Embryo Differentiation Research Articles

Influence of Incubation Period on Callus Tissues for Plant Regeneration in Ophiorrhiza pectinata Arn. Through Somatic Embryogenesis

Ophiorrhiza pectinata Arn. is an important medicinal plant reported to contain camptothecin which is used for the treatment of different types of cancers. The present study describes an efficient plant regeneration system through somatic embryogenesis. Embryogenic callus tissues raised from aseptic leaf explants were used for inducing somatic embryos under incubation of callus tissues at varying periods from 10 to 60 days. It was noticed that callus tissues incubated between 30 and 50 days in 0.5 mg l−1 2,4-dichlorophenoxy acetic acid were responded positively in half-strength Murashige and Skoog liquid media supplemented with 3 mg l−1 indole-3-acetic acid and started differentiation of globular structures in 15–20 days. Maximum differentiation of embryos was obtained in callus tissues incubated for 40 days. Clumps of embryos at different stages got differentiated into plantlets in MS media supplemented with 6-benzyl aminopurine (0.2 mg l−1) and indole-3-acetic acid (0.1 mg l−1) and produced an average of 47.92 ± 5.33 plants from 1.0 to 1.5 g callus tissues. The regenerated plantlets were successfully deflasked, hardened and established in paper cups filled with coarse sand and recorded more than 90% establishment under shade net house (50% shade) condition with adequate watering. The method of plant regeneration system developed would be useful for raising large number of plants and production of bioactive compounds.

The necessity of TEAD4 for early development and gene expression involved in differentiation in porcine embryos

TEA domain family transcription factor 4 (Tead4) is known to be important for the trophectoderm (TE) segregation in murine embryos. However, the role of TEAD4 in early development of porcine embryos is still unknown. We examined TEAD4 expression patterns and attempted to determine the functions of TEAD4 during porcine preimplantation development using RNA interference. TEAD4 mRNA was upregulated from the 2–4-cell to 8–16-cell stages and then decreased to the blastocyst stage. Nuclear localization of TEAD4 protein was detected at the 16-cell stage, as well as at subsequent developmental stages. In porcine embryos injected with TEAD4 siRNA, transformation from morula to blastocyst was inhibited. Although TEAD4 downregulation did not affect the expression levels of POU class 5 homeobox 1 (OCT-4), transcription of SRY-related HMG-box gene 2 (SOX2) was detected at high level in TEAD4-downregulated embryos. It is possible that TEAD4 contributes to blastocyst formation in porcine embryos through downregulation of SOX2 expression. Collectively, our results indicate that TEAD4 is an important factor for the preimplantation development of porcine embryos.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

Characterization of ADP-ribosyl cyclase 1-like (ARC1-like) activity and NAADP signaling during slow muscle cell development in zebrafish embryos

We recently demonstrated the requirement of two-pore channel type 2 (TPC2)-mediated Ca2+ release during slow muscle cell differentiation and motor circuit maturation in intact zebrafish embryos. However, the upstream trigger(s) of TPC2/Ca2+ signaling during these developmental processes remains unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing messenger, which is suggested to target TPC2 in mediating the release of Ca2+ from acidic vesicles. Here, we report the molecular cloning of the zebrafish ADP ribosyl cyclase (ARC) homolog (i.e., ARC1-like), which is a putative enzyme for generating NAADP. We characterized the expression of the arc1-like transcript and the NAADP levels between ~ 16 h post-fertilization (hpf) and ~ 48 hpf in whole zebrafish embryos. We showed that if ARC1-like (when fused with either EGFP or tdTomato) was overexpressed it localized in the plasma membrane, and associated with intracellular organelles, such as the acidic vesicles, Golgi complex and sarcoplasmic reticulum, in primary muscle cell cultures. Morpholino (MO)-mediated knockdown of arc1-like or pharmacological inhibition of ARC1-like (via treatment with nicotinamide), led to an attenuation of Ca2+ signaling and disruption of slow muscle cell development. In addition, the injection of arc1-like mRNA into ARC1-like morphants partially rescued the Ca2+ signals and slow muscle cell development. Together, our data might suggest a link between ARC1-like, NAADP, TPC2 and Ca2+ signaling during zebrafish myogenesis.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

Explant type and stress treatment determine the uni- and multicellular origin of somatic embryos in the tree fern Cyathea delgadii Sternb.

