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.

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.

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.

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.

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.

Somatic embryogenesis and peroxidase activity of desiccation tol-erant mature somatic embryos of loblolly pine

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.

Enhanced Somatic Embryogenesis and Plant Regeneration from Embryogenic Cultures Derived from Mature Loblolly Pine Zygotic Embryos by Suspension Culture and Medium Selection

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.

Plantlet regeneration through somatic embryogenesis in Nordmann's fir (Abies nordmanniana)

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.

Characterization of Gh SERK2 and its expression associated with somatic embryogenesis and hormones level in Upland cotton

Somatic embryogenesis （SE） is one of the most important steps during regeneration of cotton, but the molecular mechanism of SE remains unclear. SOMATIC EMBRYOGENSIS RECEPTOR KINASE （SERK） gene is known to function in SE. A homolog GhSERK2 （accession number： JF430801） was cloned from Upland cotton and characterized for its functions in SE. GhSERK2 expressed in different tissues and showed higher expression level in floral organs than vegetative ones with the highest levels in ovule and anther. GhSERK2 expressed during SE with a high level at globular embryos stage. Upon treatment with indole-3-butytic acid （IBA）, the transcription level of GhSERK2 was induced and promoted SE subsequently. A 2-day treatment of 2,4-dichlorophenoxyacetic acid （2,4-D） induced the expression of GhSERK2, but treatments of 2,4-D for longer periods sharply inhibited the GhSERK2 transcription level of embryogenic callus （EC）. The levels of hormones, including 3-indoleacetic acid （IAA）, abscisic acid （ABA）, and brassinosteroid （BR）, were increased in the initial calli induced from the over-expression of GhSERK2 cotton. Our results indicated that GhSERK2 expression was associated with induction of SE and closely related to hormone levels during tissue culture in Upland cotton, and the gene might play an important role in regeneration of cotton.

Cloning and Characterization of a Somatic Embryogenesis Receptor-Like Kinase Gene in Cotton (Gossypium hirsutum)

A novel gene, GhSERK1, was identified in cotton. It encoded a protein belonging to the somatic embryogenesis receptor- like kinase （SERK） family. The genomic sequence of GhSERK1 was 6 920 bp in length, containing a predicted transcriptional start site （TSS）. Its full-length cDNA was 2 502 bp, encoding a protein of 627 amino acids. Sequence analysis of GhSERK1 revealed high levels of similarity to other reported SERKs, as well as a conserved intron/exon structure that was unique to members of the SERK family. Expression analysis showed that GhSERK1 mRNA was present in all organs of cotton plants and at different developmental stages, but its transcripts were most abundant in reproductive organs. Compared with that of the male-fertile line, the level of GhSERK1 mRNA was lower in the anther of the male-sterile cotton line, in which the pollen development was defected. Taken together, these findings illustrated that the GhSERK1 play a critical role during the anther formation, and may also have a broad role in other aspects of plant development.

Reactive oxygen species, nitric oxide and plant cell death associated with caspase-like protease activity during somatic embryogenesis in Fraxinus mandshurica

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.

Relationship between H_(2)O_(2) accumulation and NO synthesis during osmotic stress:promoted somatic embryogenesis of Fraxinus mandshurica

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.

Studies on the Factors Affecting on Somatic Embryogenesis in Soybean

The effects of the concentration of MS micro salts, 2,4-dichlorophenoxyacetic acid (2,4-D), 3-naphthalene acetic acid (NAA), proline and adenine on callus formation and somatic embryogenesis were investigated using orthogonal design with immature cotyledons of soybean. The results showed that the role of concentration of micro salts on frequency of callus formation and somatic embryogenesis were significant. MS medium supplemented with 2,4-D, NAA, proline and adenine could stimulate callus formation. The concentration of MS micro salts possessed obvious effects on somatic embryogenesis of soybean.The category and concentration of the hormone needed were different among genotypes when embryogenesis were induced.

Exogenous Spermidine Promotes Somatic Embryogenesis of Cunninghamia lanceolata by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C.lanceolata(gymnosperm)regulate somatic embryogenesis,we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis.By applying exogenous spermidine,we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content.Based on these results,further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis.Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.

The Regulatory Roles of microRNAs and Associated Target Genes during Early Somatic Embryogenesis in Liriodendron Sino-Americanum

Somatic cells respond to considerable stress,and go through a series of phytohormone pathways,then forming an embryo.The developmental process is recorded as somatic embryogenesis(SE).One of the key components regulating SE are the microRNAs(miRNAs).Despite previous studies,it is still not clear exactly how miRNAs exert their function of regulating targets during conditionally activated early SE.Here,we use Liriodendron sino-americanum as a model system and perform a combined analysis of microfluidic chips and degradome sequencing to study this process.We identified a total of 386 conserved miRNAs and 153 novel miRNAs during early SE.According to the ANOVA test,239 miRNAs showed 12 distinct expression patterns.Through degradome sequencing,419 targets and 198 targets were identified for 136 known miRNAs and 37 novel miRNAs,respectively.Gene Ontology(GO)and metabolism pathway enrichment analysis revealed that these targets were significantly involved in oxidation-reduction processes,calmodulin-mediated signal transduction pathways and carbohydrate metabolism.The genes that were related to stress responses,phytohormone pathways and plant metabolism were identified within the targets of miR319,miR395,miR408,miR472,miR482,miR390,miR2055,miR156,miR157,miR171,miR396,miR397,miR529,miR535 and miR159.According to promoter analysis,various cis-acting elements related to plant growth and development,phytohormones response and stress response were present in the promoter of the miRNAs.The differential expression patterns of 11 miRNA-target modules were confirmed by real-time quantitative PCR.The study demonstrated that the miRNA plays an important role in the early SE process by regulating its target and then participating in carbohydrate metabolism and stress response.It also provided a valuable resource for further research in determining the genetic mechanism of SE,and then facilitating breeding programs on plants.

Comparative transcriptome study provides insights into acquisition of embryogenic ability in upland cotton during somatic embryogenesis

Background： The conversion from non-embryogenic callus （NEC） to embryogenic callus （EC） is the key bottleneck step in regeneration of upland cotton （Gossypium hirsutum）, and hinders the transgenic breeding of upland cotton. To investigate molecular mechanisms underlying acquisition of embryogenic potential during this process, comparation analysis of transcriptome dynamics between two upland cotton cultivars with different somatic embryogenesis abilities was conducted. Results： Differentially expressed genes involved in the transformation from NEC to EC were detected in the two different cultivars. Principal component analysis based on DEGs showed that the NEC tissues of the two cultivars were highly heterogeneous, whereas the derived EC tissues were similar, which suggested the homogeneousness of EC between different lines. In the highly embryogenic cultivar CCRI 24, more of these genes were down-regulated, whereas, in the recalcitrant cultivar CCRI 12, more were up-regulated. Bioinformatics analysis on these DEGs showed that the vast majority of differentially expressed genes were enriched in metabolism and secondary metabolites biosynthesis pathways. Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were enriched in both cultivars, and the associated genes were down-regulated more in CCRI 24 than in CCRI 12. We deduced that vigorous secondary metabolism in CCRI 12 may hinder primary metabolism, resulting in tardiness of cell differentiation. Interestingly, genes involved in the plant hormone signal transduction pathway were enriched in the recalcitrant cultivar CCRI 12, but not in CCRI 24, suggesting more radical regulation of hormone signal transduction in the recalcitrant cultivar. Signal transduction rather than biosynthesis of plant hormones is more likely to be the determining factor triggering NEC to EC transition in recalcitrant cotton lines. Transcription factor encoding genes showed differential regulation between two cultivars. Conclusions： Our study provides valuable information about the molecular mechanism of conversion from NEC to EC in cotton and allows for identification of novel genes involved. By comparing transcriptome changes in transformation from NEC to EC between the two cultivars, we identified 46 transcripts that may contribute to initiating embryogenic shift.

Somatic Embryogenesis and Plantlet Regeneration in Callus Cultures Derived from Mature Zygotic Embryos of Slash Pine

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.