Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we prese...Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we present an efficacious Agrobacterium tumefaciens-mediated transformation system in somatic embryos of‘Heiye'litchi.This system was developed through the optimization of key variables encompassing explant selection,A.tumefaciens strain delineation,bacterium concentration,infection duration,and infection methodology.The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1,resulting in the generation of transgenic calli.However,the differentiation of transgenic calli into somatic embryos encountered substantial challenges.To delineate the intricate molecular underpinnings of LcMYB1's inhibitory role in somatic embryo induction,a comprehensive transcriptome analysis was conducted that encompassed embryogenic calli(C),globular embryos(G),and transgenic calli(TC).A total of 1,166 common differentially expressed genes(DEGs)were identified between C-vs.-G and C-vs.-TC.Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that these common DEGs were mostly related to plant hormone signal transduction pathways.Furthermore,RT-qPCR corroborated the pronounced down-regulation of numerous genes that are associated with somatic embryo induction within the transgenic calli.The development of this transformation system provides valuable support for functional genomics research in litchi.展开更多
Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results ...Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos （SEs） are poorly understood. In this study, we showed that YUCCAs （YUCs） encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expres- sion analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-l-carboxylic acid （ACC）, or mutation of ETHYLENE-OVERPRODUCTION1 （ET01） disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 （ctrl） mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation.展开更多
We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） ...We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.展开更多
A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree （Cinnamomum camphora L.） is presented. After a subculture of 2-3 years, embryogenic calli could be...A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree （Cinnamomum camphora L.） is presented. After a subculture of 2-3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog （MS） basal medium supplemented with a range of combinations of cytokinins （BA） and auxins （2,4-D or NAA） for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations （〈 5 mg-L-1） had an effect similar to 2,4-D, whereas high concentrations （〉 5 mg·L^-1） of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-D.展开更多
Cytological,biochemical and physiological charcteristics of quiescent,high-vigor carrot somatic embryos obtained with the use of regulation culture were studied. As shown in cytological observation, quiescent somatic ...Cytological,biochemical and physiological charcteristics of quiescent,high-vigor carrot somatic embryos obtained with the use of regulation culture were studied. As shown in cytological observation, quiescent somatic embryos obtained by regulation culturing for 70 days displayed small compacted cells with thick wall, dense cytoplasm and large amount of starch granules. Biochemical tests revealed a starch content and a soluble sugar content 1.9 and 1.7 times respectively as much as those of the control embryos(normal culture) while physiological study demonstrated that the respiration was maintained stally at a very low level,only 1/4 that of the control at the end of the 70-day period. These results provide further evidences that regution culture so after the morphology, biochemistcy, physiologr and metabolism of somatic embryos that they became quiescent,highly synchronized, and had their roots ungrown, a sate facilitating safe storage.展开更多
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
Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentratio...Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.展开更多
Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identi...Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos： the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an in- creasing in sucrose concentration.展开更多
Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant reg...Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant regeneration of shea （Vitellaria paradoxa） from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium （MS） containing 3% sucrose, 0.24% Phytagel, and various concentrations of 2,4-Dichlorophenoxyacetic acid （2,4-D） after four weeks of culture in darkness. Rates of embryogenic callus induction were significantly affected by the addition of 2, 4-D to the medium. Within 28 days of culture, the highest percentage of embyogenic calli （77.61%） occurred on MS media containing 0.45 ~tM of 2,4-D in the dark. Somatic embryos were obtained by culturing embryogenic callus （in the dark） on MS medium fortified with 3% sucrose, 0.24% phytagel and devoid of growth regulators. Culturing at 16 h photoperiod restricted both the induction of embryogenic calli cultures and somatic embryos. Somatic embryos germinated, developed shoots and rooted vigorously on MS medium devoid of growth regulators. Germinated plantlets were acclimatized, successfully.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
In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus.In the present study,the expression level of callose synthase gene in embr...In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus.In the present study,the expression level of callose synthase gene in embryos of E.senticosus in response to 2,4-D,sucrose and mannitol treatments was analyzed by RT-PCR.The results show that plasmolysis pretreatment using sucrose and mannitol significantly promoted the expression of callose synthase gene.Also,the thicker cell walls of explant plasmolyzed compared with controls were observed during the somatic embryogenesis.We suggest that the callose may make the cells in epidermis separate from neighboring cells and then develop into embryogenic potential cells.展开更多