Cyathea delgadii is the first fern species for which somatic embryogenesis (SE) has recently been described. With this discovery, a new experimental model for exploration of SE was found. In this study, the effect of explant type (stipe and internode), length and diameter of the explant-donor frond, and stress treatment (by sucrose and air desiccation) on somatic embryo origin and SE efficiency was studied. In control culture, somatic embryos originated from single cells of stipe explants, whereas those induced on internodes were of multicellular origin. Although the activation of cell divisions was more abundant and the formation of somatic embryos occurred earlier in a culture of internodes than of stipe explants, the morphogenic capacity of internodes was much smaller. On their surface, the groups of competent cells formed protuberances that give rise to only three somatic embryos per internode. In contrast, almost 92% of stipe explants that were excised from the first frond, measuring 10 mm in length and less than 300 µm in diameter, produced an average of 21 somatic embryos. Stipes treated with sucrose were not able to SE. More than fourfold increase in SE efficiency was obtained on internodes by their treatment with 0.4 M sucrose for 45 min. It was achieved by changing the pathway of somatic embryo differentiation from multicellular to unicellular. These results provide an excellent basis for further research on the mechanism of SE induction associated with a single- and multi-cell proliferation, especially that both types of the embryogenic pathway can occur on the same hormone-free medium.

Effects of regional hypoxia and incubation temperature on growth, differentiation, heart mass, and oxygen consumption in embryos of the leopard gecko (Eublepharis macularius)

Oviparous reptile embryos must tolerate fluctuations in oxygen availability and incubation temperature during development. In this study, regional hypoxia was simulated by painting eggs of Eublepharis macularius with melted paraffin wax to decrease the available surface area for gas exchange by approximately 80%. Experimental and control eggs were incubated at either 28 or 34 °C and embryo mass, stage, heart mass, relative heart mass, and oxygen consumption (V̇O2) were measured at 15 and 30 days of incubation. Embryo mass from the regional hypoxia treatment was reduced by about 50% at day 15 and by about 30% at day 30 of incubation, independent of incubation temperature compared to controls. Embryo stage from the regional hypoxia treatment was reduced by about 2 stages at day 15 independent of incubation temperature but there was no effect of hypoxia treatment at day 30. Absolute heart mass was reduced by about 60% in regional hypoxia embryos sampled at day 15 while relative heart mass was increased by about 30% in regional hypoxic embryos at day 30 compared to controls, suggesting that heart mass is conserved at the expense of somatic growth. Embryo V̇O2 was affected by incubation temperature at both 15 and 30 days of incubation but not by regional hypoxia treatment. These results indicate that embryos of E. macularius possess plasticity in their capacity to respond to reduction in oxygen availability during incubation, and are able to survive and continue developing when gas exchange surface area is severely limited.

Direct somatic embryogenesis of potato [Solanum tuberosum (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO3, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO3 for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (> 174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.

An efficient method for immature embryo rescue and plant regeneration from the Calli of Ziziphus jujuba ‘Lengbaiyu’

ABSTRACTHybrid breeding of Chinese jujube (Ziziphus jujube) is limited by embryo abortion. Improving the success of embryogenic progeny and immature embryo culture techniques could increase the breeding efficiency of new jujube varieties. We optimised the current immature embryo culture techniques by using immature embryos before the globular stage of Chinese jujube variety Lengbaiyu. The optimal growth media were as follows: for 30-day-old immature embryo growth, MS + IBA 0.8 mg·L−1 + ZT 0.5 mg·L−1 + GA3 7.0 mg·L−1 + NAA 0.2 mg·L−1 + LH 0.5 mg·L−1 + 5% sucrose, with 22.22% embryonic growth rate and 0.41% plantlet formation rate; and for young embryo differentiation, MS + TDZ (0.8 mg·L−1) + IAA (0.5 mg·L−1) + sucrose (3%), with 81.12% differentiation rate. Thus, regeneration via callus induction effectively improved the efficiency of embryo rescue from young immature embryos in Lengbaiyu, which might provide new approaches for breeding Chinese jujube.

Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence.

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.

Expression profiles of Sox transcription factors within the postnatal rodent testes.

SRY-related box (Sox) transcription factors are conserved among vertebrate species. These proteins regulate multiple processes including sex determination and testis differentiation of the male embryo. Members of the Sox family have been identified in pre- and postnatal testis and are known to play an important role in sex determination (Sry, Sox9), male gonadal development, and fertility (Sox4, Sox8, Sox30). However, their expression profiles per cell types remain elusive. The objectives of this research were to characterize the expression profiles of Sox family members within adult testes using publically available datasets and to determine whether these findings are consistent with literature as well as immunofluorescence and in situ hybridization results. We have found that Sox4, Sox8, Sox9, and Sox12 are highly expressed in Sertoli cells, whereas Sox5, Sox6, and Sox30 were typically expressed in spermatocytes and spermatids. Spermatogonia were characterized by the expressions of Sox3, Sox4, Sox12, Sox13, and Sox18. Hence, these results suggest that Sox transcription factors may play different roles according to cell types of the adult mammalian testis.

