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 sys...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.展开更多
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 offsp...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展开更多
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 reg...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.展开更多
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 d...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.展开更多
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 (Rhodoph...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.展开更多
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 suspen...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.展开更多
Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a ...Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn’t result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.展开更多
The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal forc...The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%;with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h;considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h;meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.展开更多
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 transforma...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.展开更多
In the present study, it is aimed to investigate embryo and protoplast isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic process. Globular embryo...In the present study, it is aimed to investigate embryo and protoplast isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic process. Globular embryos with suspensor were fully removed by enzymatic maceration for between 100-180 min of incubation. The highest obtained embryo yields were found in Rapidase EX Color and Rapidase Vino Super, with degree of 89.2-90.3 × 103·g-1 MF and 86.4-91.3 × 103·g-1 MF, respectively. According to multicomponent maceration tests, a treatment comprising 150 min of the enzymatic reaction with 0.18% (E/S) at 50℃ was the most optimal for obtaining high embryo yields. Regarding protoplast isolation tests, enzyme combinations C (1% Cellulase “Onozuca” R-10 + 0.2% Macerozyme R-10 and 0.1% Driselase) and F (1% Cellulase “Onozuka” + 1% Macerozyme + 0.5% Driselase) after 15 hours produced the best results for protoplast isolation and were significantly different compared to other enzyme combinations. Greater protoplast yields were obtained using a rotatory system with protoplasts incubated in the dark. Also the present findings were discussed from the point of view of in vitro plant manipulation and orchid cultivation in cultural media and then their importance emphasized in molecular biological/genetical studies.展开更多
[Objective]The paper was to reveal the preparation and regeneration conditions of Ceratocystis caradoxa protoplast.[Method]Using multi-factors orthogonal test,the effects of hypha age,enzyme system,time of hydrolysis,...[Objective]The paper was to reveal the preparation and regeneration conditions of Ceratocystis caradoxa protoplast.[Method]Using multi-factors orthogonal test,the effects of hypha age,enzyme system,time of hydrolysis,hydrolysis temperature,osmotic pressure stabilizer and regeneration medium on C.paradoxa protoplast were studied.[Result]The optimum condition for preparing protoplast were conidia cultured in liguid SYM medium for 24 h,enzyme mixture of 1%driselase and 1%lytic enzyme used to digest hypha for 1.5 h,0.7 mol/L MgSO4·7H2O used as osmotic stabilizer,or individual enzyme 1%driselase used to digest hypha for 1.5 h,1.0 mol/L mannitol as osmotic stabilizer.The regeneration rate was over 40%under C.paradoxa protoplast regenerating on CM medium with mannitol and hydrolysis for 2 h.[Conclusion]Higher protoplast yield and higher regeneration rate could be obtained under above conditions,which is beneficial for transformation and further research.展开更多
This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activiti...This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of C.militaris were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as Ac-Cm.On the other hand,when A.cinnamomea and C.militaris were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as Cm-Ac.We selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental A.cinnamomea and C.militaris,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA sequencing.In comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium conditions.For gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.展开更多
This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incuba...This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield. The optimized isolation conditions were: 40% base solution 3(deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2 ·2 H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2% w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was(1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.展开更多
Compact calli derived from immature spikelet of a foxtail millet variety—Jigu 11cann’t be directly used for protoplast isolation because of its firm physical structure,and must beloosened with subculturing in M<s...Compact calli derived from immature spikelet of a foxtail millet variety—Jigu 11cann’t be directly used for protoplast isolation because of its firm physical structure,and must beloosened with subculturing in M<sub>1</sub>,M<sub>2</sub> and M<sub>3</sub> media successively and altering these media compo-sitions.The loosened calli can be selected from the regulation and used for protoplast isolationsuccessfully.Rate of protoplast division in KM<sub>8</sub>P medium was 12.3—33.5%.Calli derivedthrough protoplast division are loose and cann’t be used directly for plan regeneration because ofits soft physical structure.When they were subcultured in N<sub>6</sub>—1,N<sub>6</sub>—2,N<sub>6</sub>—3 and N<sub>6</sub>—4 media,in which the media compositions were changed,the compact calli were obtained and 129 plantletswere regenerated from them.101 plants,which grew to maturity after transplanting the plantletsinto field,exhibited sterility in some degree.Most of the subsequent lines derived from the regen-erated plants were sterile and only two lines could get normal reproduction.展开更多
Eight F<sub>1</sub>-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plan...Eight F<sub>1</sub>-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere展开更多
The progress in protoplast technology and its application in fruit breeding were summarized,and the existing problems and application prospect were also discussed.
