Efficient Plant Regeneration System from Leaf Explant Cultures of via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of . To induce embryogenic structures, fully expanded leaf explants of were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.

Heterologous overexpression of the cyanobacterial alcohol dehydrogenase confers cold tolerance to the oleaginous alga .

Temperature is an important regulator of growth in algae and other photosynthetic organisms. Temperatures above or below the optimal growth temperature could cause oxidative stress to algae through accumulation of oxidizing compounds such as reactive oxygen species (ROS). Thus, algal temperature stress tolerance could be attained by enhancing oxidative stress resistance.

Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS).

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus.

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly.

The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.

Genetic Impairment of Cellulose Biosynthesis Increases Cell Wall Fragility and Improves Lipid Extractability from Oleaginous Alga .

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene () expression would lead to protein accumulation without the accumulation of storage C polymers in .

The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp.

a homolog of mice MPV17, enhances osmotic stress tolerance.

Mutation in the human gene or the functional yeast orthologue result in mitochondrial DNA depletion. homologs are also found in plants including , but the function of these genes remain unclear. genome contains 10 homologs.

Elevated Inorganic Carbon Concentrating Mechanism Confers Tolerance to High Light in an Arctic sp. ArM0029B.

Microalgae and higher plants employ an inorganic carbon (Ci) concentrating mechanism (CCM) to increase CO availability to Rubisco. Operation of the CCM should enhance the activity of the Calvin cycle, which could act as an electron sink for electrons generated by photosynthesis, and lower the redox status of photosynthetic electron transport chains. In this study, a hypothesis that microalgal cells with fully operating CCM are less likely to be photodamaged was tested by comparing a mutant with its wild type (WT).

PtDRG1, a Desiccation Response Gene from Pyropia tenera (Rhodophyta), Exhibits Chaperone Function and Enhances Abiotic Stress Tolerance.

Pyropia are commercially valuable marine red algae that grow in the intertidal zone. They are extremely tolerant to desiccation stress. We have previously identified and reported desiccation response genes (DRGs) based on transcriptome analysis of P.

Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris.

Photosynthesis of microalgae enables conversion of light energy into chemical energy to produce biomass and biomaterials. However, the efficiency of this process must be enhanced, and truncation of light-harvesting complex (LHC) has been suggested to improve photosynthetic efficiency. We reported an EMS-induced mutant (E5) showing partially reduced LHC in Chlorella vulgaris.

An Alcohol Dehydrogenase Gene from sp. Confers Salt Tolerance in Transgenic Tobacco.

salt-responsive gene 1 () was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of -overexpressing (-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The -OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions.

Current status and perspectives of genome editing technology for microalgae.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases.

Increased lipid production by heterologous expression of AtWRI1 transcription factor in .

Background: Genetic engineering of microalgae is necessary to produce economically feasible strains for biofuel production. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not sufficiently stable and/or efficient for large-scale production of biofuels and other materials. Transcription factors (TFs) are emerging as good alternatives for engineering of microalgae, not only to increase production of biomaterials but to enhance stress tolerance.

Transcriptional Regulation of Cellulose Biosynthesis during the Early Phase of Nitrogen Deprivation in Nannochloropsis salina.

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N-), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N- stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis.

The Mechanism of Starch Over-Accumulation in High-Starch Mutants Identified by Comparative Transcriptome Analysis.

The focus of this study was the mechanism of starch accumulation in high-starch mutants. Three . mutants showing high-starch content were generated using gamma irradiation.

Transcriptome-Based Identification of the Desiccation Response Genes in Marine Red Algae Pyropia tenera (Rhodophyta) and Enhancement of Abiotic Stress Tolerance by PtDRG2 in Chlamydomonas.

Pyropia tenera (Kjellman) are marine red algae that grow in the intertidal zone and lose more than 90% of water during hibernal low tides every day. In order to identify the desiccation response gene (DRG) in P. tenera, we generated 1,444,210 transcriptome sequences using the 454-FLX platform from the gametophyte under control and desiccation conditions.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9.

Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal.

A unique digital microfluidic electroporation (EP) system successfully demonstrates higher transgene expression than that of conventional techniques, in addition to reliable productivity and feasible integrated processes. By systematic investigations into the effects of the droplet EP conditions for a wild-type microalgae, 1 order of magnitude higher transgene expression is accomplished without cell wall removal over the conventional bulk EP system. In addition, the newly proposed droplet EP method by a droplet contact charging phenomena shows a great potential for the integration of EP processes and on-chip cell culture providing easy controllability of each process.

Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii.

Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii.

Plastid and mitochondrion genomic sequences from Arctic Chlorella sp. ArM0029B.

Background: Chorella is the representative taxon of Chlorellales in Trebouxiophyceae, and its chloroplast (cp) genomic information has been thought to depend only on studies concerning Chlorella vulgaris and GenBank information of C. variablis. Mitochondrial (mt) genomic information regarding Chlorella is currently unavailable.

Comparative proteomics using lipid over-producing or less-producing mutants unravels lipid metabolisms in Chlamydomonas reinhardtii.

In this study, proteomic approach was employed to advance our understanding of genes associated with microalgal lipid production. We first generated random mutants of Chlamydomonas reinhardtii, which displayed increased or decreased lipid content, compared to that in the wild-type. Mutants were designated as CR12 and CR48.

A new Arctic Chlorella species for biodiesel production.

Microalgae are a potential resource for biodiesel production. A green alga, Chlorella sp., was isolated from Arctic sea ice, which was named ArM0029B.

Droplet electroporation in microfluidics for efficient cell transformation with or without cell wall removal.

An efficient cell transformation method is presented that utilizes droplet electroporation on a microfluidic chip. Two types of green microalgae, a wall-less mutant and a wild type of Chlamydomonas reinhardtii, are used as model cells. The PDMS-glass electroporation chip is simply composed of a flow-focusing microstructure for generating cell-encapsulating droplets and a serpentine channel for better mixing of the content in the droplet, and five pairs of parallel microelectrodes on the glass slide, without involving any expensive electrical equipment.

Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas.

Heat shock proteins and molecular chaperones are key components contributing to survival in the abiotic stress response. Porphyra seriata grows on intertidal rocks exposed to dynamic environmental changes associated with the turning tides, including desiccation and heat stress. Analysis of the ESTs of P.