Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.

Effects of different high temperatures and incubation time periods on embryogenesis in isolated microspore culture of Chinese cabbage (Brassica campestris L. ssp pekinensis)

Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The results showed that cultured microspores of Chinese cabbage developed into embryos at all the incubation temperatues from 28℃ to 37℃ ,but the best response to high temperature occured at 35℃. Among the four kinds of time periods, the highest yield of embryos was obtained at the 24h treatment. Therefore, the isolated microspore culture of Chinese cabbege ran be efficiently carried out at 35℃ for 24 hours.

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.

The paternal epigenome and embryogenesis： poising mechanisms for development

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

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.

Pollen development and multi-nucleate microspores of Populus bolleana Lauche

Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.

<i>In Vitro</i>Plant Regeneration of <i>Dendrocalamus stocksii</i>(Munro) M. Kumar, Remesh &Unnikrisnan, through Somatic Embryogenesis

Dendrocalamus stocksii is fast cultivating economically important forest crop species. National Mission of Bamboo Application (NMBA) of India has been identified in 15 industrially important bamboo species. Traditionally it was propagated through by offset cuttings and rhizome splitting which was not meeting the demand, culm cuttings needed mass material to propagate and rooting percentage mixed. Plant regeneration through somatic embryogenesis was achieved in callus cultures derived from the callus initiated through type of explants viz. leaf, leaf sheath, shoot tip, nodal shoot segments, and inter node segments from aseptic cultures. Explants were cultured on Murashige & Skoog basal media supplemented with 2,4 Dichloro diphenyle ethane 0.44 μM/L with additives (Ascorbic acid 8.8 μM/L, citric acid 4.8 μM/L Cysteine 3.02 μM/L and Glutamine 14.6 μM/L) with 3% sucrose and Agar agar 0.6%. Cultures were incubated in the dark at 25&deg;C ± 1&deg;C. Out of five types of explants nodal shoot induced callus > 80% followed by leaf sheath (60%) and no callus was induced in leaf. Various nutrient media viz. Murashige and Skoog (MS), Woody Plant Media (WP), Gamborg media (B5) and Heller’s (HE) media fortified with 2,4 D (0.2 - 1.10 μM/L) and Kinetin 0.10 μM/L were tested for high frequency callus induction. Among four nutrient media tested MS media fortified with 2,4 D (0.55 - 1.1 μM/L) 100% callus induction. Calli multiplication was carried out with various concentrations of PGR’s with 10% coconut milk. Out of these MS media 2,4 D 0.55 μM /L and 10% coconut milk concentration were found best for high frequency (80%) calli multiplication. Various combinations of α-naphthalene acetic acid (NAA) with N6-benzyiaminopurine (BAP) and kinetin were tested for embryo germination, out of which MS media supplemented with NAA 0.55 μM /L and BAP 0.22 μM /L were showed high frequency (80%) germination. Germinated plantlets carefully transferred to polybags containing potting mixture of sand soil and compost in the ratio of 40:10:50 with 10 Kg/m3 + 250 gm/m3 fungicide. Plantlets were kept 4 weeks under poly tunnel inside mist chamber followed by two weeks outside poly tunnel in mist chamber. Plants are lifted to the canopy condition directed to a week before subjected to them in the institute division nursery.

Somatic Embryogenesis in Iberian Grapevine (Vitis vinifera) Cultivars Using Carpels as Initial Explants: Protocol Establishment and Histological Evaluation

Trincadeira and Aragonez are two important grapevine cultivars in the Iberian Peninsula, used for high quality red wines production. Both cultivars are strongly affected by fungal diseases, with consequent high loses on plant productivity and fruit quality. A successful protocol for plant regeneration via somatic embryogenesis (SE) was established for both cultivars allowing further plant improvement based on gene transfer technology. Several factors were evaluated during the three different phases which characterize an SE plant regeneration protocol. The culture room temperature during the induction phase, a parameter usually accepted as standard by most researchers, proved in these trials to significantly affect the embryo induction rates. Concerning embryo conversion, it was specially affected by the embryo developmental stage, by the intensity and duration of the chilling treatment and by the supplementation of conversion culture medium with activated charcoal. The responses obtained, both for induction and conversion, proved to be highly genotype dependent. Calli structure, as well as embryo integrity, was histologically observed, allowing to characterize embryonic and non-embryonic masses and to identify abnormalities on embryo development.

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.

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.

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

Somatic embryogenesis was achieved in two oil yielding tropical tree species i.e. Simarouba glauca & Azadirachta indica using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.5 – 1.5 mg/l benzylaminopurine (BA) and 2.0 - 3.0 mg/l NAA (1-napthaleneacetic acid) or 2, 4-D (2,4-dichlorophenoxyacetic acid) and 3% sucrose. MS medium containing 1.0 mg/l BA and 2.0 mg/l NAA was noted to be the most effective in inducing friable embryogenic callus (FEC) in Simarouba glauca;the number of somatic embryos per culture varied in MS medium supplemented with 1.0 – 1.5 mg/l BA and 1.0 mg/l NAA. In Azadirachta indica, somatic embryos developed on MS medium supplemented with 0.5 mg/l BA and 1.5 – 2.0 mg/l 2,4-D which were in various shapes and sizes after the first subculture on MS medium supplemented with 0.25 mg/l abscisic acid. The somatic embryos which developed shoots were isolated and rooted in 1/2 strength MS medium supplemented with 0.25 mg/l abscisic acid and 2% sucrose. About 25% of embryos germinated within 20 days of culture in case of Simarouba glauca and 62% in Azadirachta indica. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate controlled hardening chamber.

Developmental Dynamics of Wheat (<i>Triticum aestivum</i>L.) Microspores under Culture

Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.

An Observation Data Driven Simulation and Analysis Framework for Early Stage <i>C. elegans</i>Embryogenesis

Recent developments in cutting-edge live microscopy and image analysis provide a unique opportunity to systematically investigate individual cell’s dynamics as well as simulation-based hypothesis testing. After a summary of data generation and analysis in the observation and modeling efforts related to C. elegans embryogenesis, we develop a systematic approach to model the basic behaviors of individual cells. Next, we present our ideas to model cell fate, division, and movement using 3D time-lapse images within an agent-based modeling framework. Then, we summarize preliminary result and discuss efforts in cell fate, division, and movement modeling. Finally, we discuss the ongoing efforts and future directions for C. elegans embryo modeling, including an inferred developmental landscape for cell fate, a quasi-equilibrium model for cell division, and multi-agent, deep reinforcement learning for cell movement.

Differential Effects of Cold and Heat Shock on Embryogenic Induction and Green Plant Regeneration from Wheat (Triticum aestivum L.) Microspores

Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.

Transgene Homozygosity Following Particle Bombardment of Haploid Barley Microspores

Information from previously published studies that are basic to this study is: 1) Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically

芥蓝游离小孢子培养技术优化及其在育种中的应用

以16个芥蓝品种(F1)为供体材料,比较分析了优化花蕾的消毒、小孢子的游离释放以及过滤等环节对芥蓝游离小孢子培养效果的影响。结果表明,16份参试芥蓝供体材料都具有胚胎发生能力,并全部获得了不同类型的再生植株,实现了较大规模、批量地获得芥蓝双单倍体(DH)。经田间综合评价筛选,应用DH系育成了不同熟性的芥蓝一代杂种20M4芥蓝、21M2芥蓝和22M11芥蓝。