Showing papers on "Protoplast published in 2015"
TL;DR: The results confirm the better tolerance of these hybrids to salt stress when compared to their BF15 parent, and an improved control of Na+ accumulation and a stable K+/Na+ ratio.
Abstract: Salinity is one of the major stresses threatening potato plants (Solanum tuberosum L.) by affecting their growth and yield. It leads to oxidative stress by the production of reactive oxygen species responsible for alteration of macromolecules. To improve the tolerance of potato to salt stress, we have used somatic hybridization to produce interspecific potato hybrids by protoplast fusion between the BF15 variety and the wild Solanum berthaultii species. These hybrids showed an improved tolerance to salt stress when cultivated in vitro. The present work aims to analyze the response of the hybrids to salt stress in greenhouse conditions. Thus, the development of plants and their antioxidant capacity in response to salt stress were followed. All hybrids showed better growth and stable chlorophyll content compared to those of the BF15 parent plant. Membrane lipid peroxidation, evaluated by measuring the malondialdehyde accumulation (MDA) in plant organs, showed low levels in the hybrids. Higher antioxidant enzyme activities were measured in the roots of the hybrids when compared to those of the BF15 parent. These hybrids also showed an improved control of Na+ accumulation and a stable K+/Na+ ratio. These results therefore confirm the better tolerance of these hybrids to salt stress when compared to their BF15 parent.
60 citations
TL;DR: Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis and Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.
Abstract: Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 × 105 protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.
37 citations
TL;DR: It is suggested that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism and potentially produce peptides with antimicrobial activity from originally functional proteins.
Abstract: Protein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process. We thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation “burst”, with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity. We suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide “burst” is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.
36 citations
TL;DR: The potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes is demonstrated.
Abstract: Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein–protein interaction, and the investigation of PCD-related processes.
Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein–protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.
36 citations
TL;DR: Analysis of antioxidant levels, CAT activity and CAT isoform 3 mutants indicate that peroxisome-localized CAT plays a major role in restoring ROS homeostasis.
Abstract: The ability to induce Arabidopsis protoplasts to dedifferentiate and divide provides a convenient system to analyze organelle dynamics in plant cells acquiring totipotency. Using peroxisome-targeted fluorescent proteins, we show that during protoplast culture, peroxisomes undergo massive proliferation and disperse uniformly around the cell before cell division. Peroxisome dispersion is influenced by the cytoskeleton, ensuring unbiased segregation during cell division. Considering their role in oxidative metabolism, we also investigated how peroxisomes influence homeostasis of reactive oxygen species (ROS). Protoplast isolation induces an oxidative burst, with mitochondria the likely major ROS producers. Subsequently ROS levels in protoplast cultures decline, correlating with the increase in peroxisomes, suggesting that peroxisome proliferation may also aid restoration of ROS homeostasis. Transcriptional profiling showed up-regulation of several peroxisome-localized antioxidant enzymes, most notably catalase (CAT). Analysis of antioxidant levels, CAT activity and CAT isoform 3 mutants (cat3) indicate that peroxisome-localized CAT plays a major role in restoring ROS homeostasis. Furthermore, protoplast cultures of pex11a, a peroxisome division mutant, and cat3 mutants show reduced induction of cell division. Taken together, the data indicate that peroxisome proliferation and CAT contribute to ROS homeostasis and subsequent protoplast division induction.
34 citations
TL;DR: These interspecific potato somatic hybrids with diverse cytoplasm background have potential to be employed in potato breeding programmes to widen the cultivated gene pool through conventional and molecular methods.
