Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.     

Influence of auxin on the establishment of bilateral symmetry in monocots

To study the influence of auxin on the shift from radial to bilateral symmetry during monocot embryogenesis, the fate of young wheat (Triticum aestivum L.) zygotic embryos has been manipulated in vitro by adding auxins, an auxin transport inhibitor and an auxin antagonist to the culture medium. The two synthetic auxins used, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), induced identical phenotypes. In the most severe cases, the shift from radial to bilateral symmetry was blocked resulting in continuous uniform radial growth. The natural auxin indole-3-acetic acid (IAA) induced the same phenotype. The effect of 2,4,5-T and 2,4D depended on their concentrations and on the developmental stage of the isolated embryos. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin transport inhibitor, the overall embryo symmetry was abnormal. The relative position of the shoot apical meristem in comparison with the scutellum was anomalous. The quality of shoot apical meristem and the scutellum differentiation was altered compared with normal developed embryos. No root meristem was differentiated. The effect of TIBA depends on its concentration and on the developmental stage of the isolated embryos. By contrast, 2-(pchlorophenoxy)-2-methylpropionic acid (PCIB) which is described as an auxin antagonist, has no visible direct effect on the embryonic symmetry. These observations indicate that auxin influences the change from radial symmetry to embryonic polarity during monocot embryogenesis. A model of auxin action during early wheat embryo development is proposed.     

TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium

To study the effect of auxin on direct somatic embryogenesis from leaf cultures ofOncidium `Gower Ramsey', 1-cm-long explants have been cultured in vitro testing IAA, 2,4-, quercetin, TIBA and PCIB. On a modified MS medium devoid of plant growth regulators, leaf cells of three regions (leaf tips, adaxial sides and cut ends) formed somatic embryos. After 8 weeks in culture, the frequencies of embryo-forming explants were 55, 52.5 and 30 % on leaf tips, adaxial sides and cut ends, respectively, and the numbers of embryos per dish was 89.3. Except for TIBA, other growth regulators (IAA, 2,4-, quercetin, PCIB) and their combinations tested, all retarded direct embryo formation. In the presence of 0.1 and 0.5 μM TIBA, leaf tip, adaxial sides and cuts end of explants gave almost the same embryogenic response as the control. However, 10 and 27.5 % of explants were induced to form embryos from abaxial sides, and these explants did not form embryos on cut ends. In addition, after 8?weeks in culture, TIBA at 0.5?μM highly promoted the mean numbers of embryos per dish to 134.2.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Developmental responses to UV-B radiation in common frog <Emphasis Type="Italic">Rana temporaria</Emphasis> embryos from along an altitudinal gradient

Solar ultraviolet radiation (UV) is harmful for developing amphibians. As UV increases with altitude and latitude, it is suggested that high altitude and latitude populations have evolved tolerance to high levels of UV. Using laboratory experiments, we tested the hypothesis that Rana temporaria populations from several altitudes (438–2,450 m above sea level) were adapted to UV by assessing the effects of artificial UV on embryos mortality rate, malformations, and body length at hatching. We also tested the protective role of the jelly surrounding the embryos. Without artificial UV exposure, hatching success decreased with altitude of the population. Malformation rates were low for all populations (mean 1.36%), and hatching size increased with altitude. The artificial UV (UV-B, UV-A, and visible) used was similar to the solar spectrum received at high altitude. Exposed embryos had performance similar to that of embryos without exposure: a decreased hatching success with altitude and a low malformation rate (mean 0.85%). However, hatching size did not vary with altitude, and UV-exposed embryos tended to be smaller at hatching than non-exposed embryos. Removal of the protective jelly envelope greatly decreased the performance of UV-exposed embryos: hatching success strongly decreased with altitude and embryos of the highest population (2,450 m asl) did not develop. Malformation reached 4.98%, without population differences, and hatching size of embryos without jelly was smaller than hatching size of non-manipulated embryos with no population effect. This study demonstrates that lowland jellyless embryos were less sensitive (i.e., exhibited a higher survival rate) than highland embryos and, on the other hand, that the jelly envelope was a more efficient protection for embryos in highland than in lowland populations. A trade-off hypothesis is presented to explain this difference in UV tolerance and protection among populations: in a harsh, highland environment (i.e., with a curtailed activity period), embryos have to invest in development and growth and, thus, embryo protection should be a female investment (via the jelly). In a lowland environment, embryonic growth and development are less constrained and embryos are able to invest and use protection or repair mechanisms inherited from their parents.     