The inhibitory allelopathic activity of </span><i><span style="font-family:Verdana;">Pueraria montana </span></i></span><span style="font-family:Verdana;">...The inhibitory allelopathic activity of </span><i><span style="font-family:Verdana;">Pueraria montana </span></i></span><span style="font-family:Verdana;">(Kudzu), and activities of two putative allelochemical isoflavones, puerarin and daidzein, were evaluated using the protoplast co-culture method with digital image analysis using lettuce as a recipient (DIA-PP</span><span style="font-family:""> </span><span style="font-family:""><span style="font-family:Verdana;">method). Cotyledon protoplasts of Kudzu were isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution. Optimal hormonal condition and density for growth of Kudzu protoplasts were surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce protoplasts was 50 μl liquid MS basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or 0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu at a low density (10</span><sup><span style="font-family:Verdana;">4</span></sup><span style="font-family:Verdana;">/ml). Slightly less inhibition by Kudzu on cell wall formation and yellow pigment accumulation stages of lettuce growth was also observed. Puerarin did not inhibit the growth of lettuce protoplasts at three growth stages but slightly stimulated growth at high concentrations. By contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of lettuce protoplast growth and strongly inhibited cell division at 100 μM. Daidzein might be one cause of the strong inhibitory allelopathic activity of Kudzu. Grade of inhibitory activities w</span></span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> compared with th</span><span style="font-family:Verdana;">at</span><span style="font-family:Verdana;"> of other allelopathic plants including an invader plant and their allelochemicals studied using the DIA-PP method.展开更多
An effiecent cell suspension culture system has been initiated using callus obtained frommature seeds of the Chinese Japonica variety Eryi 105 as a source.Over the last 12 months,using the'Nottingham Method',p...An effiecent cell suspension culture system has been initiated using callus obtained frommature seeds of the Chinese Japonica variety Eryi 105 as a source.Over the last 12 months,using the'Nottingham Method',protoplasts have been produced from these cell suspen-sions,and via the PEG method of transformation.45 regenerated plants were obtained fromDNA-treated protoplasts selected by 80μg/ml G418.The research of molecular analysis onthose plantlets is being carried on by Plant Genetic Manipulation Group,Department of LifeScience,University of Nottingham,U.K.展开更多
[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,ly...[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,lysozyme concentration,enzymolysis temperature and time on the protoplast formation rate and regeneration rate were investigated by single factor experiments.On this basis,with the protoplast formation rate as an index,the protoplast preparation process was optimized by an orthogonal experiment.[Results]Bacterial age and enzymolysis time had a greater impact on the protoplast formation rate,followed by enzymolysis temperature and lysozyme concentration.The optimal process for the preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasts from bacterial cells cultured for 15 h in the late logarithmic growth phase by enzymolysis with 0.8 mg/ml lysozyme at 37℃for 180 min and promote fusion with PEG6000.In order to facilitate the screening of fusion protoplasts,the empty plasmids p ET-28a and p ET-Duet were transformed into L-lysine-producing E.coli,respectively,and strains p ET-28a-lys01 and p ET-Duet-lys01 were obtained.Fusion strains were then obtained through protoplast fusion.Double-resistance KA1-10were screened on plates containing kanamycin and ampicillin,and a high-yielding fusion strain KA8,which produced L-lysine,was screened by fermentation experiments finally.[Conclusions]The results of this study provide a reference method for further improving the yield of L-lysine and other amino acid strains.展开更多
基金supported by the National Natural Science Foundation of ChinaChina (Grant Nos. 31872051, 32072528)the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。
文摘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.
