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.

Rapid Detection of Somatic Cell Count Based on Hybrid Variable Selection Method

Somatic cell count detection is the daily work of dairy farms to monitor the health of cows.The feasibility of applying near-infrared spectroscopy to somatic cell count detection was researched in this paper.Milk samples with different somatic cell counts were collected and preprocessing methods were studied.Variable selection algorithm based on hybrid strategy and modelling method based on ensemble learning were explored for somatic cell count detection.Detection model was used to diagnose subclinical mastitis and the results showed that near-infrared spectroscopy could be a tool to realize rapid detection of somatic cell count in milk.

Sex bias in FGFR3 somatic mutations in bladder cancer

Background:Strong sex disparities have been observed among patients with bladder cancer(BCa).FGFR3 is one of the most frequently mutated genes in bladder cancer,and there are inconsistencies in its frequency in male and female patients.Methods:Here,we conducted a meta-analysis comparing the FGFR3 somatic mutation frequency in men and women among 7351 patients with BCa from 18 cohorts.Results:We showed that female patients had a 1.32 times higher risk of having FGFR3 somatic mutations than males.This difference was attributed to mutations occurring at the 2 most frequently mutated sites,S249 and Y375.Additionally,nonsense mutations were more likely to be found in women,whereas indel/frameshift mutations were almost exclusively found in men;however,no difference was noted for missense mutations.Conclusions:A female sex bias in FGFR3 somatic mutationswas observed in BCa.Well-powered individual participant data analyses addressing the possible confounding effects of other factors(eg,age,ethnicity,smoking status,muscle invasiveness,and molecular subtype),as well as analyses integrating omics and functional investigations,are warranted to further validate and explain the mechanisms of the current findings.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

Udder Health Status of First Parity Dairy Cows in Early-Lactation Based on Somatic Cell Count Categories

The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.

Transcriptome‑wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle

Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

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.

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Regeneration of Diploid Intergeneric Somatic Hybrid Plants Between Microcitrus and Citrus via Electrofusion

Leaf-derived protoplasts of Rough lemon (Citrus jambhiri Lush, 2n = 2x = 18) were electrofused with embryogenic suspension protoplasts of its relative, Microcitrus papuana Swingle (2n = 2x = 18), with an intention of creating novel germplasm. Six plants were regenerated following protoplasts fusion. Cytological examination demonstrated that they were diploids with 18 chromosomes (2n = 2x = 18). RAPD (random amplified polymorphic DNA) analyses with six arbitrary 10-mer primers showed that the regenerated plants had identical band patterns to those of Rough lemon for primers OPA-07, OPAN-07, OPE-05 and OPA-08, Whereas for the other two primers, OPA-04 and OPS-13, bands specific to M. papuana could be detected in the regenerated plants. Cytological and RAPD analysis revealed that the regenerated plants were diploid somatic hybrids between M. papuana and Rough lemon. The putative hybrids were morphologically similar to Rough lemon. This is the first report on production of diploid somatic hybrid plants between citrus with its related genus via symmetric fusion.

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.

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root_tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.

cpSSR: a New Tool to Analyze Chloroplast Genome of Citrus Somatic Hybrids

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and successfully used to analyze chloroplast genome inheritance of Citrus somatic hybrids. Twenty-two previously reported cpSSR primer pairs from pine (Pinus thunbergii Parl.), rice (Otyza sativa L.) and tobacco (Nicotiana tabacum L.) were tested in Citrus, nine of which could amplify intensive PCR products by agarose gel electrophoresis. Chloroplast genome inheritance of Citrus somatic hybrids from nine fusions was then analyzed, and five of the nine pre-screened primer pairs showed polymorphisms by polyacrylamide gel electrophoresis. The results revealed the random inheritance nature of chloroplast genome in all analyzed Citrus somatic hybrids, which was in agreement with previous reports based on RFLP or CAPS analyses. It was also shown that cpSSR is a more efficient tool in chloroplast genome analyses of somatic hybrids in higher plants, compared with the conventional RFLP or CAPS analyses.

Agronomic Trait and Protein Component of F_2 Hybrid Originated from Intergeneric Somatic Hybridization Between Triticum aestivum and Agropyron elongatum

Protoplasts derived from common wheat (Triticum aestivum L,. cv. Jinan 177) were fused with UV-treated protoplasts of Agropyron elongatum. (Host) Nevski by PEG method, and fertile asymmetric somatic hybrid plants resembling wheat morphology were obtained. The F-2 hybrid plants could be divided into 3 types according to their morphology. Type I hybrids had high and loosely standing stalks with big spikes and grains. Type ii hybrids were dwarf and compact in shape with high tillering ability and smaller spikes. Type III hybrids were similar to type I as a whole but had more compact and erect spikes. All the F-2 hybrid lines were superior to wheat in seed protein content, although some difference existed between themselves. Protein analysis of immature embryos and flag leaves from hybrids by two-dimensional electrophoresis showed that they possessed characteristic proteins of both parents and some new proteins. There existed also some different kinds of proteins in different lines.

INTERSPECIFIC SOMATIC HYBRIDS IN ACTINIDIA

Protoplasts isolated from cotyledon-derived calli of Actinidia chinensis var. chinensis (2n = 2x=58) were fused by the PEG method with cotyledon-callus protoplasts of A. deliciosa var. deliciosa (2n = 6x = 174) or with mesophyll protoplasts of A. kolomikta (2n = 2x = 58), respectively. Randomly amplified polymorphic DNA (RAPD) markers and flow cytometry was used to confirm the occurrence of somatic hybrids. RAPD results with some primers surveyed indicated that one clone (A. chinensis + A. deliciosa) and four clones (A. chinensis + A. kolomikta) had RAPD banding patterns which combined the parental banding profiles. Ploidy levels of the (A. chinensis + A. deliciosa) clone were deduced as octoploid (2n = 8x), and the (A. chinensis + A. kolomikta) clones were tetraploid (2n = 4x), triploid (2n = 3x) or pentaploid (2n = 5x). The clones were confirmed as interspecific somatic hybrids in Actinidia.

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.

Production of Somatic Hybrid Plants Between Two Types of Wheat Protoplasts and the Protoplasts of Haynaldia villosa

小麦 (TriticumaestivumL .)济南 177的两种原生质体 ,一种来自快速生长的悬浮细胞 ,它们因长期继代而丧失分化能力 ,其染色体只有 2n =2 4～ 2 8;另一种来自可以再生的愈伤组织 ,其原生质体不能持续分裂。它们中任一种与UV照射过的簇毛麦原生质体融合均不能再生植株。然而当它们混合在一起作为受体时 ,能够获得再生绿色植株。细胞核基因和胞质基因的分析证明这些绿色植株是杂种。以上事实说明这两种原生质体在融合时存在某些互补的关系 ,讨论了这种融合方式的可能作用及重要性。

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.