Abstract: Solanum cardiophyllum Lindl. (PI 341233) (2n = 2x = 24, 1EBN) is a diploid wild potato species highly resistant to late blight, the most serious disease of potato. However, it poses a problem of sexual incompatibility with common potato. So to circumvent this problem, in this study, we developed interspecific potato somatic hybrids between cultivated S. tuberosum dihaploid C-13 and wild species S. cardiophyllum via protoplast fusion. Out of 26 regenerants, only 4 were confirmed as true somatic hybrids containing both parental genomes based on molecular markers and phenotypes. Somatic hybrids were identified by RAPD, ISSR, SSR, AFLP and cytoplasm (chloroplast and mitochondrial genomes) type molecular markers. Intermediate phenotypes of somatic hybrids were confirmed by leaf, flower and tuber traits. Late blight resistance of the hybrids was assessed by challenge inoculation of P. infestans under controlled conditions. Somatic hybrids were found tetraploid by flow cytometry, exhibited high pollen stainability by acetocarmine staining and formed berries and viable seeds after crossing with a common potato variety. Somatic hybrids possessed diverse cytoplasm types (W/α, W/γ and T/β) as assessed by chloroplast and mitochondrial genome-specific markers. Further, cluster analysis based on the Jaccard’s coefficient of molecular profiles generated by all above markers showed genetic distinctness in somatic hybrids and parents. Taken together, these interspecific somatic hybrids with diverse cytoplasm background have potential to be employed in potato breeding programmes to widen the cultivated gene pool through conventional and molecular methods.
31 citations
TL;DR: A modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana is described, which significantly simplifies and speeds up functional studies without compromising the yield and the viability of protplasts.
Abstract: The protoplast transient assay system has been widely used for rapid functional analyses of genes using cellular and biochemical approaches. This system has been increasingly employed for functional genetic studies using double-stranded (ds) RNA interference (RNAi). Here, we describe a modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana. This modification significantly simplifies and speeds up functional studies without compromising the yield and the viability of protoplasts. We also present the procedure for the isolation and transfection of protoplasts from mesophyll cells of an emerging model grass species, Brachypodium distachyon. Further, we detail procedures for RNAi-based functional studies of genes using transient expression of in vitro synthesized dsRNA in protoplasts.
24 citations
TL;DR: In this paper, a haploid strain of Ustilago esculenta was found to produce higher yields of protoplasts when treating with 15 mg−mL−1 lywallzyme in a sucrose-containing solution at 30°C for 3 h.
Abstract: Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia , resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia . In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL−1 lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta .
24 citations
TL;DR: Responses to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplast.
23 citations
TL;DR: This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protopleft fusion or genome editing technologies.
Abstract: This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 μM 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 × 106 protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 μM thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 μM GA3. The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.
20 citations
Journal Article•
TL;DR: This work addresses the question, if somaclonal variation may be observed in these grapevine regenerants, and chooses RAPD-PCR (WILLIAMS et al. 1993) as a method permitting easy and rapid screening for genetic differences.
Abstract: Introduction: Recently regeneration of plants from grapevine protoplasts (Vitis sp. \"Seyval blanc\") has been reported for the first time (REUSTLE et al. 1995). This way of regeneration involves prolonged tissue culture with the intrinsic potential of provoking somaclonal variation in other plant species (LARKIN and ScowcRAFT 1981). We wanted to address the question, if somaclonal variation may be observed in these grapevine regenerants. Hence RAPD-PCR (WILLIAMS et al. 1993) was chosen as a method permitting easy and rapid screening for genetic differences. This technique was applied using 60 different lOmer primers in PCR amplifications on 47 grapevine plants, each one regenerated from a single protoplast (protoclone ).
TL;DR: Although the effect of PVP on plating efficiency was not clear, the formation of microcalli from grapevine protoplasts was remarkably improved when adding PVP at ''day 0'' or ''day 7'' of cultivation.
Abstract: The effect of the addition of polyvinylpyrrolidone (PVP-40) to grapevine protoplast cultures ( Vitis sp. cv. Vidal blanc) at different stages of development (day 0, 7 and 14 of cultivation) and the influence of activated charcoal (AC) in several concentrations (0, 0.1, 0.5 and 1.0 %) on protoplast growth were studied. Both additives were investigated using different modified culture media (CPW-13, V/KM, MS-P). The application of 0.5% PVP could not prevent browning of the culture media but reduced it to a low level. Although the effect of PVP on plating efficiency was not clear, the formation of microcalli from grapevine protoplasts was remarkably improved when adding PVP at ''day 0'' or ''day 7'' of cultivation. AC could prevent browning of the media in all concentrations tested but at the same time inhibited plating efficiency. Conversely, formation of microalli occurred only with 0.5 and 1.0% AC, but at very low frequencies. A superior suitability of one of the culture media tested for grapevine protoplasts culture could not been found.