文摘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
基金Supported by Young Scientifi c Fund of Heilongjiang Province (QC2010093)Project of Daqing Scientifi c Program (sggh2009-029)
文摘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.
文摘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.
文摘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.
文摘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.
文摘Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn’t result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.
文摘The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%;with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h;considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h;meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.
基金funded by the National Natural Science Foundation of China(3190020451 and 31771862)。
文摘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.
文摘In the present study, it is aimed to investigate embryo and protoplast isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic process. Globular embryos with suspensor were fully removed by enzymatic maceration for between 100-180 min of incubation. The highest obtained embryo yields were found in Rapidase EX Color and Rapidase Vino Super, with degree of 89.2-90.3 × 103·g-1 MF and 86.4-91.3 × 103·g-1 MF, respectively. According to multicomponent maceration tests, a treatment comprising 150 min of the enzymatic reaction with 0.18% (E/S) at 50℃ was the most optimal for obtaining high embryo yields. Regarding protoplast isolation tests, enzyme combinations C (1% Cellulase “Onozuca” R-10 + 0.2% Macerozyme R-10 and 0.1% Driselase) and F (1% Cellulase “Onozuka” + 1% Macerozyme + 0.5% Driselase) after 15 hours produced the best results for protoplast isolation and were significantly different compared to other enzyme combinations. Greater protoplast yields were obtained using a rotatory system with protoplasts incubated in the dark. Also the present findings were discussed from the point of view of in vitro plant manipulation and orchid cultivation in cultural media and then their importance emphasized in molecular biological/genetical studies.
基金Supported by Key Project of Hainan Province(ZDYF2019072)Basic Scientific Research Project of CATAS(16301520190010).
文摘[Objective]The paper was to reveal the preparation and regeneration conditions of Ceratocystis caradoxa protoplast.[Method]Using multi-factors orthogonal test,the effects of hypha age,enzyme system,time of hydrolysis,hydrolysis temperature,osmotic pressure stabilizer and regeneration medium on C.paradoxa protoplast were studied.[Result]The optimum condition for preparing protoplast were conidia cultured in liguid SYM medium for 24 h,enzyme mixture of 1%driselase and 1%lytic enzyme used to digest hypha for 1.5 h,0.7 mol/L MgSO4·7H2O used as osmotic stabilizer,or individual enzyme 1%driselase used to digest hypha for 1.5 h,1.0 mol/L mannitol as osmotic stabilizer.The regeneration rate was over 40%under C.paradoxa protoplast regenerating on CM medium with mannitol and hydrolysis for 2 h.[Conclusion]Higher protoplast yield and higher regeneration rate could be obtained under above conditions,which is beneficial for transformation and further research.
基金supported by grants from the Ministry of Science and Technology of Taiwan(Grant number:MOST 106-2320-B-037008-MY2,MOST 108-2320-B-037-022-MY3,108-2811-B-037-511,and 109-2927-I-037-502)funded by the Drug Development and Value Creation Research Center,Kaohsiung Medical UniversityDepartment of Medical Research,Kaohsiung Medical University Hospital(Grant number:KMU-TC108A03-11)。
文摘This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of C.militaris were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as Ac-Cm.On the other hand,when A.cinnamomea and C.militaris were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as Cm-Ac.We selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental A.cinnamomea and C.militaris,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA sequencing.In comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium conditions.For gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.
基金Supported by the China Agriculture Research System(No.CARS-50)the Science and Technology Program of Guangdong Province of China(Nos.2016A020222023,2015B090903081)the Project of Guangdong Province Education Department(No.2017KCXTD014)
文摘This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield. The optimized isolation conditions were: 40% base solution 3(deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2 ·2 H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2% w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was(1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.
