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
The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature e...The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.展开更多
Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by syn...Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.展开更多
White, translucent, glossy mucilaginous callus was initiated from the mature zygotic embryos explants on callus induction medium with 2,4-D, BA, and kinetin in the 3-9th week of culture. This type of callus induction ...White, translucent, glossy mucilaginous callus was initiated from the mature zygotic embryos explants on callus induction medium with 2,4-D, BA, and kinetin in the 3-9th week of culture. This type of callus induction occurred at a lower fre-quency with either a-naphthaleneacetic acid (NAA) or IBA (both 8 mg/L). White, translucent, glossy mucilaginous callus was embryogenic and mainly developed from the cotyledons of the mature zygotic embryo. Somatic embryos were formed on dif-ferentiation medium. Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 mm abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG6000). Scanning electron micros-copy of desiccated somatic embryos showed that the size and external morphology of the desiccation tolerant somatic embryos recovered to the pre-desiccation state within 24-36 h, whereas the sensitive somatic embryos did not recover and remained shriveled, after the desiccated somatic embryos had been rehydrated. Peroxidase activity of desiccated somatic embryos in-creased sharply after 3 days of desiccation treatment, and desiccation tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation tolerant somatic embryos was possibly ad-vantage of catalyzing the reduction of H2O2 which was produced by drought stress, and protecting somatic embryos from oxida-tive damage.展开更多
Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 b...Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.展开更多
I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic cal...I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana （Steven） Spach. Addition- ally, the effect of ABA, PEG-4000 and different wave- lengths on the maturation of somatic embryos was tested. The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of microprop- agation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nord- manniana can be carded out, including the preceding 24-h seed disinfection with NaOCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.展开更多
Osmotic stress promotes somatic embryogenesis of Fraxinus mandshurica,which leads to accumulation of reactive oxygen species(ROS).The single pieces of cotyledons of F.mandshurica were used as explants to induce somati...Osmotic stress promotes somatic embryogenesis of Fraxinus mandshurica,which leads to accumulation of reactive oxygen species(ROS).The single pieces of cotyledons of F.mandshurica were used as explants to induce somatic embryogenesis in osmotic-stress medium.Furthermore,the hydrogen peroxide H_(2)O_(2) content of explanted cells was varied by adding exogenous H_(2)O_(2) or catalase solution to assess the effects of the exogenous H_(2)O_(2)on somatic embryogenesis,intracellular H_(2)O_(2)accumulation,and the relationship between signaling mediated by ROS or reactive nitrogen species.The results revealed that exogenous H_(2)O_(2)(100?300μmol L^(–1))increased the number of somatic embryos.On 60th day of exogenous H_(2)O_(2)(200μmol L^(–1))treatment,the number of somatic embryos of explants treated,which was 136.54%,was higher than the control.Moreover,exogenous H_(2)O_(2)(100μmol L^(–1))significantly increased the intracellular H_(2)O_(2)content and enhanced the activities of superoxidase dismutase and peroxidase.Finally,exogenous H_(2)O_(2)(100μmol L^(–1))activated the intracellular non-enzymatic pathway for nitric oxide(NO)synthesis.The somatic embryogenesis in broadleaf trees increases with the change of endogenic ROS content,and depends on the upregulation of antioxidant enzymes.Both H_(2)O_(2)and NO,as signaling molecules,were found to be involved in the process of somatic embryogenesis in broadleaf trees.In the process of exogenous H_(2)O_(2)promoting somatic embryogenesis,NO synthesis depended on non-enzymatic reactions.These results provide a scientific basis for resolving the mechanism by which ROS levels are regulated during somatic embryogenesis of broadleaf trees and establish a reasonable and efficient technology system for regulating somatic embryogenesis of trees.展开更多
