Development of the central nervous system in equine twin fetuses derived by somatic cell nuclear transfer.

Because of the growing importance of horses in leisure and several sports, somatic cell nuclear transfer (SCNT) is being used more frequently for cloning animals for performance and reproductive purposes. However, because of the need to perforate the zona pellucida during microsurgical reconstruction of the oocyte, it is possible that SCNT-derived embryos undergo premature hatching, resulting in embryo bisection and twinning. Therefore, because equine twin pregnancies often lead to abnormal embryo development and pregnancy failure, we performed a detailed comparative assessment of equine twin fetuses derived by SCNT with particular attention on the development of the central nervous system at 40 and 60 days gestation. The results of this study indicate that although cloned twin embryos show small differences in size, they do not exhibit apparent macro- or microscopic developmental discrepancies in the central nervous system, suggesting that the twining phenomenon resulting from SCNT does not affect fetal differentiation.

Use of High Performance Liquid Chromatography for the Determination of Endogenous Hormone Levels in Solanum tuberosum L. Subjected to Carbon Dioxide Enrichment of the Root Zone.

Changes in cytokinin, auxin, and abscisic acid levels were determined by high performance liquid chromatography following CO(2) enrichment of the root zone. Both trans zeatin and zeatin riboside significantly decreased in the roots 6 days after treatment with CO(2). In the leaves zeatin riboside increased whereas trans zeatin showed no significant difference from the control. Indoleacetic acid in the leaves increased dramatically for 6 days following CO(2) treatment; however, indoleacetic acid levels in the roots increased slightly after 2 days and remained higher than the control. Abscisic acid increased shortly after CO(2) treatments; after 6 days this difference was no longer evident.

Changes in Carbon Fixation, Tuberization, and Growth Induced by CO2 Applications to the Root Zone of Potato Plants.

The root systems of potato plants (Solanum tuberosum L. var. Russet Burbank) treated with CO(2) for 12 hours showed an increase in dry matter as early as 2 days after the treatment. When treated plants were allowed to grow for 3 to 6 weeks there was a substantial increase in tuberization. In addition, there was an increase in stolon length, number of tubers per stolon, and overall dry weight after the enrichment of the root zone with CO(2). Plants treated with CO(2) showed higher concentrations of malic and citric acids and of the cations Mg(2+) and Ca(2+). The effect of CO(2) was more dramatic when CO(2) was applied to the root zone than when it was applied to the shoots.

Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed.

Lettuce seeds (Lactuca sativa L.) used in this study germinated 98% at 25 C in light or dark. Their germination was completely inhibited by 0.20 m NaCl, 0.35 m mannitol, or polyethylene glycol 6000 (-7 bars) under continuous light when germination tests were made in Petri dishes. Approximately 50% germination occurred in sealed flasks due to endogenously produced C(2)H(4) and CO(2). Removal of either or both gases prevented germination. In the presence of endogenous CO(2), addition of C(2)H(4) (0.5 to 16 microliters/liter) stimulated 95 to 100% germination (after 5 days) only in the light, but the rate of germination was dependent on C(2)H(4) concentration. At 16 microliters/liter C(2)H(4), full germination occurred within 72 hours. Addition of up to 3.2% CO(2) had no adverse effect on the C(2)H(4) action. Higher concentrations or the complete absence of CO(2) reduced both rate and total germination. CO(2) alone was ineffective.Under these osmotic conditions the promotive effect of C(2)H(4) was under the control of phytochrome.

Studies on the carbon dioxide promotion and ethylene inhibition of tuberization in potato explants cultured in vitro.

Ethylene inhibited the tuberization of etiolated potato (Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro. Carbon dioxide did not overcome the C(2)H(4) inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C(2)H(4) prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin-increased tuberization of sprout sections.Three to five days of exposure to CO(2) were required to obtain promotion of tuberization of stolons cultured in vitro. Bicarbonate ion did not affect starch synthetase activity isolated from potato tubers in vitro. The evidence presented suggests that CO(2) gas rather than HCO(-) (3) or CO(2-) (3) ions in equilibrium with dissolved CO(2) was probably responsible for the stimulation. Morphological changes elicited by CO(2) and C(2)H(4) are described and the mechanism of action of both on tuberization is discussed.

Effect of Gibberellic Acid, Kinetin, and Ethylene plus Carbon Dioxide on the Thermodormancy of Lettuce Seed (Lactuca sativa L. cv. Mesa 659).

The effects of gibberellic acid and kinetin with ethylene plus carbon dioxide on the thermodormancy of lettuce seeds (Lactuca sativa L. cv. Mesa 659) at 35 C in the dark were studied. The combination of gibberellic acid plus kinetin with ethylene plus carbon dioxide was most effective in overcoming thermodormancy in these Great Lakes type seeds, alleviating any induced light requirement. Gibberellic acid action required at least a minimal level of ethylene plus carbon dioxide. Kinetin action was independent of ethylene plus carbon dioxide but interacted with the gases when the gases were added. A schematic representation of the interaction is presented.

Identification of plant hormones from cotton ovules.

An extract from 8-day-old cotton ovules (Gossypium hirsutum L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A(1), A(3), A(4), A(7), A(9), and A(13) in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine(2iP), 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine(2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-beta-d- ribofuranosylpurine (zeatin riboside) in the third fraction.

Effect of carbon dioxide and ethylene on tuberization of isolated potato stolons cultured in vitro.

Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C(2)H(4) which is by itself also inhibitory of tuberization. Furthermore, C(2)H(4) inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C(2)H(4). The antagonistic actions of CO(2) and C(2)H(4) on tuberization are discussed.

Absorption and distribution of high specific radioactivity 2-C-abscisic Acid in cotton seedlings.

High specific radioactivity (26.3 mc/mmole) racemic 2-(14)C-abscisic acid was synthesized. An aliquot of abscisic acid, 1.2 x 10(-4)m in aqueous methanolic solution, was applied to the surface of either a cotyledon or the first true leaf of 8- to 32-day-old cotton seedlings (Gossypium hirsutum L.). After various intervals (6-192 hours), the seedlings were processed for autoradiography, counting, and identification of the radioactivity. After 6 hours, radioactivity was observed moving basipetally out of the treated leaf toward the roots. Four days later, radioactivity could be detected throughout the whole seedling. After 8 days, 10% of the recovered radioactivity was found in the roots, and 80% remained in the treated leaf blade. Neither leaf type nor age had any effect on the abscisic acid movement or pattern of distribution. Isolated radioactivity from the roots was identified as abscisic acid, based on comparison with an authentic standard by thin layer chromatography, gas-liquid chromatography, or gas-liquid chromatography-mass spectrometry.

The role of phytochrome in an interaction with ethylene and carbon dioxide in overcoming lettuce seed thermodormancy.

Ethylene and CO(2) were used to control induction of germination in thermodormant lettuce seed (Lactuca sativa L.). These experiments ultimately showed that germination depends on the presence of an active form of the phytochrome. The phytochrome system is functional and stable at 35 C, a temperature which completely inhibits germination. Phytochrome responses to red or far red light and darkness showed that this inhibition of germination under light must be due to some other block(s) rather than to a direct inactivation of the phytochrome system itself. A postred radiation increase in lettuce seed germination that is not reversed by far red light was observed. The CO(2) requirement for C(2)H(4) action is not due to a change in the medium's pH; addition of C(2)H(4) plus CO(2) at the start of imbibition did not result in as much germination as when they were added several hours after imbibition. This reduction in germination, when the gases are added at the start of imbibiton, is due to CO(2).

Interaction of carbon dioxide and ethylene in overcoming thermodormancy of lettuce seeds.

The combination of ethylene with CO(2) will completely overcome the thermodormancy of lettuce (Lactuca sativa L.) seeds at 35 C. This combination is effective if it is added to seeds either at the start or after several days of imbibition. The action of ethylene is dependent upon the CO(2) level present in the atmosphere surrounding the seeds. When CO(2) is trapped by KOH the ethylene effect is essentially nil.

Sex expression in cucumber plants as affected by 2-chloroethylphosphonic Acid, ethylene, and growth regulators.

The effects of 2-chloroethylphosphonic acid (Ethrel), ethylene, and some growth retardants on sex expression of cucumber plants (Cucumis sativus L.) were investigated, with the use of a monoecious cultivar (Improved Long Green) which has a strong tendency toward maleness.Ethrel caused increased femaleness when applied at 50 milligrams per liter at the first to the third leaf stage, but when applied at the cotyledon stage it was ineffective. The later the time of application, the higher the node at which the first female flower appeared. The total number of female flowers was about the same regardless of application time. A mixture of gibberellins A(4) and A(7) caused maleness, and Ethrel caused femaleness. However, when applied in combination at the first leaf stage the interaction was not significant. It seems, therefore, that Ethrel and gibberellins are not antagonistic but rather have different sites of action, although they have opposing effects on sex expression.Ethylene caused femaleness but was far less effective than Ethrel. Alar (N-dimethylaminosuccinamic acid), CCC((2-chloroethyl)trimethylammonium chloride), Phosphon D(2, 4-dichlorobenzyl-tributylphosphonium chloride), and abscisic acid did not affect sex expression of cucumber.

Movement of (RS)-Abscisic Acid in the Cotton Explant.

The movement of abscisic acid in the cotton explant was investigated by bioassay and radioisotope techniques. The rate of movement was 20 to 30 millimeters per hour with most of the abscisic acid moving unchanged through the explant into the basal agar. The rate of movement was the same through the abscission zone as through petiole tissue. Patterns of accumulation and metabolic products are discussed.

Effects of gibberellins on abscission in cotton seedling explants.

Gibberellic acid (GA3) accelerated abscission when applied, in a wide concentration range, to excised abscission zones of cotton. Abscission was promoted equally by distal or proximal applications of from 10(-3) to 100 µg. A slight, but inconsistent, abscission retardation was obtained with distal applications of 10(-6) and 10(-7) µg.Seven different gibberellins accelerated abscission equally when applied distally at amounts of 5×10(-4) to 5×10(-1) µg per abscission zone. At 5×10(-5) µg there were great differences in effectiveness; their activities can be ranked: A3>A5≥A4>A7=A8>A1=A9.The ready translocatability of GA3 was suggested when 1.0 or 0.01 µg was applied to one petiole, and the opposite untreated petiole abscised at the same time as the treated one. However, 0.001 µg was not effective in moving across the stem and inducing abscission of the untreated petiole.The rate of abscission of petioles treated with 1.0 µg GA3 was not affected by increasing the length of the petiole from 3 to 9 mm. However, abscission of petioles treated with smaller amounts is inversely proportional to petiole length.The rate of abscission of petioles treated with GA3 decreased with increasing seedling age; there was a simultaneous increase in abscission rate of the controls.