Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.     

Peptide models of four possible insulin folding intermediates with two disulfides

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.     

Control of in vitro growth of viviparous embryo mutants of maize by abscisic acid

The plant hormone abscisic acid (ABA) is believed to play a role in the onset of developmental arrest in seeds. Embryos of the viviparous mutants of Zea mays do not undergo arrest but germinate directly on the ear. This study investigates the possibility that the mutants vp1, vp5, vp7, vp8, and vp9 are defective in some aspect of ABA action. Mutant and wild type embryos were removed from developing seeds at 18, 21, and 24 days after pollination and cultured aseptically on media containing a range of ABA concentrations. Seedlings were harvested after seven days when lengths and fresh and dry weights were recorded. The results indicate that these five viviparous mutants differ in their response to ABA. Two mutants, vp5 and vp8, exhibit the same sensitivity to growth inhibition by ABA as wild type. The remaining three mutants, however, manifest a range of decreased sensitivities with vp1 being the least sensitive, followed by vp7 and vp9.     

Mouse embryos exposed to oxygen concentrations that mimic changes in the oviduct and uterus show improvement in blastocyst rate,blastocyst size,and accelerated cell division

After in vivo fertilisation, the preimplantation embryo goes through cleavage during migration along the oviduct in mammals or the fallopian tube in a woman and ends up inside the uterus. This study investigates the effect of a protocol aimed at closely reproducing that natural oxygen concentration in the oviduct (7 % O₂ from day 1 to day 3 and 2 % from day 3 to day 5), in contrast to the concentrations (5 % or 20 %) widely used in practice in ART using morphokinetic. Female mice (BI6/CBAca) were sacrificed, and zygotes were isolated 20 h after mating and randomly allocated to three parallel groups, which were grown under high atmospheric, low, or sequential oxygen concentrations. Zygotes were cultured in GTL medium (Vitrolife) and observed by the Primovision time-lapse system. Blastocyst rate at 120 h in the sequential group (91.3 %) was significantly increased over the high (76.3 %) and low (74.4 %) groups. Blastocyst size was also enlarged in the sequential group compared to the high and low groups. Moreover, cell division in the sequential group was significantly faster at almost every cleavage stage than it was in the other groups. Notably, the duration of the interims between stages also differed significantly between the groups. This study demonstrated that, in comparison to routinely used high or low oxygen conditions, oxygen concentrations mimicking changes in the oviduct and uterus significantly improve the blastocyst rate and size and accelerate cell division at several stages as well as the interims between cleavage events.     

RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

Seed quality and seed quantity in red maple depends on weather and individual tree characteristics

Under future climate change, plant species are expected to shift their ranges in response to increasing temperatures and altered precipitation patterns. As seeds represent the single opportunity for plants to move, it is critical to quantify the factors that influence reproduction. While total seed production is clearly important, seed quality is equally as critical and often overlooked. Thus, to quantify how environmental and tree‐level characteristics affect seed quality and quantity, the reproductive output of red maple (Acer rubrum) was measured along an elevation gradient in the Monongahela National Forest, WV. A variety of individual‐level characteristics were measured (e.g., DBH, canopy area, height, and tree cores were taken to quantify growth), and seed traps were placed under seed‐bearing trees to collect samaras and quantify total seed production. A random subsample of collected seeds from each tree was micro‐CT scanned to determine embryo volume, photographed for morphology measurements, and used for germination trials. The number of seeds produced was negatively affected by frost events during flowering, and stand density. The trees with the most seeds also showed reduced growth in recent years. Only 63% of scanned seeds showed embryo development, and of those seeds—only 23% germinated. The likelihood of embryo presence increased as growth rate decreased, while embryo size increased with tree height, smaller DBH, and in areas dominated by hemlock. Both larger embryo volume and larger overall seed size increased the likelihood of germination. The results highlight the importance of including seed quality in addition to seed quantity for a more complete representation of reproductive output.     

Embryology of Clitoria ternatea (Fabaceae)

ABSTRACT

The embryology of Clitoria ternatea was studied. Anthers contain four sporangia. The anther wall comprises an epidermis, an endothecium, a middle layer and a glandular tapetum. Microspore tetrads are tetrahedral and pollen grains are shed at the 2-cell stage. The ovule is campylotropous, bitegmic and crassinucellate. The micropyle is formed by both the integuments. The megaspore tetrad is linear or T-shaped. The chalazal megaspore is functional and embryo sac development follows the monosporic Polygonum type. Endosperm development is of the nuclear type. The chalazal part of the endosperm forms a haustorium. Embryo development follows the Onagrad type.