With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo...With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.展开更多
A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a r...A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.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
Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic in...Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.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
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
Using the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel elec...Using the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel electrophoresis（SDS-PAGE） was used for the analysis of soluble proteins produced during the somatic embryogenesis of this plant. There are six polypeptides, which might play some roles in the process of somatic embryo development. Tltree polypeptides（45, 53 and 55 kD） were detected in the stages of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf. One polypeptide（ 83 kD） was specific for the stages of embryoid with eoleoptiles and embryoid with leaf. One polypeptide（37 kD） was detected in the first two stages, namely, embryogenic callus and globular embryoid. One polypeptide（35 kD） was regularly synthesized in each stage, from embryogenic callus to embryoid with leaf.展开更多
Formation of somatic embryos from non-germline cells is unique to higher plants and can be manipulated in a variety of species. Previous studies revealed that overexpression of several Arabidopsis genes, including WUS...Formation of somatic embryos from non-germline cells is unique to higher plants and can be manipulated in a variety of species. Previous studies revealed that overexpression of several Arabidopsis genes, including WUSCHEL （WUS）/PLANT GROWTH ACTIVATOR6 （PGA6）, BABY BOOM, LEAFY COTYLEDON1 （LEC1）, and LEC2, is able to cause vegetative-to-embryonic transition or the formation of somatic embryos. Here, we report that a gain-offunction mutation in the Arabidopsis PGA37 gene, encoding the MYBI18 transcription factor, induced vegetative-toembryonic transition, the formation of somatic embryos from root explants, and an elevated LEC1 expression level. Double mutant analysis showed that WUS was not required for induction of somatic embryos by PGA37/MYB118. In addition, overexpression of MYBll5, a homolog of PGA37/MYB118, caused a pga37-like phenotype. A myb118 myb115 double mutant did not show apparent developmental abnormalities. Collectively, these results suggest that PGA37/ MYB118 and MYB115 promote vegetative-to-embryonic transition, through a signaling pathway independent of WUS.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
基金supported by the National Natural Science Foundation of China(31872066 and 32272663)the Science and Technology Planning Project of Guangzhou,China(2023B01J2002)+2 种基金the Key Research and Development Program of Hainan,China(ZDYF2023XDNY052)the Seed Industry Engineering Project of Department of Agriculture and Rural Affairs of Guangdong,China(2022-NPY-00-004 and 2022-NBH00-001)the Litchi Industry Science and Technology Special Mission of Yunnan,China(202204BI090021)。
文摘Litchi has great economic significance as a global fruit crop.However,the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation.Here,we present an efficacious Agrobacterium tumefaciens-mediated transformation system in somatic embryos of‘Heiye'litchi.This system was developed through the optimization of key variables encompassing explant selection,A.tumefaciens strain delineation,bacterium concentration,infection duration,and infection methodology.The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1,resulting in the generation of transgenic calli.However,the differentiation of transgenic calli into somatic embryos encountered substantial challenges.To delineate the intricate molecular underpinnings of LcMYB1's inhibitory role in somatic embryo induction,a comprehensive transcriptome analysis was conducted that encompassed embryogenic calli(C),globular embryos(G),and transgenic calli(TC).A total of 1,166 common differentially expressed genes(DEGs)were identified between C-vs.-G and C-vs.-TC.Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that these common DEGs were mostly related to plant hormone signal transduction pathways.Furthermore,RT-qPCR corroborated the pronounced down-regulation of numerous genes that are associated with somatic embryo induction within the transgenic calli.The development of this transformation system provides valuable support for functional genomics research in litchi.
文摘Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos （SEs） are poorly understood. In this study, we showed that YUCCAs （YUCs） encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expres- sion analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-l-carboxylic acid （ACC）, or mutation of ETHYLENE-OVERPRODUCTION1 （ET01） disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 （ctrl） mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation.
基金supported by "948" Ad-vanced Forestry Technology Introduction Program (Grant No. 2006-4-73).
文摘We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.
基金supported by the Natural Science Foundation of Henan Province of China(0611033300).
文摘A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree （Cinnamomum camphora L.） is presented. After a subculture of 2-3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog （MS） basal medium supplemented with a range of combinations of cytokinins （BA） and auxins （2,4-D or NAA） for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations （〈 5 mg-L-1） had an effect similar to 2,4-D, whereas high concentrations （〉 5 mg·L^-1） of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-D.
