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.

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.

Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer

[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.

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.

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.

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.

Screening and verification of the factors influencing somatic embryo maturation of Larix olgensis

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.

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.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Role of hydrogen peroxide in stress-induced programmed cell death during somatic embryogenesis in Fraxinus mandshurica

We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.

百子莲体细胞发育受体激酶APSERK1(Somatic Embryogenesis Receptor Kinase1)基因全长克隆及表达分析

根据前期百子莲(Agapanthus praecox ssp.orientalis)转录组测序分析的结果,获得了1个与胚性能力相关的关键基因Somatic Embryogenesis Receptor Kinase1(SERK1)同源性较高的核心片段。采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法得到了百子莲SERK基因cDNA全长序列,命名为ApSERK1。序列分析表明,百子莲ApSERK1基因开放阅读框(ORF)为1800bp,编码1个含有600个氨基酸的蛋白质。氨基酸同源性比对发现,百子莲ApSERK1蛋白序列与水稻、小兰屿蝴蝶兰、铁皮石斛、椰子树和菠萝等植物蛋白序列的相似性可达到90%以上。实时荧光定量qRT-PCR结果表明,ApSERK1基因有一定时空表达差异,在百子莲的种子、小花梗中表达含量最高,且随着外源生长素浓度的增加,胚性愈伤组织的胚性能力呈现下降趋势。推测ApSERK1基因是衡量植物细胞胚性能力的重要指标。

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.

Somatic Embryogenesis in Lily Bulb Scale Cultures

Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.

Effect of Rare Earth Elements on the Induction Frequency of the Somatic Cell Embryo in The Fruit of Chinese Wolfoerry

A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.

Effect of the thin cell layer technique in the induction of somatic embryos in Pinus patula Schl. et Cham

Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option for the rapid multiplication of high-quality,genetically improved material.This study induces somatic embryogenesis in this species using the thin cell layer(TCL)technique.Two sources of explants(complete immature embryos;lTCL segments from immature embryos)were evaluated.The efficiency of TCL from longitudinal sections[lTCL]and transverse[tTCL]was evaluated.The results show using thin cell layers from immature embryos cultivated in 16 light/8 dark hours achieves induction of somatic embryos.A higher percentage of embryogenic callus was obtained when tTCL segments were used as an explant source.These results produced somatic embryos from tTCL segments of an immature embryo without germinating the seed,making the process more time efficient.In addition,this technique can be used to generate somatic embryogenesis in forest species that have low germination rates.

Breeding and Simple-rapid Regeneration Protocol for Jisheng 1 with Glandless Trait and High-frequency Somatic Embryo Production Ability

Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence of these compounds limits their usage as food and feed.To obtain a glandless cotton variety with high-frequency somatic embryo production ability,27 glandless varieties

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.

Oxidative Stress Responsive <i>SERK1</i>Gene Directs the Progression of Somatic Embryogenesis in Cotton (<i>Gossypium hirsutum</i>L. cv. Coker 310)

Somatic embryogenesis (SE) is a prominent mode of regeneration in plants. The acquisition of SE is predominantly invoked by the oxidative stress which plays an important role in signal transduction and cellular redox. Since balanced generation of oxidants is important to cellular differentiation, modulation in cell redox could be responsive to genotypic refinement for SE. To study the dynamics of cellular redox during SE, we conducted comparative expression analyses of cotton (Gossypium hirsutum), using two independently purified near-isogenic lines for the trait of SE. We interrogated expression changes in cell-signaling factor Somatic Embryogenesis Receptor Kinase (SERK) and activity of antioxidant Glutathione in different developmental stages including cotyledonary leaf, calli from different stages of regeneration of fully-regenerating (FR) and non-regenerating (NR) lines of Coker310 cultivar. At evolutionary scale, the cotton SERKs showed high sequence similarity in receptor kinase domain with diverse systems. Exclusively, SERK1 responsible for potential signaling processes during SE revealed significant expression up-regulation in the embryogenic calli of FR line. Similarly, activity of antioxidant glutathione was substantially up-regulated in embryogenic calli of FR line in comparison to its counterpart form. In contrast, calli from early-stages of regeneration of both FR and NR lines had no significant influences on the regulation of SERK and glutathione levels prior to the acquisition of embryogenesis. These results highlight that in vitro purification of FR line in cotton for enhanced regeneration potential (through SE) resulted in signaling and metabolic transformations of the manner in which cellular redox levels have become modulated.