Darryl G. Stout

Protoplast rotation in a rotating electric field: The influence of cold acclimation

SummaryAn experimental and theoretical investigation has been made of the rotation of protoplasts ofSecale cereale L. (cv Puma) in a rotating electric field for the purpose of determining the electrical properties of the protoplast plasma membrane. The dependence of the protoplast rotation rate on: (1) the rotation rate of the applied electric field; (2) the electrical conductivity of the external medium; and (3) cold acclimation or lack thereof were determined. A theoretical analysis of the rotation rate of polarizable spherical cells in a rotating electric field leads to a qualitatively similar formula to that of Arnold and Zimmermann (Z. Naturforsch.37:908–915, 1982), but it differs from this earlier work by a large numerical factor (∼180). Detailed comparisons of the observed protoplast rotation rates with the new theory show generally good agreement. The protoplast rotation measurements allow a noninvasive determination of the specific plasma membrane capacitance,cm. The average value found in the present experiments iscm=(0.56±0.08)×10−2 F/m2. Within the experimental errors, thecm values are the same for cold-acclimated and noncold-acclimated protoplasts. Determination of plasma membrane resistance from protoplast rotation measurements does not appear feasible because of the high values of the specific resistance.

Possible role of transient electric fields in freezing-induced membrane destabilization

SummaryEvidence is presented to support the hypothesis that electrical potentials generated during the freezing of aqueous solutions (the Workman-Reynolds effect) may contribute to the destabilization of the plasma membrane and cryoinjury of isolated protoplasts. Specifically. (1) electric potential diffrences of sufficient magnitude to cause lysis of the plasma membrane occur during the rapid freezing of isolated protoplasts suspended in sorbitol: (2) survival of protoplasts is inversely correlated with the magnitude of the potential difference and (3) cold acclimation increases the stability of the plasma membrane to applied electric fields. A discussion is given of the different physical phenomena though to be involved in the Workman-Reynolds effect. The basis equations for these phenomena are outlined.

Cultivar and storage effects on germination and hard seed content of alfalfa

The impermeable coat of alfalfa (Medicago sativa L.) seed can reduce germination to an extent unacceptable for commercial use. The usual method of increasing germination of lots with high proportion of impermeable or hard seeds, mechanical scarification, can damage seeds. Experiments were conducted to determine the effect of cultivar, year of production and storage conditions on germination and hard seed content in alfalfa. Experiments with four Canadian cultivars indicated a significant effect of cultivar on seed weight, germination and hard seed content in freshly harvested seed. Year of production had a greater influence on these seed traits than cultivar. Under uncontrolled storage conditions, germination of 35 alfalfa synthetics increased and hard seed content decreased with time, although not at the same rate for all synthetics. Storage at 20 °C for up to 64 mo did not significantly decrease hard seed content. At 35 °C, hard seed content decreased continuously for all cultivars (for one cultivar to ...

MIXTURES OF PERSIAN CLOVER WITH ITALIAN RYEGRASS OR BARLEY-ITALIAN RYEGRASS FOR ANNUAL FORAGE

In the interior of British Columbia, spring barley (Hordeum vulgare L.) and Italian ryegrass (Lolium multiflorum var. italicum Beck.) are intercropped with fertilizer N as a 1-yr break before reseeding irrigated alfalfa (Medicago sativa L.). Persian clover (Trifolium resupinatum L.) was seeded with barley and ryegrass or only ryegrass to determine its effect on seasonal yield and forage nutritive value. All species mixtures were grown with and without a total of 200 kg ha−1 of N to compare N2 fixation by Persian clover with N fertilizer. Averaged over 2 yr, adding Persian clover to barley-ryegrass provided a total yield that was 96% of that obtained by adding fertilizer N to barley-ryegrass. Second cut yield averaged 58% more with clover than with N. However, the grasses yielded more than the grasses with added clover in the third cut. Adding clover to ryegrass provided 94% of the total yield of adding N fertilizer. Persian clover was higher in crude protein and in vitro digestibility of dry matter than r...

Alfalfa seed germination tests and stand establishment: The role of hard (water impermeable) seed

Freeze thaw scarification has been observed to increase the germination rate of alfalfa (Medicago sativa L.) seed containing a large proportion of hard (water impermeable) seed in a 7-d laboratory germination test; however, a comparable increase in plant density is not always seen in the field. To investigate this discrepancy, a field experiment with untreated and scarified seed was carried out using cultivars with high (Apica, 35%; Barrier, 32%) and low (Apollo II, 1%; WL316, 0.3%) percentages of hard seed. Plants were counted at the three-trifoliate-leaf and 10% bloom stages in 1992, the planting year, and at 10% bloom in 1993. In the field, effects of scarification were seen only at the 10% bloom stage in 1992, increasing the plant densities of high hard seed cultivars by 17% while decreasing those of low hard seed cultivars by 10%. Two laboratory experiments were also done to determine the effect of temperature, lighting (light, shade, dark) and media (on blotter, in soil) on the germination of hard s...

