mg· l -1 glutamine, 100 mg · l -1 casein hydrolysate, 70 g · l -1 mannitol, 10 g · l -1 sucrose, 5 g · l -1 glucose, 585 mg · l -1 MES, 5.37 μmol· l -1 NAA, 2.26 μmol· l -1 2,4-D, 2.22 μmol · l -1 BA) was optimal for protoplast regeneration. Agarose-solidified medium caused a decrease in the number of cell divisions (used in C. melo ‘PI 124112’). Cultivation at 25oC resulted in a higher frequency of cell divisions (tested in C. metuliferus ).

Preparation of protoplasts from Chlorella protothecoides.

Abstract: This paper describes an enzymatic method for yielding protoplasts from the microalga Chlorella protothecoides. Four kinds of commercially available enzymes were tested. The enzymatic digestion was optimal with 2% cellulase R-10 and 1% snailase prepared in 25 mM Tris buffer (pH 6.0) containing 0.6 M D-mannitol, and the protoplast density could reach the peak after treatment at 30°C for 16 h. Nearly all liberated protoplasts were green in the presence of 0.01% phenosafranin, indicating their high viability. The regeneration rate was about 70% when 0.6 M D-mannitol was used as an osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this algal species.

A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae.

Abstract: Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1–12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.

Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species

Abstract: By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)—two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 106/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2–41 additional ones indicating elimination of C. persicum chromosomes.

FITC delivery into plant cells using magnetic single-walled carbon nanotubes.

Abstract: In this paper, fluorescein isothiocyanate (FITC) was covalently bonded with magnetic single-walled carbon nanotubes (mSWCNTs) that were purified using our previous method. To demonstrate our design, mSWCNT-FITC was delivered into plant cells (canola and carrot cells) driven by external magnetic forces. From FACS results, the FITC delivery efficiency was about 100% for both two canola and carrot protoplasts, which were further confirmed by the confocal and sectional TEM images. Some mSWCNTs were found trapped both inside the endosomes of canola protoplast and outside endosome near the nuclear membrane of carrot protoplast according to the sectional TEM images. All results showed that mSWCNT is a good delivery carrier for biomolecules.

Development of a transformation method for the nematophagous fungus Dactylellina cionopaga

Abstract: Dactylellina cionopaga is a trapping fungus that produces adhesive columns and a two-dimensional network. The factors that influence protoplast preparation and regeneration of D. cionopaga were analyzed, and poly ethylene glycol (PEG)-CaCl 2 - or Agrobacterium tumefaciens -mediated transformation was conducted to develop a transformation system for the fungus and provide a tool for studying the function of nematode infection-related genes. The results indicate that between 4.175±1.025×10 6 and 3.08±1.4×10 7 , protoplasts/ml were obtained under optimized conditions and that the protoplasts could be regenerated on potato dextrose agar (PDA), RA and IM regeneration media. D. cionopaga transformation using PEG-CaCl2 or A. tumefaciens displayed 4.2 to 11 resistant colonies/μg DNA using 10 6 protoplasts and 180-270 resistant colonies using 10 6 conidia. Molecular analysis and microscopy of randomly selected transformants showed that the target genes were integrated into the genome of D. cionopaga and that green fluorescence could be detected in transformants containing pK2-BarGFP, which carried a glufosinate ammonium resistance gene and the enhanced green fluorescence protein gene. The methods used in this study for protoplast preparation and convenient Agrobacterium -mediated transformation of D. cionopaga represent useful tools for genetic research on this nematophagous fungus. This is the first report on protoplast generation and transformation of D. cionopaga. Key words: Nematophagous fungi, Dactylellina cionopaga, Agrobacterium tumefaciens- mediated transformation, PEG-CaCl2-mediated transformation, protoplast preparation and regeneration.

Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

Abstract: For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant with desired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluate various combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Lilium ledebeourii Bioss. The results of experiment revealed that the protoplast yield was significantly affected by different treatment levels. Cellulase at 4% gave the highest numbers of protoplasts at 3.71×10 5 protoplast/g FW. Pectinase at 1% gave the highest numbers of protoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated that concentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at 0.2% for 24 h gave the highest viability. Interactions of cellulase × pectinase, cellulase × time, pectinase × time and cellulase × pectinase × treatment time were significant at P≤0.05 for protoplast number. The highest and lowest protoplast numbers were produced in media containing 4% cellulase and 1% pectinase for 24 h (6.65×105 protoplast/g FW) and 1% cellulase and 0.2% pectinase for 12 h, respectively. It's concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.

RESEARCH PAPER Protoplast fusion of Rhizopus oryzae and Rhizopus microsporus for enhanced fumaric acid production from glycerol

Abstract: Rhizopus oryzae and Rhizopus microsporus strains were screened for their ability to produce fumaric acid on glycerol as the sole carbon source in the medium. After seven days of stationary culture, fumaric acid was assayed by HPLC analysis, and maximum concentrations of 0.3% (w/v) and 0.33% (w/v) were recorded. Protoplast fusion was used to improve fumaric acid production. A selective medium for the fusant culture was composed on the basis of biochemical differences between parental strains, as examined using the Biolog FF MicroPlate TM Fungi Identification Test. Double fusion rounds led to a 1.46-fold increase in fumaric acid productivity relative to the parental strains. Individual Rhizopus fusants demonstrated a various ability to produce fumaric acid from 2.0% (w/v) of glycerol, with the most effective ones producing from 0.2 to 0.27 g@ g !1 of this acid. To date, no studies have been carried out to improve fumaric acid production by Rhizopus with the use of glycerol as the only carbon source in the medium.

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Abstract: Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.

Electrically Induced Protoplast Fusion UsingPulse Electric Fields forDielectrophoresi s1

Abstract: Theformation ofprotoplast chains insuspensions ofisolated pea(Pisum sativum cvRan1)mesophyll protoplasts induced by electric fields wasstudied. Thechain formation induced byasinewavefield (2V,peaktopeak; 500-0.1 kHz)wascompared tothat induced byanalternating pulse field (1V,amplitude; 0.1-0.4 kHz). Anincreased number ofdielectrophoretically paired protoplasts, formation ofprotoplast chains inthepresence ofCaCI2 upto5 mM,andprotoplast fusion inthepresence of3mmCaC12 were found whenthepulse field wasapplied. Thepresent results suggest thepossibility ofelectrically induced protoplast fusion atcation concentrations thatprevent fusion whensine-wave fields are applied.

Protoplast isolation and culture for somatic hybridisation of rapid cycling Brassica rapa with ‘Anand’ CMS and Brassica juncea

Abstract: Protoplasts were isolated from the young leaves of rapid cycling Brassica rapa and cotyledons and hypocotyls of 10-day-old Brassica juncea seedlings. Protoplasts were fused by 40% polyethylene glycol and cultured in modified K8p medium supplemented with 2.5 mg·l−1 isopentenyladenine (2ip), 0.5 mg·l−1 naphthaleneacetic acid, 1 mg·l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg·l−1 zeatin, 1% dimethyl sulfoxide, and 0.4 M mannitol as osmoticum. After 3 days of initial culture, 3 different culture methods were employed and evaluated. The highest plating efficiency (1.97%) was obtained with a semi-solid agarose embedding culture method. Both shoots and somatic embryos formed from protoplast culture-derived calli. The somatic embryos were derived from asymmetrically divided calli that developed progressively into deep-purple heart shapes as well as the early-torpedo and bipolar stages to finally form complete plantlets. Thirteen putative somatic hybrids were produced via somatic embryogenesis or organogenesis. Random amplified polymorphism DNA analysis was performed to identify somatic hybrids. Six regenerated plants had a chromosome number of 2n = 56 the same as the sum of B. juncea (2n = 36) and B. rapa (2n = 20) chromosomes; 2 plants had a chromosome number of 2n = 54. These regenerated plants exhibited morphology intermediate to those of their parents. The flowers of somatic hybrids exhibited a range of variation; some were normal, while others were abnormal. No pollen was produced from regenerated plants. Two plants had crinkled petal-like stamens.