Life history variation in Pisidium (Bivalvia: Pisidiidae)

Intraspecific variation in the maximum shell length of Pisidium is small in comparison with variation in the life span of the clams, from 4 mo to 4 yr. The use of shell length as a measure of size is complicated by large intraspecific variation in the weight-length relationship, a possible reflection of resource availability on the weight of the soft parts. Maximum embryo length is relatively constant (1 mm) in the majority of species but litter size is variable (1 to 40), generally increasing with parent size. The time of egg-laying is determined by the size of the clam, season, and previous episode of reproduction, while the timing of the release of embryos, depending also on embryonic growth rate, is additionally affected by temperature and oxygen conditions. In brief, the maximum adult and embryo lengths are relatively conservative traits while growth rates vary by an order of magnitude.
Reproductive effort, when measured by the ratio of litter weight to parent weight, may increase or decrease with parent length and age. Both semelparous and iteroparous populations are known in several species. We suggest that if maximum adult length can be reached by the first reproductive season (in a favourable environment) the population is semelparous, otherwise (in an unfavourable environment) the population is iteroparous. Gravid clams in semelparous populations continue to grow, and thus assimilation cannot be meaningfully partitioned into reproduction, somatic growth and maintenance.
Along a depth gradient from 8 to 65 m, maximum and mature shell lengths, embryo length and litter size varies significantly in Pisidium conventus subpopulations not distinguishable electrophoretically. Large clams in deep water produce large embryos but small litters, which is contrary to the general trend of increasing litter size with parent length. Low food availability that decreases juvenile growth rate may restrict litter size in this case.     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Application of image analysis to fed-batch cultures of somatic embryos

Summary Nutrient limitation and inhibitor accumulation have been shown to impede the development of somatic embryos of carrots in batch cultures. To improve the development of embryos, semicontinuous cultures with daily medium replenishment were performed. An image analysis system capable of identifying normal and abnormal embryos was used to facilitate the kinetic study. At a high daily medium replenishment rate of 20%, the biomass production was also higher, but not the embryo concentration. Total embryo and torpedo embryo concentrations were both higher at a 10% medium replenishment rate than at 5% and 20%. The profiles of embryo concentrations under three medium replenishment rates appeared similar until a late stage of cultivation; however, statistical analysis of the morphological features distribution revealed that significant differences were discernible earlier. For the formation of mature embryos, the optimal daily medium replenishment is judged to be in the range of 10–20%. In this study, more than 20000 embryos were classified and counted. The information on population kinetics and statistical comparison were made possible by the availability of our image analysis system.     

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.     

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae)

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.     

The morphological variability within a population of coffee somatic embryos produced in a bioreactor affects the regeneration and the development of plants in the nursery

A 1-liter bioreactor was used to obtain approximatively 800 Coffea arabica somatic embryos, 86% of which reached the `germinated' stage but with morphological heterogeneity. The population was sub-divided into three categories according to cotyledon area: 'small', 'medium' and 'large', that amounted to 32%, 36% and 4.5%, respectively. The effect of embryo morphology on plantlet conversion after direct sowing in soil and on plant development in the nursery was investigated. Somatic embryos with large cotyledons had only a 25% plantlet conversion rate, whereas somatic embryos with small to medium-sized cotyledons had conversion rates of 47% and 63%, respectively. The vigour of the aerial and root systems of regenerated plantlets at the end of the plant conversion stage was also affected as the embryos with small, medium and large cotyledon mostly regenerated small plantlets (0.5–1.5 cm), medium plantlets (1.5–2.5 cm) and large plantlets (2.5–5 cm), respectively. When transplanted in plastic bags, these 3 populations of plantlets exhibited distinct development rates. They had an initial slow growth phase, which was much longer for the small plantlets, followed by a rapid growth phase. After 40 weeks in the nursery, an analysis of the growth parameters of aerial and radical systems showed that the vigour of the plants was strongly related to the vigour of the plantlets transplanted. The heterogeneity of somatic embryos in the bioreactor affected both the plant conversion efficiency in soil and the plant growth in nursery, where it mainly resulted in retarded growth, primarily in plantlets derived from the somatic embryos with small cotyledons.     

