Protoplast Fusion Research Articles

Ontogenesis of the anastomosed laticifers of Allamanda cathartica (Apocynaceae) and the chemical nature of the stem latex.

Laticifers are secretive structures with important roles in controlling abiotic and biotic stress through the external release of viscous and bioactive latex emulsions composed of alkaloids, terpenes, flavonoids, proteins, and mucilage. Allamanda cathartica is an attractive ornamental neotropical shrub that produces abundant latex with medicinal potential. The laticifers of this species, their origins, structural types, and distribution in the primary and secondary structures of the stem were investigated, and the chemical nature of latex was determined. Anatomical, histochemical, and ultrastructural evaluations of the stem apex were performed through light and electronic microscopy. Laticifers are abundant in the primary structure, being distributed in the cortex, outer primary phloem, and pith. Their branching, anastomosing structural type develops by the dissolution of the transverse and lateral walls of precursor meristematic cells, followed by protoplast fusion. The laticifers in the secondary structure are distributed amid the axial parenchyma cells of the phloem. The latex of A. cathartica is an emulsion composed mainly of mucilage and terpenes, and it is the first time that this laticifer system has been described.

Assessment of antioxidant, antidiabetic, and antimicrobial properties of hybrid mushroom strains derived through intergeneric hybridization between Pleurotus sajor-caju and Calocybe indica

Our present study aims to evaluate the biological activities of the six hybrid mushrooms (APS) obtained through the protoplast fusion technique between two mushrooms Calocybe indica and Pleurotus sajor-caju, in our previous study. Antioxidant activity of hybridized mushroom samples was determined by the free radical scavenging method, using DPPH radical, Superoxide radical, and Hydroxyl radical. Additionally, all the mushroom extract has shown a good amount of total phenolic content, and total flavonoid content and also has potent Ferric reducing antioxidant power. The Total phenolic content, Total flavonoid content, and Ferric reducing antioxidant power were estimated. Six constituents: gallic acid, catechin, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, and cinnamic acid were identified by UHPLC. The MIC values, zone of inhibition, growth kinetic, and scanning electron microscopy (SEM) were performed to establish the antimicrobial efficacy. The tested samples exhibited antimicrobial activity against selected microorganisms Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa. A clear morphological change was observed under SEM when Bacillus subtilis and Pseudomonas aeruginosa were treated with APS-4 and APS-3 mushroom extracts. The ability to inhibit carbohydrate hydrolyzing enzymes such as α-amylase and α-galactosidase has also been studied to explore antidiabetic properties. The inhibitory activity of α-glucosidase was APS-4 reflected a very prominent result with the lowest IC50 value i.e.,118.058 ± 1.515, APS-2 and APS-6 showed almost similar IC50 values. α – galactosidase inhibitory activity of APS-4 and APS-6 exhibit better results than other strains.

Genome editing tools based improved applications in macrofungi.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.

Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

Indolizidines from Actinomycetes: An Overview of Producers, Biosynthesis and Bioactivities.

Indolizidines have long been recognized for their valuable bioactivities, their common feature being a bicyclic structure connected via a nitrogen atom. Traditionally, plants have been identified as the primary producers. However, recent discoveries have revealed that certain bacterial strains belonging to the genus of actinomycetes also possess the ability to synthesize various indolizidine-based compounds. Among these strains, Streptomyces sp. HNA39, Saccharopolyspora sp. RL78, and Streptomyces NCIB 11649 have been identified as producers of cyclizidines, characterized by their distinctive cyclopropyl moiety. Additionally, Streptomyces griseus OS-3601 synthesizes a unique class of indolizidine derivatives known as iminimycins, distinguished by their rare imine-cation structure. Protoplast fusion of a Streptomyces griseus strain with Streptomyces tenjimariensis resulted in a new indolizidine named indolizomycin. This review aims to provide an overview of known bacterial indolizidine producers, summarize current knowledge regarding the biosynthesis of cyclizidines and iminimycins, and assess their respective bioactivities.