Programmed cell death occurs in browning explants of Fraxinus mandshurica during somatic embryogenesis, but the underlying mechanism is unclear. In this study, single cotyledons of zygotic embryos of F. mandshurica we...Programmed cell death occurs in browning explants of Fraxinus mandshurica during somatic embryogenesis, but the underlying mechanism is unclear. In this study, single cotyledons of zygotic embryos of F. mandshurica were used as explants. Mitochondrial structure and function, caspase-3-like protease activity, hydrogen peroxide metabolism, and nitric oxide accumulation induced by high concentrations of sucrose and plant growth regulators were studied. The results show that plant growth regulators induced somatic embryogenesis and also promoted explant browning. High sucrose concentrations had similar effects. High concentrations of sucrose and plant growth regulators led to the accumulation of hydrogen peroxide and nitric oxide which induced changes in mitochondrial structure and function such as modifications in mitochondrial morphology, increased membrane permeability, decreased membrane potential, and the release of cytochrome c into the cytoplasm. An increase in caspase-3-like protease activity triggered programmed cell death in some browning explant cells. During somatic embryogenesis there were increased activities of superoxide dismutase, peroxidase, and catalase, which are associated with hydrogen peroxide metabolism and jointly maintain reactive oxygen species levels. Intracellular nitric oxide synthase and nitrate reductase activities were not significantly correlated with nitric oxide content. Instead, intracellular nitric oxide may be derived from non-enzymatic reactions. Our results indicate that hydrogen peroxide and nitric oxide may function as signals, playing key roles in somatic embryogenesis and programmed cell death of explant cells of F. mandshurica. The interaction between nitric oxide and reactive oxygen species determines the occurrence of programmed cell death in explant cells;somatic embryogenesis and programmed cell death are positively regulated by hydrogen peroxide. However, the regulation of nitric oxide is complex.展开更多
Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in Hippophae rhamnoides L. was achieved. The influence of basal media, carbon sources, plant growth regulators (PGRs) with dif...Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in Hippophae rhamnoides L. was achieved. The influence of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants was studied. The highest frequency of somatic embryo production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg/L kinetin (KIN) and 0.2, 0.5 mg/L indole-3-butyric acid (IAA). Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55%. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.展开更多
White, translucent, and mucilaginous embryogenic callus was initiated in cultured mature zygotic embryoexplants of two different seed sources of slash pine(Pinus elliottii) on several culture media containing auxin an...White, translucent, and mucilaginous embryogenic callus was initiated in cultured mature zygotic embryoexplants of two different seed sources of slash pine(Pinus elliottii) on several culture media containing auxin and cytokinin.Somatic proembryos were induced on media containing 2,4-D and BA. Maturatiol1 was successfully achieved on mediumsupplemented with ABA. Somatic embryos germinated into regeneration plantlets On DCR medium containing activated charcoal. HiStological observatiol1 and scanning electron inicroscopic observatiol1 showed that proembryos derived from em-bryonal suspensor mass(ESM) were formed on the surface or the inside of embryogenic callus, and the prolitbration of pro-embryos was mainly from clcavage polyembryos.展开更多
Objective: To observe the gene expression of Gankyrin during mouse embryogenesis and reveal the gene biological significance during organs and tissues formation. Methods: The expressions of Gankyrin mRNA in various or...Objective: To observe the gene expression of Gankyrin during mouse embryogenesis and reveal the gene biological significance during organs and tissues formation. Methods: The expressions of Gankyrin mRNA in various organs and tissues were detected by in situ hybridization at indicated times during embryogenesis. Results: The expression of Gankyrin mRNA in mouse day 12.5 embryo was mainly in midbrain, interbrain and endbrain; in mouse day 14.5 embryo mainly in midbrain, aorta, liver, gonad, cranium and rib; in mouse day 16.5 embryo mainly in cranium, rib and vertebra; and in mouse day 18.5 embryo mainly in cranium, rib and intestinal mucosa. Conclusion: Gankyrin gene probably participates in the development of the neural tissues (such as midbrain, interbrain and endbrain etc.), aorta, liver and gonad, intestinal mucosa and bone tissues, which may be closely associated with the function of the organs and tissues.展开更多
