Protoplast Fusion Research Articles

Improved bioethanol production using genome-shuffled Clostridium ragsdalei (DSM 15248) strains through syngas fermentation

ABSTRACTIn the present study, a genome shuffling approach was used for improvement of ethanol and acetic acid tolerance of C. ragsdalei (DSM 15248). Evaluation of metabolite production from selected shuffled strains was also conducted. Random mutagenesis was carried out using ethyl-methyl-sulfonate and UV followed by protoplast fusion. Promising fusants were selected based on enhanced ethanol and acetic acid tolerance and further evaluated for metabolite production through syngas fermentation. Strain F3-A obtained after the third round of shuffling showed ethanol production of 14.92 ± 0.75 g/L, while the parent strain showed ethanol production of 1.92 ± 0.52 g/L after seven days of syngas fermentation at 37 °C. Enhancement in 1-butanol production was also observed in shuffled strain F3-A. A seven-fold increase in ethanol concentration for shuffled strain F3-A was obtained. The study confirms the successful implementation of the genome shuffling technique for significant enhancement of metabolite production using syngas fermentation.

Improving the nematicidal potential of Bacillus amyloliquefaciens and Lysinibacillus sphaericus against the root-knot nematode Meloidogyne incognita using protoplast fusion technique

Root-knot nematodes (RKNs) are one of the major constraints of vegetable cultivation worldwide. Chemical nematicides, the primary management tool for over 50 years, have a negative impact on the environment and the ineffectiveness after prolonged use. Biological control using eco-friendly rhizosphere bacteria antagonistic to nematodes is one of the alternative approaches. The objective of this study was to improve the nematicidal activity of Bacillus amyloliquefaciens subsp. plantarum SA5 and Lysinibacillus sphaericus Amira strain against RKN Meloidogyne incognita, using the protoplast technique. Their fusants were tested for their chitinase and nematicidal activity using bioassay and greenhouse experiments. The selected fusants from the two bacterial strains were more effective in killing M. incognita J2 under laboratory conditions. Percentage mortality after 24 h of exposure were 70.85, 84.69, 95.56, 94.99, 100, and 89.46% due to the parental strains B. amyloliquefaciens and L. sphaericus and the fusants Bas3, Bas6-2, Bas8, and Bas11, respectively. There was a positive correlation between the chitinase production and the nematicidal effect of the bacterial strains. Under greenhouse conditions, Bas8 which produced the highest amount of chitinase induced the greatest reduction in nematode counts and gave the best results in shoot length and fresh and dry weights as compared to control. Chitinase production of fusant was much higher under solid-state fermentation (SSF) than submerged fermentation conditions. The recorded chitinase produced by B. amyloliquefaciens, L. sphaericus, and Bas8 were 0, 1393, and 3399 units (μg NAG/ml enzyme/h), respectively, under solid-state fermentation and 90, 85, and 143 units (μg NAG/ml enzyme/h), respectively, under submerged fermentation conditions. Protoplast fusion was a powerful technique in improving nematicidal activity. Chitinase production is an important factor in improving the nematicidal activity of such microorganisms. The obtained improved fusant could be used as a biological control agent for M. incognita.

Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity

Producing alcoholic beverages with novel flavor are desirable for winemakers. We created fermenting yeast with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Strategies of ethanol domestication, ultraviolet mutagenesis (UV) and protoplast fusion were conducted to create yeast hybrids with excellent oenological characteristic. The obtained diploid hybrid F23 showed a cell viability of 6.2% under 25% ethanol, whereas its diploid parental strains could not survive under 20% ethanol. During Chinese rice wine-making, compared to diploid parents, F23 produced 7.07%–12.44% higher yield of ethanol. Flavor analysis indicated that the total content of flavor compounds in F23 wine was 19.99–26.55% higher than that of parent wines. Specifically, F23 exhibited higher capacity in producing 2-phenylethanol, short-chain and long-chain fatty-acid ethyl-ester than diploid parents. Compared to diploid parents, F23 introduced more flavor contributors with odor activity values (OAVs) above one to Chinese rice wine, and those contributors were found with higher OAVs. Based on principal component analysis (PCA), the flavor characteristic of F23 wine was similar to each of parent wine. Additionally, sensory evaluation showed that F23 wine was highly assessed for its intensive levels in fruit-aroma, alcohol-aroma and mouthfeel. Hybrid F23 not only displayed superior flavor production and oenological performance in making Chinese rice wine, but also could act as potential “mixed-like” starter to enrich wine style and differentiation.