01 Jan 2015
TL;DR: A protoplast-based transformation system for this unique fungal species U. esculenta is reported, which was found to produce higher yields of protoplasts when treating with 15 mg mL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h.
Abstract: Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL −1 lywallzyme in a sucrose-containing solution at 30 ◦ C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta.
TL;DR: This protoplast transient expression system is suitable for studying gene expression, protein localization, and protein–protein interactions in woody plants, and would be particularly useful for studying the signaling pathway involved in the salt tolerance of P. euphratica in a homologous system, which may not even be possible using protoplasts prepared from other species.
Abstract: Populus euphratica Oliv., a species of the model woody plant genus Populus, is well known for its tolerance to salinity stress, the underlying mechanism of which is a research hotspot. Transient expression of fluorescent fusion proteins is commonly used for rapid assessment of gene functions and interactions, and thus would be useful to study the genes involved in salt tolerance in this species. Our transient gene expression protocol for P. euphratica included a simple protoplast preparation and transformation procedure from suspension cultured cells. The highest protoplast yield (8 × 107 g−1 fresh weight) with high viability (above 90 %) was obtained using an optimized enzyme mix of 4 % (w/v) cellulase R10, 0.5 % (w/v) pectinase, and 0.2 % (w/v) hemicellulase. Factors affecting protoplast transformation efficiency were also optimized: 20 μg plasmid DNA versus 105 protoplasts, and a transformation time of 20 min using PEG, which resulted in a transformation efficiency greater than 50 %. A pair of known markers was simultaneously and correctly expressed in the same P. euphratica protoplasts by co-transformation. The isolation and transformation protocol took 5 h, and results could be obtained within 24 h. This protoplast transient expression system is suitable for studying gene expression, protein localization, and protein–protein interactions in woody plants. In addition, it would be particularly useful for studying the signaling pathway involved in the salt tolerance of P. euphratica in a homologous system, which may not even be possible using protoplasts prepared from other species.
TL;DR: The hybrid varieties with superior traits developed in this study unequivocally demonstrate the significance of protoplast fusion technique in developing improved varients of macroalgae.
Abstract: Ulvophycean species with diverse trait characteristics provide an opportunity to create novel allelic recombinant variants. The present study reports the development of seaweed variants with improved agronomic traits through protoplast fusion between Monostroma oxyspermum (Kutz.) Doty and Ulva reticulata Forsskal. A total of 12 putative hybrids were screened based on the variations in morphology and total DNA content over the fusion partners. DNA-fingerprinting by inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) analysis confirmed genomic introgression in the hybrids. The DNA fingerprint revealed sharing of parental alleles in regenerated hybrids and a few alleles that were unique to hybrids. The epigenetic variations in hybrids estimated in terms of DNA methylation polymorphism also revealed sharing of methylation loci with both the fusion partners. The functional trait analysis for growth showed a hybrid with heterotic trait (DGR%= 36.7±1.55%) over the fusion partners U. reticulata (33.2±2.6%) and M. oxyspermum (17.8±1.77%), while others were superior to the mid-parental value (25.2±2.2%) (p<0.05). The fatty acid (FA) analysis of hybrids showed notable variations over fusion partners. Most hybrids showed increased polyunsaturated FAs (PUFAs) compared to saturated FAs (SFAs) and mainly includes the nutritionally important linoleic acid, α-linolenic acid, oleic acid, stearidonic acid, and docosahexaenoic acid. The other differences observed include superior cellulose content and antioxidative potential in hybrids over fusion partners. The hybrid varieties with superior traits developed in this study unequivocally demonstrate the significance of protoplast fusion technique in developing improved varients of macroalgae.
TL;DR: Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste shows promising results in terms of protoplast-to-fusant fusion and application.
Abstract: Aims
Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste.
Methods and Results
The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml−1 lysing enzymes from T. harzianum, 0·06 mg ml−1 β-glucuronidase from Helix pomatia and 1 mg ml−1 purified Penicillium ochrochloron chitinase in 0·8 mol l−1 sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE).
Conclusions
This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification.
Significance and Impact of the Study
Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.
TL;DR: In order to develop a high - efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum, incubation time, plant material type and age were studied.
Abstract: In order to develop a high - efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum , incubation time, plant material type and age were studied. Protoplasts were successfully isolated from leaves of four week - old in vitro grown plants using an enzyme mixture comprising of 2% (w/v) Cellulase Onozuka R - 10, 1.5% (w/v) Macerozyme Ono zuka R - 10 , 0.2% (w/v) Driselase and 0.1%(w/v) Pectolyase Y 23 in 0.5 M mannitol, 2.5 mM CaCl 2 .2H 2 O and 5 mM 2 (N - morpholino) - ethanesulfonic acid (MES)
TL;DR: The highest electroporation efficiency was determined by evaluating the highest protoplast survival rate under specific physical conditions and the best results were achieved in the presence of EB1 electroporated buffer.
Abstract: This article presents our further in vitro studies into the morphogenic potential of gentian cells, organs, and tissues after modification of their genome. The objective was to study the effect of electroporation and the introduction of foreign genes on the morphogenic potential of Gentiana kurroo embryogenic cell suspension protoplasts. Protoplasts were electroporated with DNA plasmids carrying nptII and bar genes. The stability of cell membranes, the contents of electroporation buffer, the length of electric pulse, the number of pulses and the strength of the electric field were studied. We determined the highest electroporation efficiency by evaluating the highest protoplast survival rate under specific physical conditions. The best results were achieved in the presence of EB1 electroporation buffer where the viability of protoplasts was 70.1%. Protoplast survival at this higher level required culture temperatures near 0EC, and a 20 μs electric pulse with an electric field of 1.0 kV/cm. After seven days of agarose embedded protoplast culture, a selective agent – kanamycin – was introduced to the medium. The cell transformation effect was improved by a long term culture of callus, regenerated somatic embryos and transformants in the presence of 50 mg/l kanamycin.
TL;DR: Osmolarity was the primary determinant for protoplast yield and viability, irrespective of osmoticum type and K was more effective than BAP in shoot regeneration, but due to synergistic blending the response was highest when both were present.
Abstract: A protocol was established for plant regeneration from leaf protoplasts of guava (Psidium guajava L.) using mixture-amount (concentration) experiments. A protoplast yield of 3.7 × 106 (viability >90 %) was obtained when 1 g leaf strips were digested in a solution of ∼0.75 M osmoticum with 6 % (w/v) enzyme containing cellulase: macerase: hemicellulase as proportion of ∼0.4: 0.5: 0.1. Protoplasts developed the maximum number of microcalli using 1.0 mg l−1 α-naphthaleneacetic acid (NAA). Maximum shoot formation (>12) via organogenesis from resulting calli was obtained using >3.4 mg l−1 PGRs containing kinetin (K): 6-benzylaminopurine (BAP) at a ratio of 0.6:0.4. Shoots were rooted in medium containing indole-3-butyric acid and plantlets were successfully acclimatized. Results of polynomial response models revealed that: (1) Osmolarity was the primary determinant for protoplast yield and viability, irrespective of osmoticum type; (2) Most of the variation in protoplast yield was driven by macerase concentration; (3) Protoplast viability was driven mainly by cellulase concentration; (4) NAA was superior to BAP for callus induction, an antagonistic proportional effect was observed when they were blended; and (5) K was more effective than BAP in shoot regeneration, but due to synergistic blending the response was highest when both were present. Overall, guava was amenable to protoplast culture and the mixture-amount design effectively characterized this protoplast system.
TL;DR: Cell wall formation in several-nucleate pollen grains of Hordeum vulgare anthers cultured in vitro was initiated at the intine and grew centripetally and branched, dividing pollen protoplast into a several-celled embryoid.
