Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,...Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.展开更多
The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryoge...The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.展开更多
Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based...Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypo-cotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.展开更多
On the MS medium supplemented with 2 ppm 2,4-D,calli were induced after 4-6weeks from the petioles of an American celery plant （Apium graveolens var.Dulce pers.cv.Florida）.Suspension culture was started from the cal...On the MS medium supplemented with 2 ppm 2,4-D,calli were induced after 4-6weeks from the petioles of an American celery plant （Apium graveolens var.Dulce pers.cv.Florida）.Suspension culture was started from the calli in a hormone-free liquid MS medium on agyratory shaker at 110 rpm,and kept at 26℃.To stimulate cell division and dedifferentiation,thesubcultures were conducted for 7 days each on the same medium.The liquid suspension containingsingle cells,cell aggregates,and somatic embryos in different stages were screened 2-3 weekslater and 1.0-1.5mm somatic embryos were obtained.These embryos were encapsulated withsodium alginate by dropping-bead method and solidified with 0.1mol CaCl<sub>2</sub>,.These synthetic seedsgerminated and developed well into seedlings in the sterilized vermiculite substrate.展开更多
基金supported by the National Key R&D Program of China (2016YFD0100306)the National Natural Science Fundation of China (31401428)+1 种基金the Fok Ying-Tong Education Foundation of China (151024)the Taishan Scholar Talent Project from China (tsqn20161018)
文摘Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.
基金This work was completed in Tohoku University,Japan
文摘The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.
文摘Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypo-cotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.
文摘On the MS medium supplemented with 2 ppm 2,4-D,calli were induced after 4-6weeks from the petioles of an American celery plant （Apium graveolens var.Dulce pers.cv.Florida）.Suspension culture was started from the calli in a hormone-free liquid MS medium on agyratory shaker at 110 rpm,and kept at 26℃.To stimulate cell division and dedifferentiation,thesubcultures were conducted for 7 days each on the same medium.The liquid suspension containingsingle cells,cell aggregates,and somatic embryos in different stages were screened 2-3 weekslater and 1.0-1.5mm somatic embryos were obtained.These embryos were encapsulated withsodium alginate by dropping-bead method and solidified with 0.1mol CaCl<sub>2</sub>,.These synthetic seedsgerminated and developed well into seedlings in the sterilized vermiculite substrate.