Preparation, purification and regeneration optimizing research of protoplasts from Rhizoctonia solani

Abstract: The aim of this work was to study the standardization of conditions to obtain and regenerate protoplasts of Rhizoctonia solani. The optimal way of harvesting 1.67 × 109protoplasts per gram from Rhizoctonia solani was to deal with 12 h aged mycelium in 0.6 M MgSO4 stabilizer (pH 5.2) combining cellulase and driselase at 35°C for 3 h. Meanwhile, the most suitable condition for protoplast regeneration had been attained. The condition was to deal with 16 h aged mycelium in 0.6 M MgSO4 stabilizer (pH 5.2) combining cellulase with driselase at 35°C for 2.5 h, and incubation at 26°C in the regeneration medium. Furthermore, a two-phase system of 0.6 M sucrose and 0.6 M mannitol was developed for separation and purification of intact protoplasts from broken protoplasts, excellent results were obtained. There was a breakthrough for protoplast technology of R. solani and other fungus was recorded in this paper. Key words: Preparation, protoplast, purification, regeneration, Rhizoctonia solani

Protoplast production and isolation from Etlingera elatior

Abstract: The technique of hybridization using plant protoplasts is widely used in plant breeding programs. The purpose of our study is to further characterize the process of protoplast isolation from the ornamental species Etlingera elatior (Jack) R. M. Smith. Protoplasts were isolated from different tissues: in vitro leaves, in vitro pseudostem, and leaves from plants cultivated hydroponically. We tested six enzymatic combinations, four incubation time periods, the rotary system (40 rpm) or steady in the dark, and three concentrations of mannitol (0.5, 0.6 and 0.7 M). The diameter and viability of obtained protoplasts were evaluated. The best source of explants used for protoplast isolation was the in vitro leaves, which yielded 22x105 protoplasts g-1 of fresh matter. The optimal incubation period was 15 hours. The in vitro leaves presented a greater viability (96%) and larger protoplasts (36.7 µm diameter). Greater yields were obtained using a rotatory system with protoplasts incubated in the dark. The best enzymatic combination was 3% Cellulase “Onozuca” R-10 + 2% Meicelase + 1% Driselase + 1% Dextran + 5 mM MES, followed by the addition of 0.6 M mannitol.

The influence of isolation stress on the (re)organization of cell walls in protoplasts of in vitro recalcitrant plants

Abstract: A study of cell-wall regeneration was conducted on protoplast cultures of four recalcitrant plant species: yellow lupin and grasspea (grain legumes), and also hyacinth and asparagus (ornamental monocots). The rate of cell wall resynthesis was investigated together with the arrangement of cellulosic fibers on the surface of protoplast-derived cells. Localization of arabinogalactan proteins in cell walls was also performed. Quick cell wall renewal occurred in grasspea and hyacinth cultures, where the percentage of protoplast-derived cells after 24h of culture accounted for 45-50%. In contrast, the presence of cell walls in half of the population was not observed in asparagus and lupin cultures until 5 and 7 days later, respectively. Grasspea, hyacinth and asparagus cells budded intensively. Moreover, the cellulosic material of the cell wall was disorganized and unevenly distributed in lupin, asparagus and hyacinth. In all the tested species arabinogalactan proteins (AGPs) were detected mainly in spherical cells with condensed cytoplasm, but were absent in the budding cells. Notwithstanding the observed differences among species, we concluded that incorrect cell wall structure/composition, together with a deregulated cell cycle might contribute to protoplast recalcitrance in the examined plants. Abnormalities could be a result of the isolation process itself and defective stress-recovering mechanisms. Subsequently, these could arrest regeneration competences and direct cells at apoptotic pathways.