Young embryos of ricy (Oryza sativa L.subsp.japonica var.Guo-xiang No.1) were cultured on MS agar medium(2,4-D 2 mg/l).Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l).After selection,the small,grain...Young embryos of ricy (Oryza sativa L.subsp.japonica var.Guo-xiang No.1) were cultured on MS agar medium(2,4-D 2 mg/l).Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l).After selection,the small,grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l,6-BA 0.2 mg/l)^1* with agarose block culture method.The protoplasts grew,divided and formed calli.After inducing differentiation,the regenerated mature plants were obtained.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
An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid...An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L kinetin (KT) and 0.2 mg/L naphthalene acetic acid (NAA), the division frequency of protoplastderived cells reached uP to over 60 %, and microcalli were obtained in 5-6 wk. Upon transferring them on agar solidified MS medium plus 2 mg/L indole-3-acetic acid (IAA), shoots were induced. After cultivating them on MS medium with or without IAA, roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal (2n=28). The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.展开更多
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 K...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.展开更多
Protoplast isolation was relevant for gene manipulation in U lva, and universal protocols have been proposed based on evaluation for various wildly collected species. However, only clonal laboratory cultures were prac...Protoplast isolation was relevant for gene manipulation in U lva, and universal protocols have been proposed based on evaluation for various wildly collected species. However, only clonal laboratory cultures were practical for genetic transformation, and whether applicability of such universal protocol existed for these artificial cultures has never been investigated. In this research, samples in different physiological states or developmental stages were tested in U. prolifera. The results proved that the protoplast yields were strongly dependent on the characteristics of samples. Neither F_v/F_m value nor chlorophyll content exhibited an ideal correlation with the protoplast yields. Alternatively, specific growth rate, coupled with developmental stage, could serve as an ef fective combined index to determine the right time for protoplast isolation. According to this instruction, here we reported the highest yields of protoplast((31.5±1.9)×10~6 cells/g f. wt.) in U. prolifera, following comparison between protocols, and further optimizations on enzyme content, incubation period, starting biomass and pretreatment. This specified protocol for artificially cultured clonal samples could meet the need for protoplast-mediated genetic transformation in U. prolifera.展开更多
Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world.Great attention has long been paid to the technique of protoplast culture of this species.Up to now, plantlet regeneration from proto...Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world.Great attention has long been paid to the technique of protoplast culture of this species.Up to now, plantlet regeneration from protoplasts of anther-derived suspension cultures of wheat has been reported for the first time. In the present study, we have obtained numerous embryoids and plantlets from protoplasts of immature inflorescence-derived calli of spring wheat.展开更多
Populus tomentosa is one special species of poplar growing in North China. Mesophyllprotoplasts were isolated from the axenic shoots and cultured in the modified KM8p liquidmedium. Protoplast-derived cells started to ...Populus tomentosa is one special species of poplar growing in North China. Mesophyllprotoplasts were isolated from the axenic shoots and cultured in the modified KM8p liquidmedium. Protoplast-derived cells started to divide after 7 days of culture. The frequencyof cell division reached about 20% in 10 days. The yellowish green calli grew compact andnodular after the hormone concentration of medium was adjusted. Shoot formation occurredwhen the protoplast--derived calli were transferred onto MS medium containing zeatin andIAA or NAA. The shoots rooted readily on 1/2 MS hormone--free medium.展开更多
Embryogenic calli were induced from the mature seeds of the hexaploid semi-winter wheat. The embryogenic cell line was established, and then the friable calli suitable for suspension were induced by adjusting the cont...Embryogenic calli were induced from the mature seeds of the hexaploid semi-winter wheat. The embryogenic cell line was established, and then the friable calli suitable for suspension were induced by adjusting the content of reduced N and changing the 2,4DAA concentration. Protoplasts were isolated from the suspension cells and cultured(?)in KM8P and some other media. A great number of microcolonies were obtained. By altering media, the microcolonies were promoted to grow further and form compact or nodule-like calli. Intact plants were regenerated through organogenesis and embryogenesis on differentiation media.展开更多
文摘Young embryos of ricy (Oryza sativa L.subsp.japonica var.Guo-xiang No.1) were cultured on MS agar medium(2,4-D 2 mg/l).Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l).After selection,the small,grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l,6-BA 0.2 mg/l)^1* with agarose block culture method.The protoplasts grew,divided and formed calli.After inducing differentiation,the regenerated mature plants were obtained.
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
文摘An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L kinetin (KT) and 0.2 mg/L naphthalene acetic acid (NAA), the division frequency of protoplastderived cells reached uP to over 60 %, and microcalli were obtained in 5-6 wk. Upon transferring them on agar solidified MS medium plus 2 mg/L indole-3-acetic acid (IAA), shoots were induced. After cultivating them on MS medium with or without IAA, roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal (2n=28). The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.
文摘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.
基金Supported by the National Natural Science Foundation of China(No.41776153)the Scientific and Technological Innovation Project financially supported by Qingdao National Laboratory for Marine Science and Technology(No.2016ASKJ02-1)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDA11020304)the Postdoctoral Application Research Program funded by Qingdao(No.2016189)
文摘Protoplast isolation was relevant for gene manipulation in U lva, and universal protocols have been proposed based on evaluation for various wildly collected species. However, only clonal laboratory cultures were practical for genetic transformation, and whether applicability of such universal protocol existed for these artificial cultures has never been investigated. In this research, samples in different physiological states or developmental stages were tested in U. prolifera. The results proved that the protoplast yields were strongly dependent on the characteristics of samples. Neither F_v/F_m value nor chlorophyll content exhibited an ideal correlation with the protoplast yields. Alternatively, specific growth rate, coupled with developmental stage, could serve as an ef fective combined index to determine the right time for protoplast isolation. According to this instruction, here we reported the highest yields of protoplast((31.5±1.9)×10~6 cells/g f. wt.) in U. prolifera, following comparison between protocols, and further optimizations on enzyme content, incubation period, starting biomass and pretreatment. This specified protocol for artificially cultured clonal samples could meet the need for protoplast-mediated genetic transformation in U. prolifera.
文摘Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world.Great attention has long been paid to the technique of protoplast culture of this species.Up to now, plantlet regeneration from protoplasts of anther-derived suspension cultures of wheat has been reported for the first time. In the present study, we have obtained numerous embryoids and plantlets from protoplasts of immature inflorescence-derived calli of spring wheat.
基金This research is granted by the National Biotechnology Programme.
文摘Populus tomentosa is one special species of poplar growing in North China. Mesophyllprotoplasts were isolated from the axenic shoots and cultured in the modified KM8p liquidmedium. Protoplast-derived cells started to divide after 7 days of culture. The frequencyof cell division reached about 20% in 10 days. The yellowish green calli grew compact andnodular after the hormone concentration of medium was adjusted. Shoot formation occurredwhen the protoplast--derived calli were transferred onto MS medium containing zeatin andIAA or NAA. The shoots rooted readily on 1/2 MS hormone--free medium.
基金This work was supported in part by the Monsando Inc., USA.
文摘Embryogenic calli were induced from the mature seeds of the hexaploid semi-winter wheat. The embryogenic cell line was established, and then the friable calli suitable for suspension were induced by adjusting the content of reduced N and changing the 2,4DAA concentration. Protoplasts were isolated from the suspension cells and cultured(?)in KM8P and some other media. A great number of microcolonies were obtained. By altering media, the microcolonies were promoted to grow further and form compact or nodule-like calli. Intact plants were regenerated through organogenesis and embryogenesis on differentiation media.