Abstract: Cell wall formation in several-nucleate pollen grains of Hordeum vulgare anthers cultured in vitro was initiated at the intine. The walls grew centripetally and branched, dividing pollen protoplast into a several-celled embryoid.
TL;DR: About twice as many protoplasts were produced from different species of fungi by involvement of P. ochrochloron chitinase than with combined commercial enzymes.
TL;DR: The grapevine was among the first plants to be cultured in vitro (1944) and Regeneration by somatic embryogenesis and organogenesis was reported in the 1970s and plantlet production from cell suspensions or callus is now a routine procedure in many laboratories.
Abstract: The grapevine was among the first plants to be cultured in vitro (1944). Regeneration by somatic embryogenesis and organogenesis was reported in the 1970s and plantlet production from cell suspensions or callus is now a routine procedure in many laboratories. Methods for isolating grapevine protoplasts have yet to be achieved. The fragmented apex technique, involving high-frequency adventitious bud formation, is a novel and efficient method for rapid multiplication of grapevines but culture of anthers and pollen has been generally unsuccessful. Micropropagation procedures for vinifera grapes, Vitis species and interspecific hybrids, including rootstocks, are all available. Seedless-seedless hybridization, involving embryo rescue in crosses with stenospermocarpic female parents, is of major significance in breeding seedless table grapes. There has been substantial progress in protoplast cell, tissue and organ culture of grapevines, but this technology is still less well developed than with some other fruit crops (notably citrus and apples). So far, tissue culture has little impact on genetic improvement. Exploitation of somaclonal variation for clonal selection is an attractive option for premium wine cultivars. There is evidence of somaclonal variation in vitro but the usefulness of this random genetic variation in viticulture is still uncertain. To date, results of field trials with vines from somatic embryos have been disappointing. The grapevine is proving to be a difficult subject for Agrobacterium -mediated genetic transformation ( A. tumefaciens and A. rhizogenes ) and microprojectile technology is another option which is being investigated.
TL;DR: From the present study, it is recommended to recommend driselase and lysing enzyme in 1 M KCl protoplasting medium and germinated conidia of F. verticillioides for the maximum production of protoplasts and 1.2 M sucrose is the best osmoticum for the regeneration of protoplast.
01 Jan 2015
TL;DR: Two major protoplast sources were evaluated in gentians, namely differentiated leaf mesophyll cells and undifferentiated callus/cell suspensions, and Totipotency exhibited by gentian protoplasts was exploited to create three different somatic hybrid combinations.
Abstract: During the last three decades, less than fifteen papers have described the results of scientific investigations in the field of gentian protoplast technology and somatic hybridization. Despite rather limited research already done on this subject, several important goals have been achieved. Protoplast-to-plant systems have been developed either for leading ornamental species or for specific medicinal plants. Two major protoplast sources were evaluated in gentians, namely differentiated leaf mesophyll cells and undifferentiated callus/cell suspensions. Plant regeneration proceeded by the two different pathways of shoot organogenesis or somatic embryogenesis. Some examples of somaclonal variation at the ploidy level were demonstrated within the pool of protoplast-derived regenerants. Totipotency exhibited by gentian protoplasts was exploited to create three different somatic hybrid combinations: intergeneric Swertia mussotii (+) Bupleurum scorzonerifolium , and interspecific Gentiana kurroo (+) G. cruciata and G. cruciata (+) G. tibetica.
TL;DR: An efficient protocol for plant regeneration was developed from protoplasts of Gentiana macrophylla Pall through somatic embryogenesis and revealed a low variation among regenerants.