A novel Trichoderma fusant for enhancing nutritional value and defence activity in chickpea.

In recent years, due to the rise in foodconsumption, much of the attention has been focused to increase the yield of the agricultural crops which resulted in compromised nutritional quality. Efforts have to be undertaken to enhance the nutritional attributes of legumes, cereals and staple food crops by increasing amino acids and mineral content. In the present study, we evaluated a protoplast fusant (H. lixii MTCC 5659) for its ability to enhance nutritional value and defence activity in chickpea. Essential amino acids; methionine (9.82mgkg-1dw), cysteine (2.61mgkg-1dw), glycine (11.34mgkg-1dw), valine (9.26mgkg-1dw), and non-essential amino acids; aspartic acid (39.19mgkg-1dw) and serine (17.53mgkg-1dw) were significantly higher in seeds of fusant inoculated chickpea. Fusant significantly improved accumulation of mineral nutrients i.e. Cu (157.73mgkg-1dw), Co (0.06mgkg-1dw), Ni (1.85mgkg-1dw), Zn (157.73mgkg-1dw) and S (16.29mgkg-1dw) in seeds. Biocontrol and defence activities of chickpea increased from 20 to 35% in fusant inoculated plants suggesting its potential to ameliorate biotic stress. To the best of our knowledge, this is the first report of an increase in amino acids and mineral content of chickpea by fusant inoculation.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Preparation and Regeneration of Protoplasts from the Ethyl Vincamine Producing Fungus CH1 (Geomyces sp.)

Vinpocetine, a semi-synthetic compound derived from the alkaloid vincamine, exhibits effective pharmacological activities for the treatment and prevention of cerebrovascular circulation and vascular cognitive disorders. Vinpocetine can be produced through a one-step chemical reaction beginning with ethyl vincamine, and a two-step chemical reaction beginning with vincamine. In our previous study, the endophytic fungus CH1, Geomyces sp., was isolated and identified as a producer of ethyl vincamine, which was first obtained by endophytic fungal fermentation. However, the production was largely limited. Fungal protoplasts are a valuable experimental tool for physiological and genetic research such as protoplast fusion, gene transfer and metabolite production. In this paper, we optimized some key factors for the preparation and regeneration of protoplasts from strain CH1. Using an enzymes mixture consisting of cellulase (2.0%, w/v), glusulase (3.0%, w/v) and driselase (1.0%, w/v) in osmotic stabilizer (0.7 mol/L NaCl), the highest yield of protoplasts (6.78×107/mL) was obtained with mycelia after 72 h at pH 5.0-6.0 by digesting for 1.5 h at 30°C. After purification of the prepared protoplasts, they were regenerated in the regeneration medium using a bilayer plate culture method.

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant ( Camellia sinensis L. O. Kuntze)

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant ( <i>Camellia sinensis</i> L. O. Kuntze)

Application of the Cre/lox System to Construct Auxotrophic Markers for Quantitative Genetic Analyses in Fusarium graminearum.

The bacteriophage P1 Cre/lox system has been utilized in diverse fungi for marker recycling and exchange, generation of targeted chromosome translocations, and targeted deletion of interstitial chromosome segments. Here we show the application of this tool in the wheat and maize pathogen, Fusarium graminearum. We explored three different ways to introduce Cre into strains with floxed genes, namely transformation with an episomal or integrative plasmid (pLC28), fusion of protoplasts of strains carrying floxed genes with strains expressing Cre by forcing heterokaryons, and crosses between strains with floxed genes and strains expressing Cre to isolate progeny in which the target genes had been deleted during the cross. We used this system for the construction of strains bearing auxotrophic markers that were generated by gene replacement with positively selectable markers followed by Cre-mediated marker excision. In addition, updated protocols for transformation and crosses for F. graminearum are provided. In combination, strains and tools developed here add to the arsenal of methods that can be used to carry out molecular genetics with F. graminearum.