Suspension cultures initiated from callus derived from petiole explants of aspen hybrid （Populus tremuloides×P. tremula） produced somatic embryos. Callus was induced on a MS medium supplemented with 5mg·L^...Suspension cultures initiated from callus derived from petiole explants of aspen hybrid （Populus tremuloides×P. tremula） produced somatic embryos. Callus was induced on a MS medium supplemented with 5mg·L^-1 2,4-D and 0.05mg·L^-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10mg·L^-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10mg·L^-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.展开更多
In this study, two different approaches of regeneration systems for explants in Eucalyptus pellita have been taken to establish an efficient di ffer entiation and regeneration protocols. The somatic embryogenesis fro...In this study, two different approaches of regeneration systems for explants in Eucalyptus pellita have been taken to establish an efficient di ffer entiation and regeneration protocols. The somatic embryogenesis from in vitro hy pocotyls and cotyledons in Eucalyptus pellita has been investigated. The typ ical three kinds of callus types were induced from hypocotyls and cotyledons culture d on the 2,4-D containing medium with BA or kinetin. High frequency (82%) plant l et production from the friable, granular embryogenic callus derived from hypocot yls has been obtained on the differentiation medium( 1μM TDZ; 1μM BA plus 01 μMNAA ) developed in this study. The relationship between the callus types form ed on different callus induction media and their corresponding differentiation r ate was further discussed. In addition, plant regeneration through organogenesis from in vitro hypocotyls, cotyledons and mature leaves in Eucalyptus pell ita ha s also been conducted with the modified basal B 5 medium supplemented with a ra ng e of combinations of BA and NAA. The highest frequency of shoot formations for t hree kinds of explants varied on the different regeneration medium, with the reg eneration rate for leaves, cotyledons and hypocotyls in ascending order: 4167% , 6667% and 8333%, respectively. The significance of the two different plant reg eneration systems was discussed. To our knowledge, this is the first repor t on the regeneration of Eucalyptus pellita.展开更多
Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-...Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-ease.The gene daf-8 plays crucial roles in larval develop-ment and dauer formation in Caenorhabditis elegans,but little is known about its orthologue in B.xylophilus.In the present molecular characterization and functional analysis of daf-8 in B.xylophilus(Bx-daf-8),RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage,peaked in the second-stage juvenile(J2),then dramatically dropped in the J3,and remained at that low level for the rest of its life.Bx-daf-8-transgenic C.elegans was employed to mimic the spatiotemporal expression of Bx-daf-8,which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis.It was observed in head neurons and tail ganglions throughout all postembryonic stages.B.xylophilus embryos were severely abnormal,and hatching rate decreased sharply after Bx-daf-8 knockdown.daf-16-1 and da-f16-2,downstream genes in the IIS pathway,also dropped sharply after Bx-daf-8 knockdown.We propose that TGFβmay crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B.xylophilus.However,knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage,indicating that dauer entry in B.xylophilus might be controlled by several genes and is more complicated than in C.elegans.Bx-daf-8 alone did not control the dauer entry in B.xylophilus,but it was indispensable for embryogen-esis,providing a potential target for suppressing population growth of B.xylophilus.展开更多
In vitro conditions of the culture media, plant growth regulators and culture containers may cause anatomical and physiological changes that have negative effects on rooting and ex vitro acclimatization of somatic pla...In vitro conditions of the culture media, plant growth regulators and culture containers may cause anatomical and physiological changes that have negative effects on rooting and ex vitro acclimatization of somatic plantlets. The control of these factors could contribute to the improvement of somatic embryogenesis systems in conifers, especially in pines. The influence of macronutrient concentrations, explant type and culture containers in Pinus radiata D. Don in vitro somatic embryo rooting were analyzed. The highest rooting percentage was observed using half-strength macronutrient concentrations, complete micronutrients and vitamins of Quoirin and Lepoivre medium. Although the use of glass culture vessels was the best to increase the efficiency of the somatic embryogenesis process in terms of rooting, the use of ventilated containers resulted in a significant increase in the percentage of plants able to be planted in the field.展开更多
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.
