An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Establishment of Protoplast Transformation System in Alternaria tenuissima Using G418 Selection Marker

[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.

Isolation of Protoplast and Ion Channel Recording in Plasma Membrane of Suspension Cells of Populus euphratica

The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.

Transformation of Metallothionein Gene into Mushroom Protoplasts by Application of Electroporation

Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.

Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

Formation Conditions of Aspergiilus oryzae Protoplast

[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic stabilizers and different concentrations of enzyme system was carried out in this study.[Result] The protoplast yield was higher when the mycelium was cultivated for 108 h,and the osmotic stabilizer was 0.8 mol/L of NaCl.With the enzymes system of 2 mg/ml of lysozyme,6 mg/ml of cellulose and 6 mg/ml of glusulase,the protoplast yield achieved its highest,which was up to 9.0×105 per ml.[Conclusion] The yield of protoplast was affected by the situation of mycelia themselves and external conditions.This study had provided a basis for the preparation of a large number of active protoplasts and the further researches on cell fusion.

Studies on Parameters Affecting Embryogenesis of Protoplasts Isolated from Suspension of Embryogenic Cells in Loblolly Pine

Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.

Research on Protoplast Preparation and Regeneration Conditions of Tremella aurantialba

[Objective] This study aimed to explore the protoplast preparation and re- generation conditions of Tremella aurantialba. [Method] Optimal combination of five factors affecting the protoplast preparation and regeneration of Tremella aurantialba was selected by using orthogonal experiment, including enzyme system, culture age, enzymolysis temperature, enzymolysis duration and osmotic stabilizer. [Result] The results showed that there were three release modes of Tremella aurantialba proto- plasts： release from top in the early period of enzymolysis, release from the side and release in situ. The optimal protoplast preparation condition was selecting mycelium cultured in liquid medium for 3 d, for enzymolysis in mixed enzyme solu- tion containing 1% of cellulase ＋1% of snailase ＋1% of lywallzyme at 36 ℃; for 4 h, with 0.6 mol/L sucrose as osmotic stabilizer, and the obtained preparation rate had reached 1.76 ×10^7 protoplasts/ml. The optimal regeneration condition was using mycelium cultured in liquid medium for 5 d, for enzymolysis in enzyme solution con- taining 1.5% of lywallzyme at 33 ℃ for 3 h, with 0.6 mol/L sucrose as osmotic sta- bilizer, and the regeneration rate had reached 0.22%. [Conclusion] This study provid- ed valuable support for protoplast fusion, ultraviolet mutation breeding and genetic engineering research.

STUDIES ON COLOR TYPE VARIANTSFROM MUTAGENIZED PROTOPLASTS OFPORPHYRA HAITANENSIS CHANG ET ZHENG& P. YEZOENSIS UEDA (RHODOPHYCEASE )

Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04-0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 6.31- 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5- 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5-2-0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced, by colchicine

A Comparison of Different Gracilariopsis lemaneiformis(Rhodophyta) Parts in Biochemical Characteristics, Protoplast Formation and Regeneration

Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.

Isolation of protoplast from callus of Populus euphratica and H^+ fluxes across plasma membrane under NaCl stress

We used callus of Populus euphratica Olive to isolate protoplasts, and IT fluxes across plasma membrane were investigated. The concentration of enzymes for protoplast isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied. High yield and viability of protoplast was achieved after 6-8 hours incubation of P. euphratica callus in enzyme solution containing 1.5% （w：v） cellulase R-10, 0.1% （w：v） pectolyase Y-23, 0.2% （w：v） macerozyme R-10, 0.05% （w：v） hemicellulase and 0.75M）.80 mol·L^-1 sorbitol. Non-invasively ion selective microelectrode technique was used to access proton fluxes in the absence and presence of NaCl （20 mmol.L-1）. Salt-induced transient net IT effiux was observed in the plasma membrane ofP. euphratica cells. The shift of IT flux response to NaC1 shock and the relevance to salt tolerance were discussed.

Enhancement of Arachidonic Acid Production by Mortierella isabellina Through Protoplast Regeneration Mutagenesis

A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme （cellulase and glusulase） at the concentration of 4%, enzymolysis temperature at 30℃ and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-screenings, M. isabellina mutant Y-69 was obtained. GC analysis showed that the yield of arachidonic acid by Y-69 （2.92 g. L-1） was 3.56 times higher than that of the wild-type strain （0.82 g.L-1）. Pass generation tests showed that the properties of Y-69 by mutation were readily inherited.

