Somatic embryogenesis is an asexual reproduction process that occurs in many plant species,including rice.This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase(SERK),Leaf...Somatic embryogenesis is an asexual reproduction process that occurs in many plant species,including rice.This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase(SERK),Leafy Cotyledon1(LEC1)and WUSCHEL-Related Homeobox4(WOX4)and also a helpful model for embryo development and clones and transformations.Here,we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice(pigmented and non-pigmented)using modified N6 media supplemented with Kinetin(2.0 mg/L)and NAA(1.0 mg/L).Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties,rice is still unexplored,especially during somatic embryo development.Moreover,for the formation of callus induction from immature embryos,2,4-D(2.0 mg/L,3.0 mg/L)was used.This study analysed the gene expression of OsSERK,OsWOX4 and OsLEC1 genes through RT-PCR analysis.Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media.This study found that rice varieties of pigmented rice(MS Pendek and Gogoniti II)and non-pigmented rice(Pandan Ungu)showed high regeneration frequency,showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14.However,the contrast with Genjah nganjuk may be effective because of other regulatory genes.RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties,which correlate with the percentage of plant regeneration,but not for Gogoniti II.In conclusion,the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.展开更多
Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultur...Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins, cytokinins and malt extract for primary callus induction. The best response for primary callus induction (90%) was obtained when MS medium was supplemented with 5 mg/l 2,4-D and 500 mg/l malt extract. Best results for embryogenic callus induction (80%) were obtained in C<sub>8</sub> medium. The induction of somatic embryos was highest when MS medium was supplemented with 1 mg/l BAP and maturation of somatic embryos occurred when MS medium was supplemented with 5 mg/l 2,4-D and 1 mg/l BAP. Maximum plantlets were regenerated (92%) from the somatic embryos on half strength MS medium with no hormones. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1).展开更多
Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence...Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence of these compounds limits their usage as food and feed.To obtain a glandless cotton variety with high-frequency somatic embryo production ability,27 glandless varieties展开更多
Betula platyphylla is a native tree species in northern China that has high economic and medicinal value.We developed an efficient protocol for the induction of somatic embryogenesis in B.platyphalla from immature zyg...Betula platyphylla is a native tree species in northern China that has high economic and medicinal value.We developed an efficient protocol for the induction of somatic embryogenesis in B.platyphalla from immature zygotic embryos and assessed the effects of explant type,genotype,and plant growth regulators(PGRs)on embryogenic callus induction.Among the various explants evaluated,embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid(2,4-D).Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction.On the 20 days after pollination,immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency(16.8%)when cultured on half-strength MS medium supplemented with 2.0 mg L^(-1)2,4-D and 0.2 mg L^(-1)6-benzylaminopurine(6-BA).The process of proliferation of embryogenic callus,somatic embryo formation,and subsequent plantlet conversion occurred under optimal culture conditions.When regenerated plants weretransplanted to soil,95%of them developed normally and grew vigorously.This somatic embryogenesis system required 3–4 months for the regeneration of B.platyphalla plantlets from immature zygotic embryos.展开更多
To determine the optimal embryogenic capacity(somatic embryo production)of the selected elite nematode-resistant genotypes of Pinus thunbergii,variables such as embryogenic tissue(ET)morphology,maternal genotype,proli...To determine the optimal embryogenic capacity(somatic embryo production)of the selected elite nematode-resistant genotypes of Pinus thunbergii,variables such as embryogenic tissue(ET)morphology,maternal genotype,proliferation rate and tissue age were analyzed.ET morphology and histological evaluation of the proliferation stage showed a decrease in filamentous clump and protuberant surfaces and a decline in the acetocarmine-staining area,which indicates a decrease in somatic embryo production(SEP).Variations in cell physiology during the prolifera-tion stage showed that SEP was positively correlated with soluble sugars and proteins,but negatively correlated with starch,peroxidase,and superoxidase.In addition,SEP was significantly(p<0.001)affected by maternal genotype,tis-sue age and proliferation rate.Moreover,SEP was positively correlated with proliferation rate(r=0.98,p<0.001),but negatively correlated with tissue age(r=−0.95,p<0.001).In general,the results suggest that SEP could be assessed in ET proliferation stages by the apparent cell morphology,histology,proliferation rate and tissue age,which provides novel insights for evaluating the ET maturation capacity(number of somatic embryos)during the proliferation stage of P.thunbergii somatic embryogenesis.展开更多
Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for r...Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.展开更多
Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced...Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced from it. The existing cultivars are highly polyploidy and heterozygous in nature. It is propagated through air layering and marcottage methods and storability is very low. Synthetic seeds can be stored for a long time and its genetic constitution could remain the same. For germplasm maintenance and clonal propagation, synthetic seeds can be used. Somatic embryogenesis has been reported from anther or embryogenic suspension culture in various species of litchi. Regeneration via organogenesis and somatic embryogenesis from zygotic embryos has also been reported in certain species. Developing a methodology for getting somatic embryogenesis with a high frequency from zygotic embryos which is available once in a year, would be particularly useful for genetic improvement of litchi. Cotyledonary stage somatic embryos developed from zygotic embryos were encapsulated in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7% agar medium containing 3% sucrose concentration in NN basal medium (half strength of major and minor salts) with 1 mg·l<sup>-1</sup> of gibbrellic acid. Percentage germination and plantlet development for ESEs was higher than that of non encapsulated embryos (NSEs). In comparison to different hormones, gibberellic acid has a significant influence on the germination rate of ESEs after one week of dehydration was seen maximum at 9% sucrose and abscisic acid (1 mg·l-1</sup><sup></sup>) in half strength of major and minor salts in Nitsch and Nitsch medium resulting in extended storage up to 90 days without loss in germination potential and capability to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil to obtain a full grown plant.展开更多
A precocious flowering system of regenerants in asparagus (Asparagus officinalis) was achieved by treatment with a chemical inducer. Somatic embryos withered completely by being processed for 8 - 12 days with 200 μM ...A precocious flowering system of regenerants in asparagus (Asparagus officinalis) was achieved by treatment with a chemical inducer. Somatic embryos withered completely by being processed for 8 - 12 days with 200 μM n-propyl N-(3,4-dichloro-phenyl)carbamate that had been dissolved in distilled water. In contrast, precocious flowering occurred at an extremely low rate (3.4%) when somatic embryos were processed in carbamate dissolved in Murashige and Skoog’s liquid medium. To encapsulate the female and male embryos, we surveyed the optimum conditions of viscosity and concentration of sodium alginate for encapsulating the seeds, and we screened the values of 80 - 120 cps and 2% - 3%, respectively. The synthetic seeds produced also withered when they were processed with the carbamate dissolved in distilled water. However, when Murashige and Skoog’s liquid medium was used for the solvent, the flowering frequency of the synthetic seeds was enhanced (13.3%). Based on our morphological and histological observations, female and male regenerants that were processed with the carbamate solution produced individual flower organs. The conversion of sex expression did not occur. A precocious flowering system would allow a significant reduction in the time required for perennial seedlings to flower and can, therefore, save time required for further experiments that employ floral homeotic mutants.展开更多
文摘Somatic embryogenesis is an asexual reproduction process that occurs in many plant species,including rice.This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase(SERK),Leafy Cotyledon1(LEC1)and WUSCHEL-Related Homeobox4(WOX4)and also a helpful model for embryo development and clones and transformations.Here,we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice(pigmented and non-pigmented)using modified N6 media supplemented with Kinetin(2.0 mg/L)and NAA(1.0 mg/L).Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties,rice is still unexplored,especially during somatic embryo development.Moreover,for the formation of callus induction from immature embryos,2,4-D(2.0 mg/L,3.0 mg/L)was used.This study analysed the gene expression of OsSERK,OsWOX4 and OsLEC1 genes through RT-PCR analysis.Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media.This study found that rice varieties of pigmented rice(MS Pendek and Gogoniti II)and non-pigmented rice(Pandan Ungu)showed high regeneration frequency,showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14.However,the contrast with Genjah nganjuk may be effective because of other regulatory genes.RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties,which correlate with the percentage of plant regeneration,but not for Gogoniti II.In conclusion,the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.
文摘Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins, cytokinins and malt extract for primary callus induction. The best response for primary callus induction (90%) was obtained when MS medium was supplemented with 5 mg/l 2,4-D and 500 mg/l malt extract. Best results for embryogenic callus induction (80%) were obtained in C<sub>8</sub> medium. The induction of somatic embryos was highest when MS medium was supplemented with 1 mg/l BAP and maturation of somatic embryos occurred when MS medium was supplemented with 5 mg/l 2,4-D and 1 mg/l BAP. Maximum plantlets were regenerated (92%) from the somatic embryos on half strength MS medium with no hormones. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1).
文摘Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence of these compounds limits their usage as food and feed.To obtain a glandless cotton variety with high-frequency somatic embryo production ability,27 glandless varieties
基金supported by the National Key Research and Development Programme of China(No.2017YFD0600603)the 111 Project(No.B16010)the Heilongjiang Touyan Innovation Team Program(Tree Genetics and Breeding Innovation Team)。
文摘Betula platyphylla is a native tree species in northern China that has high economic and medicinal value.We developed an efficient protocol for the induction of somatic embryogenesis in B.platyphalla from immature zygotic embryos and assessed the effects of explant type,genotype,and plant growth regulators(PGRs)on embryogenic callus induction.Among the various explants evaluated,embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid(2,4-D).Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction.On the 20 days after pollination,immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency(16.8%)when cultured on half-strength MS medium supplemented with 2.0 mg L^(-1)2,4-D and 0.2 mg L^(-1)6-benzylaminopurine(6-BA).The process of proliferation of embryogenic callus,somatic embryo formation,and subsequent plantlet conversion occurred under optimal culture conditions.When regenerated plants weretransplanted to soil,95%of them developed normally and grew vigorously.This somatic embryogenesis system required 3–4 months for the regeneration of B.platyphalla plantlets from immature zygotic embryos.