基金supported by the National Key R&D Program of China(2017YFD0600600).
文摘The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.
基金supported by the Fundamental Research Funds for the Central Universities of China(2572018BW02)the National Natural Science Foundation of China (31400535 and 31570596)+1 种基金the Innovation Project of State Key Laboratory of Tree Genetics and Breeding (2016C01)the National Key R&D Program of China (2017YFD0600600)。
文摘Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.
文摘White, translucent, glossy mucilaginous callus was initiated from the mature zygotic embryos explants on callus induction medium with 2,4-D, BA, and kinetin in the 3-9th week of culture. This type of callus induction occurred at a lower fre-quency with either a-naphthaleneacetic acid (NAA) or IBA (both 8 mg/L). White, translucent, glossy mucilaginous callus was embryogenic and mainly developed from the cotyledons of the mature zygotic embryo. Somatic embryos were formed on dif-ferentiation medium. Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 mm abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG6000). Scanning electron micros-copy of desiccated somatic embryos showed that the size and external morphology of the desiccation tolerant somatic embryos recovered to the pre-desiccation state within 24-36 h, whereas the sensitive somatic embryos did not recover and remained shriveled, after the desiccated somatic embryos had been rehydrated. Peroxidase activity of desiccated somatic embryos in-creased sharply after 3 days of desiccation treatment, and desiccation tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation tolerant somatic embryos was possibly ad-vantage of catalyzing the reduction of H2O2 which was produced by drought stress, and protecting somatic embryos from oxida-tive damage.
文摘Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.
基金supported by research topic DS No.3414 of the Ministry of Science and Higher Education and funded from international corporation Vitroflora
文摘I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana （Steven） Spach. Addition- ally, the effect of ABA, PEG-4000 and different wave- lengths on the maturation of somatic embryos was tested. The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of microprop- agation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nord- manniana can be carded out, including the preceding 24-h seed disinfection with NaOCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.
基金supported by the National Natural Science Foundation of China(31570596 and 31400535)the Fundamental Research Funds for the Central Universities(2572018BW02)+1 种基金the Innovation Project of State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University,2016C01)the National Key R&D Program of China(2017YFD0600600)。
文摘Osmotic stress promotes somatic embryogenesis of Fraxinus mandshurica,which leads to accumulation of reactive oxygen species(ROS).The single pieces of cotyledons of F.mandshurica were used as explants to induce somatic embryogenesis in osmotic-stress medium.Furthermore,the hydrogen peroxide H_(2)O_(2) content of explanted cells was varied by adding exogenous H_(2)O_(2) or catalase solution to assess the effects of the exogenous H_(2)O_(2)on somatic embryogenesis,intracellular H_(2)O_(2)accumulation,and the relationship between signaling mediated by ROS or reactive nitrogen species.The results revealed that exogenous H_(2)O_(2)(100?300μmol L^(–1))increased the number of somatic embryos.On 60th day of exogenous H_(2)O_(2)(200μmol L^(–1))treatment,the number of somatic embryos of explants treated,which was 136.54%,was higher than the control.Moreover,exogenous H_(2)O_(2)(100μmol L^(–1))significantly increased the intracellular H_(2)O_(2)content and enhanced the activities of superoxidase dismutase and peroxidase.Finally,exogenous H_(2)O_(2)(100μmol L^(–1))activated the intracellular non-enzymatic pathway for nitric oxide(NO)synthesis.The somatic embryogenesis in broadleaf trees increases with the change of endogenic ROS content,and depends on the upregulation of antioxidant enzymes.Both H_(2)O_(2)and NO,as signaling molecules,were found to be involved in the process of somatic embryogenesis in broadleaf trees.In the process of exogenous H_(2)O_(2)promoting somatic embryogenesis,NO synthesis depended on non-enzymatic reactions.These results provide a scientific basis for resolving the mechanism by which ROS levels are regulated during somatic embryogenesis of broadleaf trees and establish a reasonable and efficient technology system for regulating somatic embryogenesis of trees.