Comparative Analysis of Genetic Diversity Among Bacillus Thuringiensis and Bacillus Sphaericus and Their Fusants Using Molecular Markers

Ten genetically stable fusants strains were obtained as a result of protoplast fusion technique between Bacillus sphaericus (Bs) and Bacillus thuringiensis (Bt), six of them produced from, Bs GHAI Bt 66 Ia experiment (fusants no. 1, 2, 16, 26, 31 and 34) and four fusants produced from Bs GHAI Bt I977experiment (fusants no.1, 2, 17 and 25). Two molecular marker systems, four ISSR primers (Inter-Simple Sequence Repeat) and seven SCoT primers (Start Codon Targeted Polymorphism) were employed for detection of genetic variation between the parents and their fusants. Four ISSR primers and seven SCoT detected 55and 97 amplicons, among which 92.696% and 81.948% bands were polymorphic respectively. The genetic similarity values among the species were calculated based on ISSR and SCoT profiles which ranged from 0.50 to 0.83 and 0.59 to 0.90 respectively. Based on the marker analysis, the three parents and the ten fusants were clustered into three major groups for ISSR primer and SCoT primers. Cluster analysis were performed to construct a dendrogram of the Bacillus Strains and their fusants and also reveals high genetic variation among the parents and their fusants. It could be concluded that each type of the two molecular marker approaches of DNA analysis could identify the different between the parents, and all of their fusants under investigation have probably originated from closely related ancestors and possess high degree of genetic similarity.

Isolation, identification, and improving nematotoxicity of rhizobacterial strains against Meloidogyne incognita

Background and objectives Root-knot nematodes, Meloidogyne spp., have been reported to cause severe losses to field, horticultural, and medicinal plants throughout the world. Chemical nematicides are routinely applied for protection of the aforementioned crops. Residues of these toxic nematicides are carried to the final consumed products, which can be quite hazardous to human health on consumption. Biocontrol by using antagonistic organisms to inhibit the pathogens and reduce diseases is an ecofriendly management tool. This study was planned to isolate, identify, and improve or increase the activity of two isolated rhizobacterial strains known to have nematicidal properties − based on preliminary studies − using protoplast fusion technique and assessing the nematicidal potential of the fusants against root-knot nematode, Meloidogyne incognita J2. Materials and methods Certain rhizobacterial colonies isolated from the rhizosphere soil of cucumber and banana plants were identified by 16S rDNA to Pseudomonas aeruginosa (accession number LC187271) and Bacillus licheniformis (accession number LC187270). They were manipulated using protoplast fusion technique to improve their nematicidal potentials. The fused protoplasts generated and 10 fusant isolates were assessed against M. incognita J2. Results and conclusion All the tested fusants exhibited increase in their nematicidal activity than their parents against root-knot nematode, M. incognita J2, under laboratory conditions. The percentages of nematode mortality after 72 h of exposure were 85 and 86% by B. licheniformis and P. aeruginosa suspensions, respectively, whereas the percentages of mortality caused by fusants ranged between 91 and 99% as compared with control. The obtained results indicated that protoplast fusion technique is a successful tool to enhance the lethal effect of the isolated rhizobacteria strains against root-knot nematode. The genetically engineered bacteria can play a dual role under field conditions as a biocontrol agent against nematode and subsequently improve plant productivity.

English

The genus of Trichoderma is widely applied for the biocontrol of phytopathogenic fungi in agriculture sector. Moreover, Trichoderma species are also excessively exploited in different industrial purposes due to their production of important lytic enzymes such as chitinases, glucanases and proteases. Several genetic improvement trials are carried out for maximizing the role of Trichoderma as biological control agents via mutation, protoplast fusion, and genetic transformation. This review highlights the mode of action of Trichoderma against pathogenic fungi, potential applications in different fields of life, and the recent genetic improvement trials for increasing the antagonistic abilities of this fungus as biological control agent. Key words: Trichoderma, antagonism, lytic enzymes, genetic improvement.

Construction of novel cold-tolerant strains of Volvariella volvacea through protoplast fusion between Volvariella volvacea and Pleurotus eryngii

Volvariella volvacea, an edible mushroom that grows at high temperature and humidity, has attracted growing attention because of its high nutrient value, short cropping duration, unique aroma and texture. However, it is difficult to store fresh because it is highly perishable in low-temperature. To expend its cultivation region and season, this study aims to improve the low-temperature tolerance ability and the biological efficiency of this macrofungus. For this purpose, interfamily hybridization between V. volvacea and Pleurotus eryngii was studied through PEG-mediated protoplast fusion technique. Two low-temperature resistant strains, namely, VP1 and VP2 were successfully obtained through protoplast fusion with biological characteristics screening and molecular determination. The hybrid strain VP1 showed a higher biological efficiency (31.53%), compared with the 9.38% in the parental strain. Fruiting bodies of both VP1 and VP2 had a longer storage life than that of control at 16 °C. The size and amplified fragments length polymorphism (AFLP) pattern of fusants were similar, but differed from the parental V138 species, which implied that fusants are novel strains. This research has achieved interfamily protoplast fusion, which provides an effective way and the theoretical basis for the distant hybridization breeding of edible fungi.