文摘Cytological,biochemical and physiological charcteristics of quiescent,high-vigor carrot somatic embryos obtained with the use of regulation culture were studied. As shown in cytological observation, quiescent somatic embryos obtained by regulation culturing for 70 days displayed small compacted cells with thick wall, dense cytoplasm and large amount of starch granules. Biochemical tests revealed a starch content and a soluble sugar content 1.9 and 1.7 times respectively as much as those of the control embryos(normal culture) while physiological study demonstrated that the respiration was maintained stally at a very low level,only 1/4 that of the control at the end of the 70-day period. These results provide further evidences that regution culture so after the morphology, biochemistcy, physiologr and metabolism of somatic embryos that they became quiescent,highly synchronized, and had their roots ungrown, a sate facilitating safe storage.
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Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.
基金supported by the Foundation for Science and Technology Research and Development Program of Hebei Province (09225511)the Project of the Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of the Ministry of Education, Beijing Forestry University, China (05– 04)
文摘Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos： the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an in- creasing in sucrose concentration.
文摘Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant regeneration of shea （Vitellaria paradoxa） from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium （MS） containing 3% sucrose, 0.24% Phytagel, and various concentrations of 2,4-Dichlorophenoxyacetic acid （2,4-D） after four weeks of culture in darkness. Rates of embryogenic callus induction were significantly affected by the addition of 2, 4-D to the medium. Within 28 days of culture, the highest percentage of embyogenic calli （77.61%） occurred on MS media containing 0.45 ~tM of 2,4-D in the dark. Somatic embryos were obtained by culturing embryogenic callus （in the dark） on MS medium fortified with 3% sucrose, 0.24% phytagel and devoid of growth regulators. Culturing at 16 h photoperiod restricted both the induction of embryogenic calli cultures and somatic embryos. Somatic embryos germinated, developed shoots and rooted vigorously on MS medium devoid of growth regulators. Germinated plantlets were acclimatized, successfully.
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).
基金supported by the 948 Program of State Forestry Administration (No2009-4-26)co-sponsored by SRF for ROCS and the National Natural Science Foundation (No 30972390) of China
文摘In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus.In the present study,the expression level of callose synthase gene in embryos of E.senticosus in response to 2,4-D,sucrose and mannitol treatments was analyzed by RT-PCR.The results show that plasmolysis pretreatment using sucrose and mannitol significantly promoted the expression of callose synthase gene.Also,the thicker cell walls of explant plasmolyzed compared with controls were observed during the somatic embryogenesis.We suggest that the callose may make the cells in epidermis separate from neighboring cells and then develop into embryogenic potential cells.
基金supported by the National High-tech R&D Program(863 Program)of China(2013AA102704)
文摘With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.
文摘A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.
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
文摘Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.
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 National Natural Science Foundation of China(No 30570170)Science and Technology Department of Jinlin Province, P.R.China(No 20050218)+1 种基金Jingyue Zone of Changchun City, P.R.China(No 2005D007) the Program for Changjiang Scholars and Innovative Research Team(PCSIRT) in Universities of P. R.China
文摘Using the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel electrophoresis（SDS-PAGE） was used for the analysis of soluble proteins produced during the somatic embryogenesis of this plant. There are six polypeptides, which might play some roles in the process of somatic embryo development. Tltree polypeptides（45, 53 and 55 kD） were detected in the stages of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf. One polypeptide（ 83 kD） was specific for the stages of embryoid with eoleoptiles and embryoid with leaf. One polypeptide（37 kD） was detected in the first two stages, namely, embryogenic callus and globular embryoid. One polypeptide（35 kD） was regularly synthesized in each stage, from embryogenic callus to embryoid with leaf.
文摘Formation of somatic embryos from non-germline cells is unique to higher plants and can be manipulated in a variety of species. Previous studies revealed that overexpression of several Arabidopsis genes, including WUSCHEL （WUS）/PLANT GROWTH ACTIVATOR6 （PGA6）, BABY BOOM, LEAFY COTYLEDON1 （LEC1）, and LEC2, is able to cause vegetative-to-embryonic transition or the formation of somatic embryos. Here, we report that a gain-offunction mutation in the Arabidopsis PGA37 gene, encoding the MYBI18 transcription factor, induced vegetative-toembryonic transition, the formation of somatic embryos from root explants, and an elevated LEC1 expression level. Double mutant analysis showed that WUS was not required for induction of somatic embryos by PGA37/MYB118. In addition, overexpression of MYBll5, a homolog of PGA37/MYB118, caused a pga37-like phenotype. A myb118 myb115 double mutant did not show apparent developmental abnormalities. Collectively, these results suggest that PGA37/ MYB118 and MYB115 promote vegetative-to-embryonic transition, through a signaling pathway independent of WUS.
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.