Multiplication of apical and axillary shoots of coffee arabica using various types of cytokinins

Abstract In-vitro shoot multiplication presents a promising avenue for replicating the genetic characteristics of parent plants, commonly utilized in various plant biotechnological techniques such as mutation replication, protoplast fusion, anther culture, and genetic engineering. The primary aim of this research was to assess the impact of different cytokinins on the proliferation of apical and axillary shoots in Arabica coffee multiplication. A Completely Randomized Factorial Design was employed, considering two factors: explant source and cytokinin type. Explants were sourced from both apical and axillary branches of AS2K Arabica coffee plantlets. Three cytokinins, namely 6-Benzyl adenine purine (BAP), isopentenyl adenine (2-IP), and kinetin, were incorporated into the Murashige and Skoog (MS) 1962 basal medium at concentrations of 0, 1, 2, and 3 mg/l. The findings revealed significant effects of all three cytokinins on shoot number, plantlet height, and leaf growth, albeit without influencing root development. Notably, axillary shoot-derived explants demonstrated a more pronounced effect on shoot count compared to those from apical shoots. Among the cytokinin treatments assessed, BAP at a concentration of 2 mg/l in the MS medium yielded optimal results for propagating the AS2K variety of Arabica coffee, thus representing the recommended medium for this purpose.

Intermolecular Gene Conversion for the Equalization of Genome Copies in the Polyploid Haloarchaeon Haloferax volcanii: Identification of Important Proteins.

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO_2528, encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4, which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii. Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.

Enhanced ε‑poly‑L‑lysine production in Streptomyces species by combining interspecific hybridization with multiple antibiotic resistance.

To improve the ε-PL production in wild-type strains of Streptomyces. albulus, Streptomyces. noursei, Streptomyces. rochei and Streptomyces. yunnanensis, the interspecific hybridization based on protoplast fusion was first performed. Two-species hybridizations failed to obtain hybrids with significant increase in ε-PL production, but four-species hybridizations succeed in acquiring many high-yield hybrids. 16S rDNA homology alignment and RAPD confirmed that the hybrid HX17 was restructured by integrating gene fragments from S. albulus and S. rochei with S. noursei as the carrier. S. noursei HX17 was subsequently suffered from mutagenesis and genome shuffling combining with multiple antibiotic resistance, and a mutant S. noursei GX6 was obtained with ε-PL yield of 2.23g/L in shake-flask fermentation. In fed-batch fermentation, the ε-PL production of GX6 reached 47.2g/L, which was increased by 95.6% to 136.8% over the wild parents. Ribosomal genes associated with antibiotics were sequenced and majority of mutant strains had mutations at different sites, indicating that the increase of antibiotic resistance was strongly associated with them. This research proved that combining interspecific hybridization with multiple antibiotic resistance was as an effective approach to rapidly improve the ε-PL production in Streptomyces species.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity

BackgroundLignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.ResultsThe current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.ConclusionThe results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Microfluidic Single-Cell Study on Arabidopsis thaliana Protoplast Fusion-New Insights on Timescales and Reversibilities.

Plant cells are omnipotent and breeding of new varieties can be achieved by protoplast fusion. Such fusions can be achieved by treatment with poly(ethylene glycol) or by applying an electric field. Microfluidic devices allow for controlled conditions and targeted manipulation of small batches of cells down to single-cell analysis. To provide controlled conditions for protoplast fusions and achieve high reproducibility, we developed and characterized a microfluidic device to reliably trap some Arabidopsis thaliana protoplasts and induced cell fusion by controlled addition of poly(ethylene glycol) (PEG, with a molecular weight of 6000). Experiments were conducted to determine the survival rate of isolated protoplasts in our microfluidic system. Afterward, PEG-induced fusion was studied. Our results indicate that the following fusion parameters had a significant impact on the fusion efficiency and duration: PEG concentration, osmolality of solution and flow velocity. A PEG concentration below 10% led to only partial fusion. The osmolality of the PEG fusion solution was found to strongly impact the fusion process; complete fusion of two source cells sufficiently took part in slightly hyper-osmotic solutions, whereas iso-osmotic solutions led to only partial fusion at a 20% PEG concentration. We observed accelerated fusion for higher fluid velocities. Until this study, it was common sense that fusion is one-directional, i.e., once two cells are fused into one cell, they stay fused. Here, we present for the first time the reversible fusion of protoplasts. Our microfluidic device paves the way to a deeper understanding of the kinetics and processes of cell fusion.

Using protoplast fusion to improve biocontrol ability of Fusarium oxysporum against Egyptian broomrapes (Phelipanche Aegyptiaca)

Using protoplast fusion to improve biocontrol ability of Fusarium oxysporum against Egyptian broomrapes (Phelipanche Aegyptiaca)

METHODS OF GENETIC TRANSFORMATION: MAJOR EMPHASIS TO CROP PLANTS

Advancements in gene transfer technology have indeed opened up exciting possibilities for more effectively manipulating the genetic makeup of live organisms, ranging from microorganisms to plants and animals. Direct and indirect transformations are the two basic types of gene transfer techniques. Indirect method comprises Agrobacterium mediated method as it involves intermediate host between gene of interest and target and this method is most opted one out of all present. Direct gene transformation methods, on the other hand, do not involve the use of an intermediate host organism. Instead, they rely on physical means to transfer genes between cells. Biolistic transformation uses high-velocity particles to deliver DNA into target cells, while microinjection and macroinjection involve the direct injection of DNA into cells. Protoplast fusion combines the genetic material of two different cells by fusing their protoplasts. Natural methods for gene transfer encompass mechanisms that occur naturally in various organism, includes transposition, conjugation, phage and retroviral transductions and bacterial transformation. Chemical techniques utilize chemical agents to facilitate gene transfer, such as calcium phosphate-mediated transformation, polyethylene glycol (PEG)-mediated transformation, DEAE (Diethylethanolamine)-Dextran-mediated transformation. Genes can be also being transferred using electrical techniques such as electroporation and electrofusion. Crop improvement and trait improvement are now being hastened by the fast-rising number of sequenced plant genomes, information from functional genomics data to understand gene function, innovative gene cloning, and tissue culture techniques. Despite being indispensable, its progress is still hindered by the fact that many plant species and agricultural genotypes exhibit low transformability or are resistant to established tissue culture and regeneration conditions. Here, we review the techniques employed in plant transformation and provide a concise overview of their evolution in agricultural crops, from their first inception to present time.

High-Value Utilization of Tea Forest Resources: Breeding Eurotium cristatum Strains to Enhance Lovastatin Yields in Anhua Dark Tea

The tea plant is a vital strategic forest resource in China. Dark tea produced from its leaves is an indispensable health-promoting product in western China due to its unique lipid-lowering function. Eurotium cristatum is the dominant strain in Fuzhuan brick tea (a variety of Anhua dark tea) and could produce many functional components, including lovastatin, a lipid-lowering compound. In this study, the lovastatin yield of dark tea was improved by breeding Eurotium cristatum using the protoplast fusion method. The experiments were carried out by inducing a fusion between inactivated Eurotium cristatum JH1205 and Monascus CICC5031. Among the 92 fusants screened the HPLC method, four strains (A4, A36, A54, and A76) with higher lovastatin production (more than three times as high) were obtained. The A76 strain had the highest lovastatin yield, which was 23.93 μg/mL. The location of the tea forest strongly influenced the lovastatin yield of loose dark tea. The strain bred in this study improved the lovastatin yield of loose dark tea by more than three times when compared to wild Eurotium cristatum. These results are promising for the development of tea forest resources.

Interspecies hybridization between Auricularia cornea cv. Yu Muer and Auricularia heimuer cv. Bai Muer through protoplast fusion.

Color variations in cultivated edible mushrooms present novel and potentially valuable alternatives to the research and cultivation industries. We collected, identified, and domesticated a white strain of Auricularia cornea and a white strain of Auricularia heimuer from China. However, due to an unstable phenotype and stricter requirements on environment and management technology, the production and utilization of Auricularia heimuer cv. Bai Muer make slow progress. Outcrossing is an essential means to broaden the intraspecific genetic resources to expand the gene pool and compensate for the limitations of related species hybridization. In this study, interspecies hybridization between Auricularia cornea cv. Yu Muer and Auricularia heimuer cv. Bai Muer was conducted using polyethylene glycol (PEG)-induced double-inactivated protoplast fusion. Apart from the functional complementation of double-inactivated protoplasts, the hybrids were characterized by colony morphology, antagonistic test, primordial morphology, and polymerase chain reaction (PCR) fingerprinting. The results suggested that the hybrids and their parents showed significant differences in their colony morphology. Moreover, positive barrage reactions were observed between each parent and hybrid. Inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) profile analysis of fusants and parents depicted that fusants contained polymorphic bands, which indicated the rearrangement and deletion of deoxyribonucleic acid (DNA) in the fusants. Yellowish-white primordia were obtained from two hybrids. Protoplast fusion may reinforce the genetic potential and provide an ideal alternative for breeding albino Auricularia.