基金This work was supported by the Fundamental Research Funds for the Central Universities(2572018BW02)the Innovation Project of State Key Laboratory of Tree Genetics and Breeding(2016C01)+1 种基金the National Key R&D Program of China(2017YFD0600600)the National Natural Science Foundation of China(31400535 and 31570596).
文摘Programmed cell death occurs in browning explants of Fraxinus mandshurica during somatic embryogenesis, but the underlying mechanism is unclear. In this study, single cotyledons of zygotic embryos of F. mandshurica were used as explants. Mitochondrial structure and function, caspase-3-like protease activity, hydrogen peroxide metabolism, and nitric oxide accumulation induced by high concentrations of sucrose and plant growth regulators were studied. The results show that plant growth regulators induced somatic embryogenesis and also promoted explant browning. High sucrose concentrations had similar effects. High concentrations of sucrose and plant growth regulators led to the accumulation of hydrogen peroxide and nitric oxide which induced changes in mitochondrial structure and function such as modifications in mitochondrial morphology, increased membrane permeability, decreased membrane potential, and the release of cytochrome c into the cytoplasm. An increase in caspase-3-like protease activity triggered programmed cell death in some browning explant cells. During somatic embryogenesis there were increased activities of superoxide dismutase, peroxidase, and catalase, which are associated with hydrogen peroxide metabolism and jointly maintain reactive oxygen species levels. Intracellular nitric oxide synthase and nitrate reductase activities were not significantly correlated with nitric oxide content. Instead, intracellular nitric oxide may be derived from non-enzymatic reactions. Our results indicate that hydrogen peroxide and nitric oxide may function as signals, playing key roles in somatic embryogenesis and programmed cell death of explant cells of F. mandshurica. The interaction between nitric oxide and reactive oxygen species determines the occurrence of programmed cell death in explant cells;somatic embryogenesis and programmed cell death are positively regulated by hydrogen peroxide. However, the regulation of nitric oxide is complex.
文摘Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in Hippophae rhamnoides L. was achieved. The influence of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants was studied. The highest frequency of somatic embryo production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg/L kinetin (KIN) and 0.2, 0.5 mg/L indole-3-butyric acid (IAA). Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55%. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.
文摘White, translucent, and mucilaginous embryogenic callus was initiated in cultured mature zygotic embryoexplants of two different seed sources of slash pine(Pinus elliottii) on several culture media containing auxin and cytokinin.Somatic proembryos were induced on media containing 2,4-D and BA. Maturatiol1 was successfully achieved on mediumsupplemented with ABA. Somatic embryos germinated into regeneration plantlets On DCR medium containing activated charcoal. HiStological observatiol1 and scanning electron inicroscopic observatiol1 showed that proembryos derived from em-bryonal suspensor mass(ESM) were formed on the surface or the inside of embryogenic callus, and the prolitbration of pro-embryos was mainly from clcavage polyembryos.
文摘Objective: To observe the gene expression of Gankyrin during mouse embryogenesis and reveal the gene biological significance during organs and tissues formation. Methods: The expressions of Gankyrin mRNA in various organs and tissues were detected by in situ hybridization at indicated times during embryogenesis. Results: The expression of Gankyrin mRNA in mouse day 12.5 embryo was mainly in midbrain, interbrain and endbrain; in mouse day 14.5 embryo mainly in midbrain, aorta, liver, gonad, cranium and rib; in mouse day 16.5 embryo mainly in cranium, rib and vertebra; and in mouse day 18.5 embryo mainly in cranium, rib and intestinal mucosa. Conclusion: Gankyrin gene probably participates in the development of the neural tissues (such as midbrain, interbrain and endbrain etc.), aorta, liver and gonad, intestinal mucosa and bone tissues, which may be closely associated with the function of the organs and tissues.