Abstract: An efficient protocol for plant regeneration was developed from protoplasts of Gentiana macrophylla Pall. through somatic embryogenesis. Viable protoplasts were isolated from cell suspensions derived from young seedling leaves in an enzyme solution containing 2 % Cellulase Onozuka R-10, 0.5 % Macerozyme R-10, 0.5 % Hemicellulase, and 0.4 M sorbitol with a yield of 6.2 × 106 protoplasts g−1 fresh weight. Liquid, solid–liquid double layer (sLD) and agar-pool (aPL) culture systems were used for protoplast culture. The frequency of protoplast cell divisions and colony formations in aPL culture were significant (p < 0.05) higher than those in liquid and sLD cultures. The highest division frequency and plating efficiency were 37.7 % and 16.5 %, respectively, in aPL culture supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.5 mg l−1 6-benzylaminopurine (BA). Protoplast-derived microcalli obtained from aPL culture system were transferred to solid MS medium with a reduced concentration of 2,4-d (0.5 mg l−1) to promote the formation of embryogenic calli. Somatic embryos developed into plantlets on MS medium supplemented with 2 mg l−1 BA at a rate of 51.3 %. RAPD analysis of G. macrophylla revealed a low variation among regenerants.
Journal Article•
TL;DR: It is demonstrated that grapevine protoplast regeneration is possible using embryogenic tissue induced on leaf disks of Vitis vinifera L. cv.
Abstract: The availability of a protoplast-to-plant system would offer new perspectives for breeding of disease resistant grapevines. Grapevine, known to be recalcitrant for tissue culture experiments as many other perennial species, has been successfully regenerated from nodes and meristems from a wide range of varieties, whereas the regeneration of anthers, ovules, or leaf and petiole tissue (STAMP and MEREDITH 1988, CHENG and REISCH 1989, MARTINELLI et al. 1993) is restricted to few genotypes. Although intensive efforts have been made during the last decade to develop a regeneration protocol for grapevine protoplasts, only cell division and formation of microcalli could be achieved (LEE and WETZSTEIN 1988, Mn et al. 1991, REusTLE and ALLEWELDT 1990, U1 et al. 1990). In our recent experiments, we could now demonstrate that grapevine protoplast regeneration is possible. . Materials and methods: For protoplast isolation, embryogenic tissue (somatic embryos in different stages of development), which has been induced on leaf disks of Vitis vinifera L. cv. Seyval blanc (HARsT and ALLEWELDT 1994) was used as donor tissue. The isolation protocol was carried out according to REUSTLE and NATTER (1994). After over-night digestion and subsequent purification, the derived protoplasts were resuspended in 0.65 M mannitol (pH 5.8). Protoplast density was adjusted to 0.5 x 106 cells per ml mannitol. For cultivation, protoplasts were immobilized in Na-alginate (2 %) applying a modified method of KARESCH et al. (1991). Protoplasts, embedded in the alginate layers ( density of 0.25 x 10 per ml) were transferred into petri dishes (5 cm 0) with 2 ml of several media and cultivated at 24-26 oc in the dark. As culture media a modified CPW-13 medium (FREARSON et al. 1973) with 0.54 M glucose and NN-69 medium (NITSCH and NITSCH 1969) with 0.6 M glucose, were used for the initial cultivation step. Each medium was supplemented with 1.0 % PVP-40. During the initializing period, two hormone treatments i) 1 ppm 2,4-D and 0.5 ppm BAP or ii) 4.0 ppm NOA and 0.9 ppm TDZ were tested. As control treatment, a hormone-free variant was used. After 4 weeks, alginate layers were transferred to hormone-free NN-69 medium (for 4 weeks) containing 0.4 M glucose and 1 % PVP-40, followed by a further subcultivation in the same medium but with reduced glucose concentration (0.2 M).
Journal Article•
TL;DR: Polyethylene glycol (PEG)-mediated protoplast fusion between Pleurotus florida and Lentinus squarrosulus successfully developed twelve pfls somatic hybrids using double selection screening method, proved by the colony morphology, mycelial growth, hyphal traits, fruit body parameters and RFLP profiling of rRNA-ITS region.
