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
Bacillus thuringiensis(Bt)toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops.The discovery of endophytic Bt strains opened new perspectives f...Bacillus thuringiensis(Bt)toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops.The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sap‐sucking insects,such as the Asian citrus psyllid Diaphorina citri Kuwayama(Hemiptera:Liviidae),a vector of“Candidatus Liberibacter spp.,”associated with citrus huanglongbing(HLB).In this study,translocation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil‐drench,and their systemic pathogenicity to D.citri nymphs were investigated.The pathogenicity of three wild‐type Bt strains against D.citri third‐instar nymphs was demonstrated.Among the 10 recombinant strains tested(each of them harboring a single cry or cyt gene),3 can be highlighted,causing 42%–77%and 66%–90%nymphal mortality at 2 and 5 d after inoculation,respectively.The isolation of Bt cells from young citrus shoots and dead nymphs,and PCR performed with specific primers,confirmed the involvement of the Bt strains in the psyllid mortality.This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D.citri nymphs that fed on these soil‐drench inoculated seedlings.The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D.citri and the identified genes can be used for the production of transgenic Bt citrus.展开更多
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This study was financed in part by the Coordenacao de Aperfeigoamento de Pessoal de Nivel Superior-Brasil(CAPES)and by the Brazilian Agricultural Research Corporation(EMBRAPA-grant number 03.13.03.006.00.00)。
文摘Bacillus thuringiensis(Bt)toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops.The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sap‐sucking insects,such as the Asian citrus psyllid Diaphorina citri Kuwayama(Hemiptera:Liviidae),a vector of“Candidatus Liberibacter spp.,”associated with citrus huanglongbing(HLB).In this study,translocation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil‐drench,and their systemic pathogenicity to D.citri nymphs were investigated.The pathogenicity of three wild‐type Bt strains against D.citri third‐instar nymphs was demonstrated.Among the 10 recombinant strains tested(each of them harboring a single cry or cyt gene),3 can be highlighted,causing 42%–77%and 66%–90%nymphal mortality at 2 and 5 d after inoculation,respectively.The isolation of Bt cells from young citrus shoots and dead nymphs,and PCR performed with specific primers,confirmed the involvement of the Bt strains in the psyllid mortality.This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D.citri nymphs that fed on these soil‐drench inoculated seedlings.The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D.citri and the identified genes can be used for the production of transgenic Bt citrus.