Interspecific Hybridization between Ganoderma lingzhi and G. resinaceum by PEG-Induced Double-Inactivated Protoplast Fusion

Ganoderma lingzhi is an important medicinal fungus, and it is particularly important to select strains with high yields and active substance contents. In this study, protoplasts of G. lingzhi were thermally inactivated to destroy intracellular enzyme proteins and preserve DNA. The DNA of G. resinaceum was damaged by ultraviolet (UV) radiation, and other components of the protoplasm except DNA were preserved. Then, the protoplast was induced using polyethylene glycol (PEG) for fusion. The results showed that the optimal thermal inactivation conditions for G. lingzhi were 30 min in a 45 °C water bath, and the optimal UV inactivation conditions for G. resinaceum were 70 s of irradiation using a 20 W UV lamp at a vertical distance of 15 cm. Antagonistic tests, internal transcribed space (ITS) and mitochondrial DNA identification, intersimple sequence repeat (ISSR) molecular markers and morphology were used to distinguish the parents from the fusants. Four true fusants were obtained, and the yield was 2.5%. The fruiting body yield of the fusants was significantly higher than that of G. lingzhi, and the polysaccharide and triterpene contents of the RAD-64 fusant were significantly higher than those of G. lingzhi. The results presented in this paper show that protoplast fusion technology can effectively improve G. lingzhi varieties and support the breeding of new varieties.

Protoplast Isolation and Fusion in Amomum Subulatum Roxb

Amomum subulatum Roxb. is an important spice and cash crop of Himalayan region of Indian sub-continent. An attempt has been made to isolate and fuse protoplast from the in-vitro grown seedling of Amomum subulatum Roxb. Off the various enzyme combination tried, the one containing the enzyme combination of 2% cellulase “Ozonuka” RS (Yakult Pharmaceutical Ind. Co. Ltd, Japan) 1% Macerozyme R-10 (Yakult Pharmaceutical Ind. Co. Ltd, Japan) and 1 % Hemi-cellulase (Sigma-Aldrich, USA) prepared in 10.93% D- Mannitol (Himedia, India) and 0.055% calcium chloride yielded protoplast of 2 x 104/g of leaf tissue. Protoplast fusion was achieved using 20% Polyethylene Gylcol (PEG 4000) but in small quantity added to the mixed protoplast. To develop a successful somatic hybrid of commercial importance further research is required.

Phenotypical characterization of Portulaca umbraticola: A non-conventional edible ornamental crop

Purslanes are frequently used in Brazil as potted plants and for landscaping because of their beautiful colored flowers. Despite their commercial importance, the genetic diversity within ornamental purslane remains largely unknown. Thus, a complete characterization of ornamental purslane resources is essential for its utilization since genetic variability is indispensable for the efficient development of new cultivars. This study aimed to evaluate genetic variability and recommend accessions to start a genetic breeding program for purslane (Portulaca umbraticola). Twenty purslane accessions were collected in public places in Areia City and Santa Rita City, Paraiba State, Brazil. The experiment was conducted in a greenhouse. An entirely randomized experimental design with 20 accessions and 5 replicates was utilized. Plants were evaluated for quantitative and qualitative traits. Data were submitted to analysis of variance and grouped by Scott–Knott’s criteria (p ≤ 0.05). The accessions were grouped using Tocher’s method based on the generalized Mahalanobis distance. Principal component analysis was also used to analyze the genetic divergence. There were significant differences (p ≤ 0.05) for all evaluated traits except for internode distance, plant height, and leaf length. The Scott–Knott criteria clustered the accessions into two groups for all traits except days to flowering (five groups). Tocher’s grouping based on Mahalanobis distance allowed the accessions to be separated into eight clusters. The genetic diversity observed in this study was supported by the consonance between the Tocher cluster method and the PC analyses. The traits that most contributed to genetic divergence among accessions were days to flowering (70.37%), flower length (6.10%), leaf width (3.89%), branch number (3.57%), and stem length (3.36%). Considering the criteria for use in outdoor spaces, wider plants with several large colored flowers, thicker stems, greener leaves, and earliness were interesting. Accessions 5, 7, 13, 14, 16, 18, and 20 should be selected as parents for further breeding programs. However, if the objective is to produce potted plants, dwarf plants with bigger leaves are desired, and accession 1 should be selected. This will create greater variability in segregating populations, which will increase the overall quality of ornamental use of this species. For those accessions with no reproductive verticils but with other excellent attributes, for example, high chlorophyll content breeding strategies, such as induced mutations or protoplast fusion, can be used.

Floral morphology of 'Anand' rapid cycling Brassica oleracea .

Abstract The 'Anand' type of cytoplasmic male sterility (CMS) was introduced to Brassica oleracea through protoplast fusion (Cardi and Earle, 1997), providing a source of male sterility that is of value in producing F1 hybrid varieties of this species without the cold sensitivity of 'Ogura' cytoplasm. In this species, as in others in which it has been introduced, 'Anand' mitochondria are associated with flower abnormalities. The best lines have been used by commercial breeders. This paper describes 'Anand' flower abnormalities in further detail. Rapid cycling brassica with 'Anand' cytoplasm may have promise as a model system for research on floral patterning. Materials and Methods. Three lines of rapid cycling B. oleracea carrying the 'Anand' cytoplasm and one wild type were grown. The flower morphology of 'Anand' lines was classified as good in F25-F-1A, fair in F25-F-6B, and poor in F23-F-1B (Cardi and Earle, 1997). Seedlings were started in an indoor growth room, but were maintained in a naturally lit greenhouse at 20° night, 25° day during flowering. Five flowers on 5 plants of each line were examined. Results. Morphological abnormalities affected all floral parts (Table 1). There was substantial variation among flowers on individual plants and among lines but little within lines. Conclusions. "Good" flowers have large petals that push open the sepals before the carpel elongates. Female fertility could be reduced due to occlusion of the stigma or physical damage caused by the stigma pressing against the end of a sepal and kinking the style. Female fertility in some flowers was unlikely because the carpel suture failed to fuse. In some cases the edge of the split carpel was sepal-like, consistent with the absence of SEPALLATA expression. The stigma on a sepal represents the converse. Stamens were most strongly affected, with outer stamens most reduced. This whorl is the one most frequently suppressed in flowering plants (Masters, 1869). The number of stamens was reduced to varying degrees. First to be lost was one or both of the pair outside the petals. Then the outer pair inside the petals was affected. One or both of these stamens could either be lost or doubled to give 3 to 5 stamens. Again a positional cue for stamens appears to be weakened. Anther development was often limited, the expected and desired consequence of the Anand genotype. Some undehisced " good " anthers contained pollen. There is variation even among the lines initially described as " good ". Further selection among these may help identify those that are most promising for further use in hybrid production.

Early selection of carrot somatic hybrids: a promising tool for species with high regenerative ability

BackgroundSince its discovery, somatic hybridization has been used to overcome the sexual barriers between cultivated and wild species. A combination of two somatic cells might provide a novel set of features, often of agronomical importance. Here, we report a successful approach for production and selection of interspecific somatic hybrid plants between cultivated and wild carrot using dual-labelling of protoplasts and an early selection of fused cells via micromanipulator. Both subspecies used in this study are characterised by a very high regenerative ability in protoplast cultures. Thus, a precise and effective method of hybrid selection is essential to assure the development and regeneration of much less numerous heterokaryons in the post-fusion cell mixture.ResultsElectrofusion parameters, such as alternating current and direct current, were optimised for an efficient alignment of protoplasts and reversible membrane breakdown followed by a cell fusion. Four hundred twenty-nine cells emitting green–red fluorescence, identified as hybrids, were obtained. Co-culture with donor-derived protoplasts in the alginate feeder layer system stimulated re-synthesis of the cell wall and promoted cell divisions of fusants. Somatic embryogenesis occurred in hybrid-derived microcalli cultures, followed by plant regeneration. Regenerated hybrids produced yellowish storage roots and leaves of an intermediate shape between cultivated and wild subspecies. The intron length polymorphism analysis revealed that 123 of 124 regenerated plants were hybrids.ConclusionsThe developed protocol for protoplast fusion and an early selection of hybrids may serve as an alternative to combining genomes and transferring nuclear or cytoplasmatic traits from wild Daucus species to cultivated carrot.

Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion

BackgroundSomatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion. MethodWe employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion. ResultsFlow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17–1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion. Purpose and significanceOverall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.