Abstract: Polyethylene glycol (PEG)-mediated protoplast fusion between Pleurotus florida and Lentinus squarrosulus successfully developed twelve pfls somatic hybrids using double selection screening method. Hybridity was proved by the colony morphology, mycelial growth, hyphal traits, fruit body parameters and RFLP profiling of rRNA-ITS region. ANOVA is used for phenotypic data analysis of the pfls hybrids and parents. Out of twelve, only six pfls hybrid could produce basidiocarp on paddy straw substrate in sub-tropical climate and showed phenotypic resemblance to the P. florida parent. PCA analysis showed maximum positive correlation between each phenotypic variable for all strains examined in which the highest influential variables were found between cell width and yield. The amplified rRNA-ITS bands were ranged from ~ 620 bp to 700 bp in size in all the samples tested. Restriction enzymes AluI, HinfI, HpaII and HaeIII were found to be highly polymorphic after digestion of rRNA- ITS products for all the pfls hybrids and parents. A total of 96 RFLP fragments were was produced for all in which HaeIII showed the maximum (34) and HpaII showed the minimum (16) restriction fragments in gel. The digested fragments were ranged from ~ 160 bp to 680 bp in size.
TL;DR: Examination of respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic and non-photosynthetic cells from fully expanded leaves of warm-developed and cold-developed broad bean indicates that epidermal cells make a small but significant contribution to overall leaf respiration which increases twofold in the cold.
Abstract: In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.
Patent•
11 Nov 2015
TL;DR: In this paper, a Dichotomomyces cejpii FS110 protoplast and a preparation and conversion method thereof were revealed, and a preliminary foundation was laid for establishing the genetic operation system of Dichotomyomyces cepsi FS110 and promoting the metabolic engineering reconstruction.
Abstract: The invention discloses a Dichotomomyces cejpii FS110 protoplast and a preparation and conversion method thereof. Enzymolysis is conducted on Dichotomomyces cejpii FS110 mycelium through a mixed enzyme system with lywallzyme, cellulose and helicase so that the Dichotomomyces cejpii FS110 protoplast can be obtained. With hygromycin B as the screening mark, a carrier pAN7-1 with an external gene can be introduced into the Dichotomomyces cejpii FS110 protoplast through the protoplast conversion method, and therefore a genetic operation system of the Dichotomomyces cejpii FS110 protoplast can be established, and a preliminary foundation is laid for establishing the genetic operation system of Dichotomomyces cejpii FS110 and promoting the metabolic engineering reconstruction of the Dichotomomyces cejpii FS110, and finally the yield and types of high-activity metabolites in the Dichotomomyces cejpii FS110 are increased through the genetic engineering means.
TL;DR: The effective use of the reporter gene gfp was reported to confirm the vigorous growth as a reproducible selection for the somatic hybrids between Solanum tuberosum dihaploid line and the wild species S. chacoense, confirming that vigorous growth can be used to select the fusion products between these two species.
Abstract: Somatic hybridization based on protoplast fusion is a biotechnological tool enabling multiple resistance genes or traits to be transferred from wild relatives into crops. A key step for successful regeneration of hybrids is the selection of the heterokaryons. Selection can be done on specific culture media, by using intrinsic cellular or fluorescent markers, or specific traits of the fusion products, such as vigorous growth. More vigorous growth is one of the most common used criteria, since it does not require the use of mutants and allows faster selection of putative somatic hybrids. However, vigorous growth does not occur in all fusion combinations and often additional approaches have to be used (molecular markers, flow cytometry, chromosome counts, etc.) which are more costly and time consuming. There is, therefore, a need for the development of a faster, simpler, more cost-effective and reliable method for the selection of the heterospecific fusion products. Our study reports the effective use of the reporter gene gfp to confirm the vigorous growth as a reproducible selection for the somatic hybrids between Solanum tuberosum dihaploid line and the wild species S. chacoense. In vitro shoots of transgenic S. chacoense expressing the reporter gene gfp and a wild type dihaploid line of potato were used to isolate the mesophyll protoplasts. Electrofusion of mesophyll protoplasts and standard culture methods were applied. The S. chacoense gfp+ protoplasts were unable to regenerate and when parental protoplast and putative somatic hybrid cultures were monitored, the GFP was expressed and visualized microscopically only in hybrid regenerants showing vigorous growth. The results confirmed that vigorous growth can be used to select the fusion products between these two species. This strategy might be applied to other fusion combinations to improve somatic hybrid selection.