基金The work was supported by the National Key Research and Development Projects(2018YFD0600203,2017YFD0600104)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)Doctorate Fellowship Foundation of Nanjing Forestry University.
文摘To determine the optimal embryogenic capacity(somatic embryo production)of the selected elite nematode-resistant genotypes of Pinus thunbergii,variables such as embryogenic tissue(ET)morphology,maternal genotype,proliferation rate and tissue age were analyzed.ET morphology and histological evaluation of the proliferation stage showed a decrease in filamentous clump and protuberant surfaces and a decline in the acetocarmine-staining area,which indicates a decrease in somatic embryo production(SEP).Variations in cell physiology during the prolifera-tion stage showed that SEP was positively correlated with soluble sugars and proteins,but negatively correlated with starch,peroxidase,and superoxidase.In addition,SEP was significantly(p<0.001)affected by maternal genotype,tis-sue age and proliferation rate.Moreover,SEP was positively correlated with proliferation rate(r=0.98,p<0.001),but negatively correlated with tissue age(r=−0.95,p<0.001).In general,the results suggest that SEP could be assessed in ET proliferation stages by the apparent cell morphology,histology,proliferation rate and tissue age,which provides novel insights for evaluating the ET maturation capacity(number of somatic embryos)during the proliferation stage of P.thunbergii somatic embryogenesis.
基金The authors thank Dr.Manzhu Bao(Huazhong Agricultural University,Wuhan,China),Dr.Hibrand-Saint Oyant L.(INRA,Agrocampus-Ouest,Universitéd’Angers,Beaucouzé,France)and Dr.Fabrice Foucher(INRA,78026 Versailles Cedex,France)for their excellent suggestions.We are also grateful to Dr.Wenxue Li and Dr.Hongqiu Wang(Chinese Academy of Agricultural Sci-ences,Beijing,China)for assistance with the experiments.This work was supported by grants from National Natural Science Foundation of China(Grant No.31522049)Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects(Grant No.CEFF-PXM2019_014207_000032).
文摘Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.
文摘Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced from it. The existing cultivars are highly polyploidy and heterozygous in nature. It is propagated through air layering and marcottage methods and storability is very low. Synthetic seeds can be stored for a long time and its genetic constitution could remain the same. For germplasm maintenance and clonal propagation, synthetic seeds can be used. Somatic embryogenesis has been reported from anther or embryogenic suspension culture in various species of litchi. Regeneration via organogenesis and somatic embryogenesis from zygotic embryos has also been reported in certain species. Developing a methodology for getting somatic embryogenesis with a high frequency from zygotic embryos which is available once in a year, would be particularly useful for genetic improvement of litchi. Cotyledonary stage somatic embryos developed from zygotic embryos were encapsulated in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7% agar medium containing 3% sucrose concentration in NN basal medium (half strength of major and minor salts) with 1 mg·l<sup>-1</sup> of gibbrellic acid. Percentage germination and plantlet development for ESEs was higher than that of non encapsulated embryos (NSEs). In comparison to different hormones, gibberellic acid has a significant influence on the germination rate of ESEs after one week of dehydration was seen maximum at 9% sucrose and abscisic acid (1 mg·l-1</sup><sup></sup>) in half strength of major and minor salts in Nitsch and Nitsch medium resulting in extended storage up to 90 days without loss in germination potential and capability to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil to obtain a full grown plant.
文摘A precocious flowering system of regenerants in asparagus (Asparagus officinalis) was achieved by treatment with a chemical inducer. Somatic embryos withered completely by being processed for 8 - 12 days with 200 μM n-propyl N-(3,4-dichloro-phenyl)carbamate that had been dissolved in distilled water. In contrast, precocious flowering occurred at an extremely low rate (3.4%) when somatic embryos were processed in carbamate dissolved in Murashige and Skoog’s liquid medium. To encapsulate the female and male embryos, we surveyed the optimum conditions of viscosity and concentration of sodium alginate for encapsulating the seeds, and we screened the values of 80 - 120 cps and 2% - 3%, respectively. The synthetic seeds produced also withered when they were processed with the carbamate dissolved in distilled water. However, when Murashige and Skoog’s liquid medium was used for the solvent, the flowering frequency of the synthetic seeds was enhanced (13.3%). Based on our morphological and histological observations, female and male regenerants that were processed with the carbamate solution produced individual flower organs. The conversion of sex expression did not occur. A precocious flowering system would allow a significant reduction in the time required for perennial seedlings to flower and can, therefore, save time required for further experiments that employ floral homeotic mutants.