Generality of the growth kinetics of the average individual cell in different bacterial populations.

The kinetics of growth of all the cells in a population is reflected in the shape of the size distribution of the population. To ascertain whether the kinetics of growth of the average individual cell is similar for different strains or growth conditions, we compared the shape of normalized size distributions obtained from steady-state populations. Significant differences in the size distributions were found, but these could be ascribed either to the precision achieved at division or to a constriction period which is long relative to the total cell cycle time. The remaining difference is quite small. Thus, without establishing the pattern itself, it is concluded that the basic course of growth is very similar for the various Escherichia coli strains examined and probably also for other rod-shaped bacteria. The effects of differences in culture technique (batch or chemostat culture), growth rate, and differences among strains were not found to influence the shape of the size distributions and hence the growth kinetics in a direct manner; small differences were found, but only when the precision at division or the fraction of constricted cells (long constriction period) were different as well.     

The relationship between changes in cell wall composition and the establishment of polarity in Fucus embryos

Changes in the appearance and location of fucoidin in the cell walls of Fucus embryos were related to embryo development. Fucoidin was not present in the cell wall until 10–14 hr after fertilization, when the embryos began to incorporate fucoidin preferentially into a localized area of the wall. At this time the site of rhizoid initiation was determined; that is, the embryos had undergone axis commitment. Germination of the single-celled embryo occurred between 12 and 16 hr, after fertilization, with all cell walls from germinated embryos showing fucoidin localization at the rhizoid end of the cell. The percentage of embryos with localized fucoidin at the time of axis fixation equaled the percentage of embryos that subsequently germinated. Culturing the embryos in sea water plus 0.8 M sucrose prevented the outgrowth of the rhizoid, but not the localization of fucoidin in the wall or axis commitment. Cycloheximide, nitroprusside, cytochalasin B, sulfate-free sea water, high levels of Ca²⁺, and a breakdown product of TIBA all prevented rhizoid growth and the specific localization of fucoidin. In addition, axis commitment could not be demonstrated in the presence of these inhibitors. DTNB, PCMBS, TIBA, HgCl₂, Mg²⁺ were ineffective as reversible inhibitors of rhizoid initiation. The authors propose that the fixation of axis commitment is accompanied by localized changes in the cell wall involving the incorporation of fucoidin as a structural component of the wall.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars

Different pretreatments were given to anthers of barley before culturing, and their effects assessed on the frequency of embryos and green doubled haploid plants produced. Mannitol pretreatment was better than cold pretreatment for some low responding cultivars. Optimal concentration of mannitol for pretreatment depended on cultivar. Low responding genotypes needed a higher concentration of mannitol than responsive ones. The addition of Ficoll to liquid medium increased the number of embryos and green plants. The influence of the growth regulators 2,4-D and TIBA was assayed using ten cultivars of barley grown in Spain. The anti-auxin TIBA gave good embryo production with some of the low responding cultivars. Two row-type cultivars always produced higher number of embryos and green plantlets than six row-type. The application of these modifications to 10 F₁ hybrids with potential agronomic value, allowed the production of almost 1000 doubled haploid plants from only 3500 anthers. Up to two doubled haploid plants per flower were produced from the cross Monlon × Sonja. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos

Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1.10 +/- 0.06 g; Day 6, 1.15 +/- 0.06 g; Day 8, 1.41 +/- 0.08 g; P less than 0.05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development. The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.