Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

Genotypic effects on accelerated propagation of oil palm breeding materials selected(Elaeis guineensis jacq.)using somatic embryogenesis

Vegetable oil production from oil palm(Elaeis guineensis Jacq.)is an important industry due to the rising demand every year.The somatic embryogenesis culture can propagate oil palm duplicate as parent plant,which can be selected as breeding material to produce new planting germplasm with high production or disease resistance.This study aims to evaluate the genotypic effect of somatic embryogenesis,while immature leaflets were employed as explants.The culture used embryo induction medium based on Murashige and Skoog(MS)modifications that contained 5 mg/L Naphthalene Acetic acid(NAA)and 0.5 mg/L Benzyl Amino Purine(BAP).The genotypic effect was statistically significant in the percentage of callus induction,producing somatic embryos,and germination embryos.In this study,we successfully cloned thirteen oil palm genotypes(GE-02,GE-03,GE-06,GE-07,GE-09,GE-23,GE-24,GE-27,GE-28,GE-32,GE-33,GE-34,and GE-35),with the highest number of somatic embryos formed on GE-27 with a percentage of 70.1%.The cloning was successful in accelerating the propagation of oil palm for materials breeding programs to create new varieties with high production and disease resistance.It is necessary to observation the performance of these clones in the field in terms of mantle flower appearance.

Histological Study on Soybean Somatic Embryogenesis

Soybean somatic cell could induce the development of embryoid which was similar to embryo morphologically and structurally. Somatic embryogenesis system of soybean was used to conduct genetic transformation of soybean because of its several advantages such as higher transformational efficiency, beetter synchronism and fewer plant chimeras among transgenic plants. After infected with agrobacterium tumefaciens,the initiation, differentiation and development of young cotyledon embryogenic cell of soybean which was cultured on selective culture medium with kanamycin were investigated through histological study. The result showed that somatic embryo was differentiated in non-bud differentiation way. The embryogenic cells were differentiated from epidermis of explant or cells in 1 layer or 2 layers, with the division of embryogenic cells and degradation and disorganization of surrounding cells, the embryogenic cells would form embryoid with analogous suspensor structure. Later, globular embryoid would extrude from epidermis then developed into heart-shape embryo. The experiment was expected to provide theoretical reference for the construction of high transformational system of using plant somatic embryogenesis induced by young cotyledon of soybean.

Stress Treatments and DNA Methylation Affected the Somatic Embryogenesis of Citrus Callus

The evaluation on the callus embryogenesis capacity of 15 genotypes of citrus showed that stress treatments were conducive to somatic embryogenesis and could enhance the recovery of the missed capacity of embryogenesis for some genotypes. Randomly amplified polymorphic DNA (RAPD) and methylation sensitive amplified polymorphism (MSAP) analysis indicated that there existed significant differences in DNA methylation status between the callus capable of producing somatic embryoids and that which missed the embryogenesis capacity of the same genotype Newhall navel orange ( Citrus sinensis Osb. cv. Newhall). The DNA methylation level of the former was lower than that of the latter. However, RAPD profiles did not show any difference between these two kinds of callus.

Study on the Effect of Osmosis-regulating Substances and Organic Appendices on Somatic Embryogenesis in Wheat

[Objective] The study aimed to reveal the effect of osmosis-regulating substances and organic appendices on somatic embryogenesis in wheat. [Method] The suitable concentration combination of appendices was optimized by adding different concentrations of osmosis-regulating substances including mannitol, sorbitol and organic appendices such as Gln, CH and LH, into the somatic embryogenesis in wheat. [Result] The mannitol or sorbitol lower than 40 g/L was helpful for improving somatic embryogenesis; there was no significant difference in the induction rate of somatic embryogenesis when 300-500 mg/L Gln、CH or LH was respectively added into the induced medium, while somatic embryogenesis could be enhanced dramatically in the presence of 500 mg/L Gln together with 300 mg/L CH. [Conclusion] Somatic embryogenesis could be improved to some extent by different concentrations of osmosis-regulating substances and organic appendices, which laid foundation for establishing a more perfect system of somatic embryogenesis in wheat.

Embryogenesis,Germination,Structure and Cotyledon Dimorphism of Zea mays Embryo

A series of new cognitions on the morphogenesis of maize ( Zea mays L.) embryo have been obtained with scanning electron microscopy and semi-thin section techniques. 1. The proembryo. The proembryo from zygotic cell divisions may be divided into three parts: proper, hypoblast and suspensor. The suspensor is short and small, and only exists transiently. As to the hypoblast there is a growth belt, which promotes elongation of the hypoblast. Eventually the upper portion of the hypoblast contributes to the formation of the coleorhiza and the remainder dries up, sticking to the end of the coleorhiza. 2. The maize embryo possesses dorsiventrality and cotyledon dimorphism. During early proembryo stage, the dorsiventrality appears in the proper of the embryo. On the ventral side, the cells are small with dense cytoplasm and few vacuoles. On the dorsal side, the cells are larger with lower cytoplasmic density and have more vacuoles. During later proembryo stage, the proper develops into two parts: the ventrum and the dorsurn. The ventrum rises up from the center of the ventral side. The dorsurn is composed of the marginal area of the ventral side and the whole dorsal side of the proper. During young embryo development, the ventrum differentiates into the coleoptile, apical meristem, hypocotyl, radicle and the main part of the coleorhiza. What is more important, the emergence of coleoptile primordium and radicular initials occur at the axis of the proper, then the coleoptile primordium expands from its two ends toward left and right to form a ring, and the endogenous radicular initials expand in all directions to form a conical radicular tip. All these morphogenetic activities of the ventrum follow a bilateral symmetrical pattern. The dorsurn forms the scutellum. primordium. Then the scutellum primordium, expands rapidly toward the left, right, front and back, while thickening itself, so as to make all components originating from the ventrum become hidden in the longitudinal groove of the scutellum. Lastly, the left and right lateral scales emerge from the edges of the longitudinal groove and expand toward the central line of the axis. As a consequence, morphologically, the bilateral symmetry of the ventral side of the embryo is revealed entirely. Morphogenetically, the coleoptile primordium and apical meristem in maize are similar to the coleoptile (apical cotyledon) and apex formation of the nice embryo, so the coleoptile of the maize embryo can also be considered as an apical cotyledon. The scutellum is a lateral cotyledon. These dimorphic cotyledons of the maize embryo originate from the dorsiventrality of the proper. 3. The true morphological structure of the maize embryo is recognized and its developmental stages are established. A maize embryo is a hypocotyl, in which the apical part is the shoot apex (or plumule) with the coleoptile, the central part consists mainly of the hypocotyl with a lateral cotyledon (scutellum), and the basal part is the radicle with coleorhiza. The left and right lateral scales derived from the scutellum overlap at the ventral side, leaving only two little pores at both ends of the seam from which the coleoptile and coleorhiza can be seen. The four sequential stages of maize embryonic development are as follows: (1) proembryo, stage. This stage covers a period from zygotic cell division to the appearance of the dorsum and ventrum. (2) ventrum rapid differentiation stage. (3) scutellum rapid expansion stage. (4) lateral scale development stage (or embryonic envelope formation stage). 4. To obtain a median longitudinal section perpendicular to the ventral surface is crucial for recognizing the genuine morphological structure of the maize embryo.

Fluctuations of Endogenous Hormones in Isolated Rice Embryos During Embryogenesis and Early Stages of Germination

Fluctuations of levels of several endogenous plant hormones in isolated rice ( Oryza sativa ssp. japonica) embryos during early and mid-embryogenesis and early stages of germination were studied by enzyme-linked immunosorbent assay (ELISA). Embryos were collected at different days after pollination (DAP) and different days after imbibition (DAI) of mature seeds. The contents of gibberellin(1) (GA(1)), abscisic acid (ABA), isopentenyladenine and isopentenyladenosine ( iPAs), zeatin and zeatin riboside ( ZRs) were immunochemically assayed. The GA(1) level was the highest among all hormones tested. The variations of GA(1) levels were opposite with the ABA levels, with some exceptions. During early and mid-embryogenesis, the levels of GA(1) and ABA were the highest at 4 DAP. From 8 to 18 DAP, GA(1) level declined, whereas the ABA level increased. During germination, GA(1) level increased at 2 DAI whereas simultaneously the ABA content decreased. The highest ratio of GA(1)/ABA was observed at 2 DAI The levels of iPAs and ZRs were maxima in the embryos at 4 DAP, decreased to a very low level and maintained constant thereafter. Our results provide further evidence that GA(1) plays an important role in the early stages of embryo development and germination. The balance between GA(1) and ABA, rather than their absolute contents, controls these processes throughout the development, whereas iPAs and ZRs may play important roles in early embryogenesis. The use of isolated embryos as starting material avoids the usual interferences with other tissues such as the endosperm. In addition, this is the first report dealing with the hormonal balance of early-embryos in rice.

Enzyme Solutions and H_2O_2 on Somatic Embryogenesis of Fraxinus mandshurica Rupr. (Oleaceae)

[Objective] This study aimed to explore the effects of treatments with three types of exogenous oxidase solutions and H2O2.solution on the somatic embryogenesis of Fraxinus mandshurica Rupr. （Oleaceae）. [Method] The immature zygotic cotyledons were treated with PPQ （polyphenol oxidase） solution, GQD （glucose oxidase） solution, SOD （superoxide dismutase） solution and H202 （hydrogen peroxide） at different concentrations to explore the effects on the growth, browning and somatic embryogenesis on cotyledon explants in the somatic embryogenesis of F. mandshurica. Through comparative analysis on the effects of different treatments on somatic embryogenesis of F. mandshurica, the relationship between explants browning and somatic embryogenesis was uncovered during the somatic embryogenesis of F. mandshurica. [Result] H2O2 treatment not only advanced the explants browning, but also inhibited the growth and somatic embryogenesis of explants; different concentrations of PPQ promoted the growth and browning of explants, as well as improving the incidence of somatic embryogenesis; both GOD and SOD treatment could raise the explants browning rate; when somatic embryogenesis of explants treated with enzyme solutions advanced, the incidence of somatic embryogenesis was low; however, when the disparity of the incidence of somatic embryogenesis between 30 and 60 d treatments reached its peak, the incidence of somatic embryogenesis was also high. [Conclusion] The results of this study provide basis for raising the incidence and improving the status of somatic embryogenesis of F. mandshurica, as well as optimizing the somatic embryogenesis system of F. mandshurica.

Embryogenesis and Development of Isolated Microspore in Chinese Cabbage

[Objective] The aim was to observe embryogenesis and development of isolated microspores in Chinese cabbage. [Method] Chinese cabbage F 1 hybrids were used as the experimental materials, and optical microspore was employed to observe the embryogenesis and development of isolated microspores. [Result] Cells swelled after heat shock treatment, which was the critical factor of embryoid induction. Three pathways equal division, unequal division and germination of microspores were discovered to lead to the embryogenesis from isolated microspores after swelling. Microspore could grow directly to embryoid through germination path way. Equally divided microspores formed the original embryos after successive multiple equal divisions. Original embryos could develop into cotyledon-shaped embryos via globular, heart-shaped and torpedo-shaped embryos. The large one of the two cells from unequally divided microspores continued to divide and finally formed a polar embryoid. [Conclusion] The study will provide cytological basis for high induction frequency and embryoid of Chinese cabbage.

Effect of plant growth regulators on direct somatic embryogenesis in camphor tree(Cinnamomum camphora L.)from immature zygotic embryos and embryogenic calli induction

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.

Studies on Parameters Affecting Embryogenesis of Protoplasts Isolated from Suspension of Embryogenic Cells in Loblolly Pine

Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.

Embryogenesis and Development of Isolated Microspores in Raphanus sativus

[Objective] The aim was to observe embryogenesis and development of isolated microspores of Raphanus sativus.[Method] By using Chinese radish variety Shandong Huaye Xinlimei as the experimental materials,the caryocinesia features and development of isolated microspores in radish were studied.[Result] Two types of cell division appeared in the isolated microspore：unequal division and equational division.The unequal divided microspores had two different size nucleuses,while the equational divided microspores had two same size nucleuses.The equational divided microspores developed to 4-cell structure,multi-cell structure,ball-shaped embryos,heart-shaped embryos,torpedo-stage embryos and cotyledon-stage embryos.[Conclusion] This result will provide cytological basis for the isolated microspore culture of radish.

Identification of Z-OTU protein during zebrafish oogenesis and early embryogenesis

Zebrafish（Danio rerio） Z-OTU,containing OTU and TUDOR domains,was predicted to be a member of OTU-related protease,a family of the deubiquitylating enzymes（DUBs）.A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA.Here,we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis.After prokaryotic expression,the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU,which was used for immunohistochemistry in zebrafish ovaries and embryos.Interestingly,obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis.In stage I oocytes,z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle.In addition,its protein was distributed during entire oogenesis,while mRNA was not detected in oocytes at stage IV or mature oocytes.The z-otu mRNA disappeared after midblastula transition（MBT） and its protein gradually decreased after this stage.We inferred that Z-OTU protein,like other OTU-related protease with DUB activity,was required for germinal vesicle breakdown of oocytes during meiosis,germinal vesicle migration,and embryo cleavage maintenance.