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of Carica papaya.

Efficient protocols for somatic embryogenesis of papaya (Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C. papaya. To study the effects of PEG on somatic embryogenesis of C. papaya, we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG(control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C. papaya somaticembryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.

Progress in understanding the regulation and expression of genes during plant somatic embryogenesis: A review

Based on the previous available documents involving molecular events during plant somatic embryogenesis, this report aims to review the advances that have been made for the past several years in the area of molecular mechanism of plant somatic embryogenesis. To begin with, studies suggest that the induction and differentiation of embryos from somatic tissue directly or via callusing involves the interaction of various cellular and molecular factors. Several intra-and extracellular proteins such as germins and germins-like proteins (GLPs), lipid transfer proteins (LTPs), heat shock proteins (Hsps) and late embryogenesis abundant proteins (LEA) are known to regulate the induction of somatic embryos from the somatic cell. Simultaneously, regulation and expression of specific genes such as house-keeping genes OsIAA in rice, hormone-responsive genes Dcarg-1, Dchsp-1, DcECP31, DcEMB1 in carrot and AtECP63, MtSERF1 in Arabidopsis have been identified to play key roles during the process of somatic embryogenesis. These genes are known to express differentially for synthesis of new proteins during induction and development of somatic embryo. Additionally, several transcription factors like Leafy Cotyledon (LEC) genes, Agamous-like15 (AGL15) gene, Ethylene-Responsive Element-Binding Protein (EREBPs), Knotted1-like homeobox (KNOX) proteins and RWP-RK group of plant-specific transcription factors are equally known that efficiently control the molecular events of somatic embryogenesis. Further, it is also now established that epigenetic factors like DNA methylation, histone deacetylation/methylation and microRNAs also influence the molecular mechanism of plant somatic embryogenesis.

Gene expression profiling in embryonic chicken ovary during asymmetric development.

The reproductive system in female birds arises as bilateral asymmetrical anlagen, excluding the birds of prey. Earlier, histological and messenger RNA (mRNA) expression profile studies of several genes related to gonadal sex differentiation in chicken embryos tried to elucidate the query of this asymmetry in a scattered manner. To understand the matter precisely, we have focused on mRNA expression of a cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) in second half of the embryonic days (E10-E18). The established role of leptin in development of the embryo and its expression in the embryonic ovary also drove us to check leptin receptor (LEPR) expression in the ovary. Increased expression of FSHR and CYP19A1 in the left ovary compared with that in the right ovary was identified (P<0.05), promoting preferential left ovarian development and functionality. Significant high expression (P<0.05) of the apoptotic genes in the right ovary were also involved here. Leptin probably has no direct influence on ovarian asymmetry as no significant variation in gonadal mRNA expression of LEPR was observed within the same experimental days. We propose that asymmetric expression of this cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) leads to the development of dimorphic gonads during embryogenesis.

Morphological and transcriptomic effects of endocrine modulators on the gonadal differentiation of chicken embryos: The case of tributyltin (TBT)

Morphological malformations induced by tributyltin (TBT) exposure during embryonic development have already been characterized in various taxonomic groups, but, nonetheless, the molecular processes underlying these changes remain obscure. The present study provides the first genome-wide screening for differentially expressed genes that are linked to morphological alterations of gonadal tissue from chicken embryos after exposure to TBT. We applied a single injection of TBT (between 0.5 and 30 pg as Sn/g egg) into incubated fertile eggs to simulate maternal transfer of the endocrine disruptive compound. Methyltestosterone (MT) served as a positive control (30 pg/g egg). After 19 days of incubation, structural features of the gonads as well as genome-wide gene expression profiles were assessed simultaneously. TBT induced significant morphological and histological malformations of gonadal tissue from female embryos that show a virilization of the ovaries. This phenotypical virilization was mirrored by altered expression profiles of sex-dependent genes. Among these are several transcription and growth factors (e.g. FGF12, CTCF, NFIB), whose altered expression might serve as a set of markers for early identification of endocrine active chemicals that affect embryonic development by transcriptome profiling without the need of elaborate histological analyses.

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake, Natrix natrix L. (Lepidosauria, Serpentes).