文摘Suspension cultures initiated from callus derived from petiole explants of aspen hybrid （Populus tremuloides×P. tremula） produced somatic embryos. Callus was induced on a MS medium supplemented with 5mg·L^-1 2,4-D and 0.05mg·L^-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10mg·L^-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10mg·L^-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.
文摘In this study, two different approaches of regeneration systems for explants in Eucalyptus pellita have been taken to establish an efficient di ffer entiation and regeneration protocols. The somatic embryogenesis from in vitro hy pocotyls and cotyledons in Eucalyptus pellita has been investigated. The typ ical three kinds of callus types were induced from hypocotyls and cotyledons culture d on the 2,4-D containing medium with BA or kinetin. High frequency (82%) plant l et production from the friable, granular embryogenic callus derived from hypocot yls has been obtained on the differentiation medium( 1μM TDZ; 1μM BA plus 01 μMNAA ) developed in this study. The relationship between the callus types form ed on different callus induction media and their corresponding differentiation r ate was further discussed. In addition, plant regeneration through organogenesis from in vitro hypocotyls, cotyledons and mature leaves in Eucalyptus pell ita ha s also been conducted with the modified basal B 5 medium supplemented with a ra ng e of combinations of BA and NAA. The highest frequency of shoot formations for t hree kinds of explants varied on the different regeneration medium, with the reg eneration rate for leaves, cotyledons and hypocotyls in ascending order: 4167% , 6667% and 8333%, respectively. The significance of the two different plant reg eneration systems was discussed. To our knowledge, this is the first repor t on the regeneration of Eucalyptus pellita.
基金The work was supported by the National Natural Science Foundation of China(31870633,31700565,31670652,31570638 and 31270688)National Key Research and Development Plan of China(2017YFD0600102-03).
文摘Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-ease.The gene daf-8 plays crucial roles in larval develop-ment and dauer formation in Caenorhabditis elegans,but little is known about its orthologue in B.xylophilus.In the present molecular characterization and functional analysis of daf-8 in B.xylophilus(Bx-daf-8),RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage,peaked in the second-stage juvenile(J2),then dramatically dropped in the J3,and remained at that low level for the rest of its life.Bx-daf-8-transgenic C.elegans was employed to mimic the spatiotemporal expression of Bx-daf-8,which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis.It was observed in head neurons and tail ganglions throughout all postembryonic stages.B.xylophilus embryos were severely abnormal,and hatching rate decreased sharply after Bx-daf-8 knockdown.daf-16-1 and da-f16-2,downstream genes in the IIS pathway,also dropped sharply after Bx-daf-8 knockdown.We propose that TGFβmay crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B.xylophilus.However,knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage,indicating that dauer entry in B.xylophilus might be controlled by several genes and is more complicated than in C.elegans.Bx-daf-8 alone did not control the dauer entry in B.xylophilus,but it was indispensable for embryogen-esis,providing a potential target for suppressing population growth of B.xylophilus.
基金funded by MINECO(Spanish Government)project(AGL2013-4700-C4-2R)DECO(Basque Government)
文摘In vitro conditions of the culture media, plant growth regulators and culture containers may cause anatomical and physiological changes that have negative effects on rooting and ex vitro acclimatization of somatic plantlets. The control of these factors could contribute to the improvement of somatic embryogenesis systems in conifers, especially in pines. The influence of macronutrient concentrations, explant type and culture containers in Pinus radiata D. Don in vitro somatic embryo rooting were analyzed. The highest rooting percentage was observed using half-strength macronutrient concentrations, complete micronutrients and vitamins of Quoirin and Lepoivre medium. Although the use of glass culture vessels was the best to increase the efficiency of the somatic embryogenesis process in terms of rooting, the use of ventilated containers resulted in a significant increase in the percentage of plants able to be planted in the field.