Plant Regeneration of Sweet Potato via Somatic Embryogenesis from Different Explants

[Objective] This study aimed to regenerate plants of sweet potato (Ipomoea batatas) cultivar Xushu22 via somatic embryogenesis, using leaf and shoot apex as explants. [Method] The leaf and shoot apex of Xushu 22 were separately cultured on MSB medium and MSD medium. The induced embryogenic calluses were then cultured on MS medium. The regeneration frequency of leaf and shoot apex explants were respectively calculated. [Result] The average frequency of leaf explants developing somatic callus was 95.69% compared to 30.56% in case of shoot apex explants. There were different types of morphogenic structures in the process of somatic embryo development. Leaf explants gave a high regeneration frequency to 60.61%, while the regeneration frequency of shoot apices was 22%. In addition, no morphological variations were observed in the regeneration plants. [Conclusion] Leaf explant was better than shoot apices in plant regeneration of Xushu22 via somatic embryogenesis.

The paternal epigenome and embryogenesis： poising mechanisms for development

The scope of paternal contributions during early embryonic development has long been considered limited. Dramatic changes in chromatin structure throughout spermatogenesis have been thought to leave the sperm void of complex layers of epigenetic regulation over the DNA blueprint, thus leaving the balance of that regulation to the oocyte. However, recent work in the fields of epigenetics and male factor infertility has placed this long-held, and now controversial dogma, in a new light. Elegant studies investigating chromatin and epigenetic modifications in the developing sperm cell have provided new insights that may establish a more critical role for the paternal epigenome in the developing embryo. DNA methylation, histone tail modifications, targeted histone retention and protamine incorporation into the chromatin have great influence in the developing sperm cell. Perturbations in the establishment and/or maintenance of any of these epigenetic marks have been demonstrated to affect fertility status, ranging in severity from mild to catastrophic. Sperm require this myriad of chromatin structural changes not only to serve a protective role to DNA throughout spermatogenesis and future delivery to the egg, but also, it appears, to contribute to the developmental program of the future embryo. This review will focus on our current understanding of the epigenetics of sperm. We will discuss sperm-specific chromatin modifications that result in genes essential to development being poised for activation early in embryonic development, the disruption of which may result in reduced fecundity.

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

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.

The MADS-box transcription factor CmAGL11 modulates somatic embryogenesis in Chinese chestnut(Castanea mollissima Blume)

Somatic embryogenesis(SE)is an effective approach of in vitro regeneration that depends on plant cell totipotency.However,largely unknown of molecular mechanisms of SE in woody plants such as Chinese chestnut(Castanea mollissima Blume),limits the development of the woody plant industry.Here,we report the MADS-box transcription factor Cm AGL11 in Chinese chestnut.Cm AGL11 transcripts specifically accumulated in the globular embryo.Overexpression of Cm AGL11 in chestnut callus enhanced its SE capacity,and the development of somatic embryos occurred significantly faster than in the control.RNA-seq results showed that Cm AGL11 affects the expression of several genes related to the gibberellin,auxin,and ethylene pathways.Moreover,the analysis of DNA methylation status indicated that the promoter methylation plays a role in regulation of Cm AGL11 expression during SE.Our results demonstrated that Cm AGL11 plays an important role in the SE process in Chinese chestnut,possibly by regulating gibberellin,auxin,and ethylene pathways.It will help establish an efficient platform to accelerate genetic improvement and germplasm innovation in Chinese chestnut.

Foregut duplication cysts:A report of two cases with emphasis on embryogenesis

Duplication cyst of the stomach with a pseudostratifie columnar ciliated epithelium is extremely rare.We de scribe two cases of these cysts,with emphasis on the immunophenotype and embryogenesis.The first patien was a 29-year-old man who presented with crampin abdominal pain in his left lower quadrant.The secon patient was a 26-year-old woman who had a history over several years,of chronic epigastric abdominal pai radiating to her back.Both lesions were surgically re moved.They showed the same histomorphology.Th cysts were lined by a pseudostratified respiratory ep thelium with ciliated cells.The first cyst was connecte to the stomach,while the second cyst was not connect ed.Both cysts expressed thyroid transcription factor(TTF-1) and surfactant.In this report,we explore th possible embryogenesis of these lesions in the light o TTF-1 and surfactant expression.

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg·L^-1. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g· L^-1 glutamine and 0.5 g·L^-1 asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g·L^-1 glutamine, 0.5 g·L^-1 asparagine, and 6.0 g·L^-1 agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

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.