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
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
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
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 an...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.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
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
Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation te...Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia (3 d) of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L-1 MgSO_4 + 10 mmoI·L-1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase: more than 106 protoplasts mL-1 were obtained. When collected 3y density gradient centrifugation, the concentration of protoplasts can reach 107-108 protoplasts mL-1, an amount suitable enough for experiments of transformation in fungi. For every 10_5 protoplasts, about 15-25 protoplasts can egenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora solate.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
We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized an...We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized and germinated. The immature leaves were cut into 3-5 mm pieces when the third or forth leaf appeared. Leaf pieces were inoculated on MS medium with 2,4-D 4 mg/1, NAA 2mg/1 and IAA Img/1. After 2 wk culture, calli were induced and subcultured once or twice for multiplication. 3-5 g calli were transferred to the modified MS liquid medium with 2,4-D 2 mg/1 and KT 0.5mg/1 for suspension culture. Embryogenic cell suspension was established after 2 mo culture. The effect of the growth period of suspension cells on the展开更多
Mature-embryo-derived calli or japonica rice (Oryza sativa L)Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisractory yield of 5. 20...Mature-embryo-derived calli or japonica rice (Oryza sativa L)Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisractory yield of 5. 20×106~8. 96× 106 protoplasts/g FW(fresh weight).The remaining three callus lines Initiated from seeds of cryopreserved- callus-derived plants had rooty calli, resulting in low yield or protoplasts and a large number or isolated banana- shape intact cells. Viability or protoplasts ranged 87.46% ~94.15%.The average size or Protoplasts was 207. 49~379. 04μm2 in different callus lines.Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts.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
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.
基金The National Science Foundation Project under contract No.2007FY210500the National Department Public Benefit Research Foundation of China under contract No.200805075+2 种基金the Province Science and Technology in the Guangdong Project under contract Nos 2010B060200010 and 2010B020201015the Science Expenditure in the Hainan Project under contract No.11-20410-0015the National Natural Science Foundation of China under contract Nos 41206106 and 41222038
文摘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.
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 the National Key Project of Scientific and Technical Supporting Programs funded by the Ministry of Science & Technology, China (No. 2012BAC01B05-3)
文摘Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia (3 d) of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L-1 MgSO_4 + 10 mmoI·L-1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase: more than 106 protoplasts mL-1 were obtained. When collected 3y density gradient centrifugation, the concentration of protoplasts can reach 107-108 protoplasts mL-1, an amount suitable enough for experiments of transformation in fungi. For every 10_5 protoplasts, about 15-25 protoplasts can egenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora solate.
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.
文摘We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized and germinated. The immature leaves were cut into 3-5 mm pieces when the third or forth leaf appeared. Leaf pieces were inoculated on MS medium with 2,4-D 4 mg/1, NAA 2mg/1 and IAA Img/1. After 2 wk culture, calli were induced and subcultured once or twice for multiplication. 3-5 g calli were transferred to the modified MS liquid medium with 2,4-D 2 mg/1 and KT 0.5mg/1 for suspension culture. Embryogenic cell suspension was established after 2 mo culture. The effect of the growth period of suspension cells on the
文摘Mature-embryo-derived calli or japonica rice (Oryza sativa L)Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisractory yield of 5. 20×106~8. 96× 106 protoplasts/g FW(fresh weight).The remaining three callus lines Initiated from seeds of cryopreserved- callus-derived plants had rooty calli, resulting in low yield or protoplasts and a large number or isolated banana- shape intact cells. Viability or protoplasts ranged 87.46% ~94.15%.The average size or Protoplasts was 207. 49~379. 04μm2 in different callus lines.Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts.