Flow Cytometric Characteristics of Sperm Cells Isolated from Pollen of Zea mays L

Sperm cells have been isolated from pollen of maize (Zea mays L.) and purified with Percoll density centrifugation. Their flow cytometric characteristics were determined on a FACScan flow cytometer with the fluorescent dyes, fluorescein diacetate and propidium iodide. Freshly isolated sperm cells appeared as a dot cluster on the forward scatter and side scatter dot plot. This dot cluster contained 85 to 95% of the 10 thousand counts collected. More than 98% of cells from the cluster were fluorescein diacetate positive, with no propidium iodide positivity, indicating high cell viability. After 5 hours in 15% (w/v) sucrose at room temperature (23°C), scattering properties, cell number, and percentage of fluorescein diacetate-positive cells remained the same. In contrast, Brewbaker and Kwack salts in 15% sucrose resulted in the emergence of a new cell population, as well as a decrease in cell number at 5 hours. Further investigations with individual components of the Brewbaker and Kwack salts showed that calcium was mainly responsible for the deleterious effects. These results demonstrate the utility of flow cytometry as a tool to determine viability and to monitor morphological changes of plant sperm cells and to challenge current views on the ability of Brewbaker and Kwack salts to maintain viability of isolated sperm cells.     

Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose

Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K(1)a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (C(ox)). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (micro) was a Monod function of C(ox). Specific oxygen uptake rate was proportional to mu by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state C(OX) depended only on D, while steady-state biomass concentration was controlled by both D and K(1)a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of micro. The dying portion increased with E and X.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

The effects of ear removal on senescence and metabolism of maize

Ears were removed from field grown maize (Zea mays L.) to determine the effects on senescence and metabolism and to clarify conflicting literature reports pertaining to these effects. Ears were removed at three days after anthesis and comparisons were made of changes in metabolism between eared and earless plants until grain of the eared plants matured as judged by black layer formation.     

Availability of reduced N and carbohydrates for ear development of maize

Changes in dry weights, reduced N, nitrate, and nitrate reductase activity of various plant parts of the above ground vegetation (stover) and ears of field grown maize were measured at intervals between anthesis and grain maturity. Nonstructural carbohydrate contents were also measured in some instances. Changes in dry weight and reduced N content were used to approximate net in situ photosynthetic and nitrate assimilation activities and to determine whether the availability of photosynthate or reduced N was limiting grain production.     

Endocrine profiles and embryo quality in the PMSG-PGF2alpha treated cow

Plasma progesterone and LH concentrations around estrus were determined for both PMSG treated (experimental animals) and non-treated (control animals) dairy cows and heifers of the Holstein Friesian and Jersey breeds, and these hormone profiles were related to the embryo quality. Most experimental animals experienced an increase in progesterone concentrations following PMSG treatment and an abrupt decrease to values below 3 nmol/l after PG injection. The mean (+/-SE) intervals from prostaglandin treatment to estrus were 46.9+/-1.8 h and 64.5+/-4.8 h for experimental and control animals, respectively. At the onset of heat the progesterone concentration in experimental animals with optimal embryo quality (group I) was significantly lower (p<0.01) than in experimental animals which yielded unfertilized eggs (group II) (1.2+/-0.1 versus 3.9+/-0.8 nmol/l) and significantly higher than the level in the control group (0.6+/-0.1 nmol/l). Following estrus the progesterone profiles in all 3 groups were studied and the length of the superovulatory cycle was measured to 26.0+/-4.8 days. The preovulatory LH surge occurred sooner after prostaglandin injection in experimental (41 h) than in control animals (65 h). The LH surge in group I occurred within a narrow range and reached a higher average level than group II (24.2+/-2.2 ng/ml and 16.3+/-3.7 ng/ml, respectively). The control group attained an even higher LH surge (31.8+/-8.8 ng/ml) than did the experimental animals. The data presented in this experiment indicate that plasma levels of progesterone and LH in PMSG-PGF(2)alpha treated animals are related to embryo or egg quality.     

Post-partum release of prostaglandin F(2alpha) and uterine involution in the cow

Peripheral plasma levels of the main blood plasma metabolite of PGF(2alpha) (15-keto-13,14-dihydro-PGF(2alpha)) and progesterone were investigated during the immediate, post-partum period in 59 normally calving cows. Uterine involution was monitored by weekly rectal palpations. The levels of the prostaglandin metabolite were high at parturition and remained thereafter elevated for periods varying up to 7-23 days. Uterine involution was completed during periods ranging from 16-53 days. According to the clinical findings, the animals were divided into three groups. Group A comprises 46 animals which had an uncomplicated, puerperal period. A significant (p<0.001) correlation between the duration of elevated prostaglandin levels and the time for completed uterine involution (Y=29.6 - 1.3 (X - 13.5)) was found for these animals. Group B animals (n=8) had periods of varying length with uterine discharge during the first 30 days post-partum. When compared to group A animals, the animals in group B had comparatively longer periods of prostaglandin release and also longer periods for completion of uterine involution. Group C animals (n=5) at times had palpable, thin-walled, cystlike structures in the ovaries during the first 30 days post-partum. In this group of animals, the periods of high prostaglandin levels, as well as for the completion of uterine involution, were similar to those for the animals in group A. Progesterone levels remained low during the immediate post-partum period and in no case were elevated levels found until the prostaglandin release had ceased.