文摘Compact calli derived from immature spikelet of a foxtail millet variety—Jigu 11cann’t be directly used for protoplast isolation because of its firm physical structure,and must beloosened with subculturing in M<sub>1</sub>,M<sub>2</sub> and M<sub>3</sub> media successively and altering these media compo-sitions.The loosened calli can be selected from the regulation and used for protoplast isolationsuccessfully.Rate of protoplast division in KM<sub>8</sub>P medium was 12.3—33.5%.Calli derivedthrough protoplast division are loose and cann’t be used directly for plan regeneration because ofits soft physical structure.When they were subcultured in N<sub>6</sub>—1,N<sub>6</sub>—2,N<sub>6</sub>—3 and N<sub>6</sub>—4 media,in which the media compositions were changed,the compact calli were obtained and 129 plantletswere regenerated from them.101 plants,which grew to maturity after transplanting the plantletsinto field,exhibited sterility in some degree.Most of the subsequent lines derived from the regen-erated plants were sterile and only two lines could get normal reproduction.
文摘Eight F<sub>1</sub>-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere
基金Supported by National Agro-industry(Apple Industry)Technology Research System/China Agriculture Research System of Apple(nycytx-27)
文摘The progress in protoplast technology and its application in fruit breeding were summarized,and the existing problems and application prospect were also discussed.
文摘The inhibitory allelopathic activity of </span><i><span style="font-family:Verdana;">Pueraria montana </span></i></span><span style="font-family:Verdana;">(Kudzu), and activities of two putative allelochemical isoflavones, puerarin and daidzein, were evaluated using the protoplast co-culture method with digital image analysis using lettuce as a recipient (DIA-PP</span><span style="font-family:""> </span><span style="font-family:""><span style="font-family:Verdana;">method). Cotyledon protoplasts of Kudzu were isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution. Optimal hormonal condition and density for growth of Kudzu protoplasts were surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce protoplasts was 50 μl liquid MS basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or 0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu at a low density (10</span><sup><span style="font-family:Verdana;">4</span></sup><span style="font-family:Verdana;">/ml). Slightly less inhibition by Kudzu on cell wall formation and yellow pigment accumulation stages of lettuce growth was also observed. Puerarin did not inhibit the growth of lettuce protoplasts at three growth stages but slightly stimulated growth at high concentrations. By contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of lettuce protoplast growth and strongly inhibited cell division at 100 μM. Daidzein might be one cause of the strong inhibitory allelopathic activity of Kudzu. Grade of inhibitory activities w</span></span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> compared with th</span><span style="font-family:Verdana;">at</span><span style="font-family:Verdana;"> of other allelopathic plants including an invader plant and their allelochemicals studied using the DIA-PP method.
文摘An effiecent cell suspension culture system has been initiated using callus obtained frommature seeds of the Chinese Japonica variety Eryi 105 as a source.Over the last 12 months,using the'Nottingham Method',protoplasts have been produced from these cell suspen-sions,and via the PEG method of transformation.45 regenerated plants were obtained fromDNA-treated protoplasts selected by 80μg/ml G418.The research of molecular analysis onthose plantlets is being carried on by Plant Genetic Manipulation Group,Department of LifeScience,University of Nottingham,U.K.
基金Supported by the Focus on Research and Development Plan in Shandong Province(2019JZZY011003,2020CXGC010603)National Natural Science Foundation of China(31801527)。
文摘[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,lysozyme concentration,enzymolysis temperature and time on the protoplast formation rate and regeneration rate were investigated by single factor experiments.On this basis,with the protoplast formation rate as an index,the protoplast preparation process was optimized by an orthogonal experiment.[Results]Bacterial age and enzymolysis time had a greater impact on the protoplast formation rate,followed by enzymolysis temperature and lysozyme concentration.The optimal process for the preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasts from bacterial cells cultured for 15 h in the late logarithmic growth phase by enzymolysis with 0.8 mg/ml lysozyme at 37℃for 180 min and promote fusion with PEG6000.In order to facilitate the screening of fusion protoplasts,the empty plasmids p ET-28a and p ET-Duet were transformed into L-lysine-producing E.coli,respectively,and strains p ET-28a-lys01 and p ET-Duet-lys01 were obtained.Fusion strains were then obtained through protoplast fusion.Double-resistance KA1-10were screened on plates containing kanamycin and ampicillin,and a high-yielding fusion strain KA8,which produced L-lysine,was screened by fermentation experiments finally.[Conclusions]The results of this study provide a reference method for further improving the yield of L-lysine and other amino acid strains.
