Plantlet regeneration via somatic embryogenesis in anther callus of Vitis latifolia L.

Anthers of Vitis latifolia L. (wild grape) cultured on Nitsch and Nitsch medium supplemented with 20 μM 2,4-D and 9 μM BAP produced callus after 4–6 weeks. Subculture of callus onto Nitsch and Nitsch medium containing 10 μM NAA produced somatic embryos within 6 weeks. On growth regulator-free Nitsch and Nitsch basal medium somatic embryos converted to plantlets in 6–8 weeks. One gram of callus produced more than 400 somatic embryos with 13.7% being converted to complete plantlets, which were subsequently established in soil. Regenerated plants were found to have mixoploid populations of cells, 2n = 38 and n = 19. Received: 23 May 1998 / Revision received: 21 September 1998 / Accepted: 10 October 1998     

Kinetics of the reaction of yolk cell surface in the loach to puncture and mechanic deformation

Circumferential and radial components of the yolk cell surface movements were measured in the loach embryos at the late blastula stage within 40–50 min after puncture or indentation by an obliquely directed glass rod. The yolk cell surface was preliminarily marked by coal particles. It was shown that even closely located regions of the surface differed markedly in the rate and direction of their movements. In the vicinity of puncture, the yolk cell surface at first contracted in both circumferential and radial directions and then widened, but did not reach the initial values. In more remote areas, this surface continued to contract in the circumferential direction, but was extended in the radial direction. The degree of its contraction along different radii was unequal. The reaction to oblique indentation was anisotropic: the closest area of the yolk cell surface, located along the direction of indentation, contracted in both circumferential and radial directions and formed a fold “leaking” onto the rod, while the opposite area contracted in the circumferential direction, but extended in the radial direction. A conclusion was drawn that the yolk cell surface is a multivariant mechanosensitive system. Its active responses to mechanical influences obey the same patterns as multicellular embryonic tissues.     

Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.     

Hibernation behavior of Rana lessonae and R. esculenta in their natural habitat

We studied the hibernation behavior of the water frog Rana lessonae and its hybridogenetic associate R. esculenta in their natural habitat during three successive winters. Animals caught in pitfall traps at a fenced pond were individually marked with PIT tags and some (n=36) were additionally equipped with radio transmitters. Of the animals caught, 85% left the fenced pond for hibernation. More R. esculenta remained inside the fenced area compared to R. lessonae. R. lessonae emigrated earlier in autumn and came back later in spring than R. esculenta, but the distance to their hibernation sites did not differ. Both species left the fenced pond earlier in the year when ambient temperatures were lower. All radio-tracked animals hibernated in woodland, 3–7 cm below the surface in soil, under moss, fallen leaves or small branches. Soil temperatures at the actual hibernation sites were significantly higher than at randomly chosen control sites. A surprising finding was that most frogs changed their hibernation sites during winter, and often more than once. Movements were more frequent in the warmer first half of the winter than in the cooler second half, but some animals were active even on days with mean temperatures below 1°C. These results show that both species do not spend the whole winter torpid in one particular hibernation site but move around, especially at higher temperatures. Most of the animals lost weight during the winter, and the weight loss was greater in females than in males and higher in warm than in cold winters. To what extent weight loss and survival is influenced by the chosen hibernation sites and the amount of movement during winter, and whether this contributes to the differences in species and sex ratios found in mixed populations, needs more investigation. Received: 4 August 1999 / Accepted: 15 November 1999     

Production of isolated somatic embryos from sunflower thin cell layers

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.Abbreviations NAA 1-naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-benzylamino-purine - MS Murashige and Skoog medium - B5 Gamborg medium     

Contribution of midbody channels to embryogenesis in the mouse

Summary We have examined the persistence of midbody channels during the second, third, and fourth cleavage cycles of the mouse using immunofluorescence to map the distribution of midbody microtubule bundles in intact embryos. Electron microscopy showed these bundles to be a characteristic feature of midbodies throughout the interphase period. In recently-divided embryos at each cleavage stage the number of midbodies was half the number of blastomeres, and declined towards zero as the next cleavage approached. This indicated to us that the only midbodies present in each stage were those which had arisen in the immediately-preceding division. Of those blastomeres which were in mitosis at the time of fixation, less than 4% were connected via a midbody to another blastomere, demonstrating that persistence of midbodies beyond a single cleavage cycle is a rare event. We conclude that midbody channels in our embryos are likely to connect only pairs of sister blastomeres because midbodies do not persist through multiple cleavage cycles. Midbody channels cannot, therefore, be regarded as providing extensive cell coupling in advance of the onset of gap junctional communication.     

Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.     

Establishment and maintenance of friable,embryogenic maize callus and the involvement of L-proline

Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659–668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473–497, plus 0.5 mg 1^-1 thiamine hydrochloride and 150 mg 1^-1 L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium containing inorganic elements of Murashige and Skoog (1962), plus 0.5 ml 1^-1 thiamine hydrochloride and 150 mg 1^-1 L-asparagine monohydrate - N6 medium of Chu et al. (1975) Paper No. 13,904, Scientific Journal Series Minnesota Agricultural Experiment Station     

Early appearance of catecholaminergic neurons in the central nervous system of precocial and altricial avian species

Summary The early appearance of catecholaminergic neurons, as revealed by fluorescence histochemistry, has been determined in the central nervous system of quail, pheasant, and pigeon embryos. The first neuronal assemblies displaying specific fluorescence are the locus coeruleus and the nucleus subcoeruleus ventralis. Taking into account the differences in the length of the prehatching period of these three avian species, the first catecholamine-containing neurons appear earlier in the precocial quail and pheasant than in the altricial pigeon.Investigation supported by grants from the Italian National Research Council (CNR) No 83.02058.04 (R.G.) and No 83.00492.04 (G.C.P.).