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron-dense crystalline-like core that was polygonal in shape and an electron-dense outer zone. The A granules had a spherical electron-dense eccentrically located core and a moderately electron-dense outer zone. The D granules were filled with a moderately electron-dense non-homogeneous content. The PP granules had a spherical electron-dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.

Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos.

SummaryTo interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

Application of saponin on differential staining examination in animal blastocysts

Although there are several ways such as karyotyping to evaluate the quality and normality of embryos, the counting of total cell in blastocyst after the differential staining has been used as a simple indicator for quality of culture systems and normality of embryo itself. This differential staining method was regarded as a basic technique of early developmental biology of mammals, and it helps the scientific community to understand the signals regulatingmorphological events of early developmental process. The present study was undertaken to develop a simple and fast differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using saponin as a permeabilizing agent without using species-specific antibodies and complements. The prestained blastocyst with SYTO-13 (green) was exposed to saponin solution for propidium iodide (PI) permeation into TE cells and examined for the differential staining patterns. Three dimensional confocal microscopy was used to demonstrate the process of successful staining and showed the high impact on saponin treatment. Although the fluorescent images of blastocysts showed that one or two cell of TE stained to yellowish green, ICM was protected from saponin/PI mixture with the short exposure time of SYTO-13 pre-stained blastocysts. The total stainingprocedure did not exceed 30 min before examination under epi-fluorescence or confocal microscope. These results clearly demonstrate that saponin could be used as substituent molecule instead of species-specific antibodies and complements in differential staining examination for the first differentiation of mammalian embryos.

Inhibition of Histone H3K9 Methylation by BIX-01294 Promotes Stress-Induced Microspore Totipotency and Enhances Embryogenesis Initiation.

Microspore embryogenesis is a process of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced in vitro by stress treatments and widely used in plant breeding for rapid production of doubled-haploids, but its regulating mechanisms are still largely unknown. Increasing evidence has revealed epigenetic reprogramming during microspore embryogenesis, through DNA methylation, but less is known about the involvement of histone modifications. In this study, we have analyzed the dynamics and possible role of histone H3K9 methylation, a major repressive modification, as well as the effects on microspore embryogenesis initiation of BIX-01294, an inhibitor of histone methylation, tested for the first time in plants, in Brassica napus and Hordeum vulgare. Results revealed that microspore reprogramming and initiation of embryogenesis involved a low level of H3K9 methylation. With the progression of embryogenesis, methylation of H3K9 increased, correlating with gene expression profiles of BnHKMT SUVR4-like and BnLSD1-like (writer and eraser enzymes of H3K9me2). At early stages, BIX-01294 promoted cell reprogramming, totipotency and embryogenesis induction, while diminishing bulk H3K9 methylation. DNA methylation was also reduced by short-term BIX-01294 treatment. By contrast, long BIX-01294 treatments hindered embryogenesis progression, indicating that H3K9 methylation is required for embryo differentiation. These findings open up new possibilities to enhance microspore embryogenesis efficiency in recalcitrant species through pharmacological modulation of histone methylation by using BIX-01294.

Hormonal and morphological changes during seed development of Cypripedium japonicum.

With the goal of evaluating the factors that may be influencing the reproductive success of an endangered, terrestrial orchid Cypripedium japonicum Thunb., the anatomical and morphological characteristics of seed development were assessed, as were the levels of endogenous hormones to investigate the possible relationship between hormones and seed development. The results showed that morphological characteristics of seeds were changing from milky white and filiform to brown and fusiform. The development of ovule that initiated on the fertile valves only began after pollination, and a mature embryo sac was produced at 40days after pollination (DAP). Fertilization occurred 50 to 60DAP, and the newly formed zygote divided into two unequal cells that became the embryo proper and the suspensor. Seeds reached maturity at about 115DAP, at which point they consisted only of a globular embryo and two layers of seed coat, without endosperm. The suspensor was lost, and only lipid particles and a large number of starch grains distributed in the cytoplasm. The peak of gibberellic acid (GA3), zeatin riboside (ZR), and indole acetic acid (IAA) occurred in a time sequence, as 20, 30, and 40DAP. There was a highly significant positive correlation among them (P<0.01). Abscisic acid (ABA) content increased at 60DAP and reached the maximum at 115DAP. ABA contents were significantly negatively correlated with the three hormones above (P<0.05). It indicated that the lack of endosperm and suspensor tissues, delayed differentiation of embryos, and high levels of ABA may lead to the difficulty of germination.