<i>In Vitro</i>Bioassay of Allelopathy in Four Bamboo Species;<i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku;Phyllostachys bambusoides cv. Madake;P. nigra cv. Hachiku;Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

Studies on the isolation and culture of protoplasts from Kappaphycus alvarezii

In this study, protoplasts were successfully isolated from Kappaphycus alvarezii using snail enzymes, abalone enzymes and cellulase. The optimum enzymic ratio was fixed to be 20% of abalone enzyme, 12% of cellulase and the osmotic stabilizer was 2.0 mol/L glucose. The optimum enzymic hydrolysis conditions were found to be dark enzymolysis at 30°C continuing for 4.0 h. The resultant density and yield of protoplasts achieved 32.60×10^4 mL-1, 65.20×10^4 g-1 tissue for Kappaphycus alvarezii. Finally, under the temperature of 20°C, light intensity of 1 500–2 000 lx and photoperiod of 12 h/d, two developmental pathways were investigated：（1） callus-like cell mass and regenerated plantlet occurred on protoplast;（2） young shoots and calluslike cell mass occurred in tissue blocks after enzymolysis.

Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Improvement of chromium biosorption through protoplast electrofusion between Candida tropicalis and Candida lipolytica

Protoplasts from Candida tropicalis and Candida lipolytica were fused under an optimized electrofusion （electrical pulse strength 6 kV/cm, pulse duration time 40μs and pulse times 5） and then regenerated on YEPD media for achieving new genotypes with higher chromium loading capacity. A target fusant RHJ-004 was screened out by its chromium resistance and chromium-sorbing capacity tests for further research. The comparative study of applicability shows that the fusant has better performance than its parent strains in respect of solution pH, biomass concentration and chromium loading capacity. Especially for treating low concentration Cr（VI） （〈20 mg/L）, above 80% chromium is sequestered from the aqueous phase at pH 1-9. Atomic force microscopy （AFM） visualizes the distribution of chromium on the binding sites of the cells, suggesting that the altered surface structure and intracellular constitutes of the fusant associate with its increased biosorption capacity. The rapid biosorption processes of chromium foUow the Langmuir model well.

Plants regenerated from mesophyll protoplasts of white mulberry

Morus alba(white mulberry) mesophyll protoplasts were isolated from leaves of 30-45 day old sterile shoots,with protoplast yields of 2.5 x 107 g-1/F.W. after purification. The protoplasts were cultured in a modified K8P liquid medium containing 0.2 mg/L 2,4-D(2,4- Dichlorophe-noxy acetic acid), 1 mg/L NAA(Naphthyl acetic acid) and 0.5 mg/L BA(6-benzylaminopurine). A low plating density (5 x 104/ml) proved to be favourable to the division of protoplast-derived cells. The first divisioll occurred 4 days after culture, and the division frequency reached 24% at 10 days. A number of cell colonies and microcalli formed in 6 weeks. The microcalli were transferred onto MSB medium with 0.5 mg/L NAA and 0.5 mg/L BA for further proliferation. Shoot formation was initiated when the calli of 3-4 mm in size were transferred onto MSB differentiation medium with 0.1 mg/L NAA and 1 mg/L BA. The frequency of shoot formation was 35%. The shoots of 4-5 cm in height were excised from the callus and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA. After transplantation into pots, the regenerated plants grew vigorously in the phytotron.

Research on protoplast preparation and culture in Gladiolus hybridus Hort

Protoplasts prepared from the aseptic leaves of Rose Supreme in Gladiolus hybridus Hort. were cultured. The yield of protoplasts with vigour was the highest under the following conditions： 1.5% cellulase Onzuka RS, 0.5% pectinase Y-23, 0.6 mol· L^-1 mannitol, pH 5.8, digestion time 2 h, and temperature 27℃. After isolation and purification, protoplasts were cultured in solid and liquid combined medium, which included KM8P, 0.6 mol· L^-1 mannitol, 1.0 mg· L^-1 2, 4-D, 0.2 mg· L^-1 NAA and 0.2 mg· L^-1 KT. The division of the protoplasts first emerged in medium after cultured 4 days, and emerged only a few times.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of <i>Prunus yedoensis</i>Using Protoplast Co-Culture Bioassay Method

Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated. Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.

Optimization of protoplast isolation,transformation and its application in sugarcane (Saccharum spontaneum L.)

Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.