Stable, fertile somatic hybrids between Sinapis alba and Brassica juncea show resistance to Alternaria brassicae and heat stress

Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of > 38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.

Progress in somatic hybridization research in potato during the past 40 years

Somatic hybridization has been used in potato to overcome the sexual barriers between the cultivated (Solanum tuberosum L.) and wild species. To date hundreds of inter/intra-specific somatic hybrids have been produced via protoplast fusions using 23 Solanum species and characterized for multiple traits such as agronomic, disease/pest resistance, salinity, frost and others. With increasing success in recovery of fusion products, somatic hybrids have been exploited in potato genetics, breeding and genomics studies. Here, we report on progress in somatic hybridization research in potato during the past 40 years.

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild-type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling

Mitochondrial Genome of Callus Protoplast Has a Role in Mesophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate, symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyll-parent-type cybrids. It suggested that mitochondrial DNA (mtDNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis (L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein (GFP) gene, in an effort to elucidate whether mtDNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids’ with mtDNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mtDNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

In vitro biotechnological approaches on Vanilla planifolia Andrews: advancements and opportunities

In vitro biotechnological advancement of Vanilla plays a major role in germplasm conservation, genetic engineering, accelerated clonal multiplication and production of disease-free plants with enviable aromatic properties. Several attempts have been taken place for the establishment of efficient in vitro protocol for Vanilla in the past few decades. Optimization of various conditions during different phases of micropropagation, for instance development of in vitro aseptic cultures, multiple shoot regeneration, rooting and acclimatization of the plantlets are discussed in this review. In addition to basic micropropagation techniques, various other in vitro biotechnological applications such as clonal fidelity assessment, genetic transformation, synthetic seed technology and cryopreservation are also highlighted. Apart from the existing data, applied aspects like embryo rescue, mutation breeding, genetic engineering, protoplast fusion, somaclonal variation, in vitro enhancement of vanillin production through cell suspension culture, hairy root culture or bioreactors and cryopreservation need to be investigated further. Overall, the current review gives a synopsis on progress and prospect of in vitro culture of Vanilla.

Regeneration of cell suspension derived Apium graveolens L. protoplasts

Cytoplasmatic male sterility (CMS), which can be achieved by protoplast fusion and regeneration, has potential to greatly facilitate hybrid breeding of celery (Apium graveolens L.). Therefore as a first step we developed a simple and efficient protoplast isolation and regeneration protocol for three commercial A. graveolens varieties (green and white celery and celeriac). To this end, cell suspensions from independent cell lines of open pollinated cultivars and inbred lines were initiated as a source for protoplast isolation. Comparative analyses showed that culturing was most successful in modified Kao and Michayluk liquid medium supplemented with 0.3 mg l−1 2,4-D. The cytokinin type (TDZ or zeatin) and concentration had no significant effect on regeneration efficiency. Microcalli were obtained within 15 days to 5 weeks after protoplast isolation. Supplementing the culture medium with 25% conditioned medium increased microcolony formation for some of the cultured lines. Plants were obtained within 2 months of microcallus culturing and these were all diploid, suggesting genetic inheritance consistency. The efficiency of regeneration mainly depended on the specific genotype, with outcrossing genotypes displaying high heterogeneity in regeneration responses whereas inbred lines did not regenerate. The protocol presented here enables to implement protoplast fusion in celery breeding.

Setting up Schizosaccharomyces pombe Crosses/Matings.

Here we provide methods for setting up standard crosses with Schizosaccharomyces pombe strains. All strain genotypes and pedigrees should be recorded in a laboratory strain book. Matings between two haploid strains of interest are induced on solid medium poor in nitrogen. Usually, sporulation agar (SPA) plates are preferred, but for difficult matings it is advisable to try several mating media in parallel because one medium might allow for more efficient mating. Protoplast fusion can be used to produce zygotes from sterile mutants that fail to mate.

Protoplast fusion technology and its application in genetic improvement of plants: A review

Protoplast fusion technology and its application in genetic improvement of plants: A review