Effect of storage temperature and time on viability of rhizobia on lime-coated alsike clover ( Trifolium hybridum ) seed

Seed is often stored in warehouses where the temperature may drop below freezing or increase to 40°C depending on the time of year. Survival of rhizobia on lime-coated alsike clover ( Trifolium hybridum L.) seed stored in polypropylene bags was monitored under various temperature regimes ranging from –10 to 35 °C at Agriculture Canada Range Research Station, Kamloops, British Columbia, Canada during 1990 and 1991. Rhizobia were applied ata range of initial concentrations. Seed was inoculated with a peat-based clover inoculant (‘B’ inoculant, Nitragin Ltd, Milwaukee, Wisconsin, USA), and then given a commercial polymer-based lime coat (GNR™, Grow Tec Ltd, Nisku, Alberta, Canada). Rhizobia died continuously at all temperatures within the range —10 to 35°C. The dependence of Iog 10 (number of viable rhizobia/seed) on storage time was best described by a linear equation: Iog10(viable rhizobia/seed) = a + b (time). Coefficient a providedan estimate of the initial concentration of rhizobia. Coefficient b provided a measure of how rapidly rhizobia died. The death rate of rhizobia was the same during storage at 5 or 20 °C, but increased at a storage temperature of 35 °C. Storage at freezing temperatures did not increase the rate of rhizobial death but repeated freezing and thawing resulted in an increase. As the rate of rhizobial death was similar at constant temperatures from — 10 to 20 °C, temperature requirements are not stringent. Nevertheless, some temperature control is required to maximize the legal storage life of preinoculated coated seed, which in this study was estimated to be 96 days.

Growth and development of pinegrass in interior British Columbia.

Pinegrass (Calumugrostis rubescens Buckl.) is an important source of forage on forested and clearcut ranges in interior British Columbia. The vegetative growth and development of this infrequently flowering grass was documented. This information is required to improve our understanding of pinegrass grazing resistance, and in turn, of its grazing management. Numbers of tillers me2 and number of leaves per tiller were counted at intervals during the growing seasons of 1978 and 1979. Leaf blade area was measured at intervals during 1978 and 1979. Tiller height was recorded during 1978,1979, and 1982, while shoot weight was recorded at intervals during 1982. Pinegrass had up to 4 leaves per tiller, but on average only 3.2 leaves were present by the time growth ceased in July. Total leaf blade area was reached in July, and is largely comprised of 2 leaves. Total leaf blade area(y) was predicted from tiller height (x): y = 0.39375+ 0.051604x + 0.00419223~2 (I?2 = 0.97). A large proportion of leaf blade area was dead by the end of July. Tiller weight reached a maximum in July; it increased during May to July owing to an increase in number of leaves, leaf area, and specific weight of leaves. Growth analysis indicated that net assimilation rate (NAR), and relative growth rate (RGR) were high in mid-May and then gradually decreased to zero in July. NAR and RGR of pinegrass appeared typical for C3 plants. Pinegrass (Calumugrostis rubescens Buckl.) provides 50% of the forage production within the interior Douglasfir (Pseudorsuga menziesii Mirb.) zone of British Columbia (McLean et al. 1969), and about 6 million hectares of this zone are used for summer grazing (Tisdale and McLean 1957). Single year simulated grazing studies have shown that pinegrass is most sensitive to herbage removal during July (Freyman 1970, Stout et al. 1980). A study involving successive years of simulated grazing showed that stand vigor, measured as number of tillers m-‘, decreased each year by an amount that depended upon the intensity of the herbage removal (Stout et al. 1981). Clipping biweekly during the summer growth period to a height of 5 cm caused the stand vigor to decrease by one-half after each year of clipping, whereas clipping biweekly during the summer growth period to a height of 10 cm caused the stand vigor to decrease by one-half only after 3.7 years. In British Columbia, pinegrass became unpalatable by mid-August (McLean 1967) and the quality of pinegrass was adequate for rapid growth of yearly steers or for maintaining weanling calves only until 1 August (McLean et al. 1969). This study was conducted to characterize the growth and development of pinegrass. Such basic knowledge will increase our understanding of the simulated grazing studies and the forage quality studies that have already been conducted. In addition, this knowledge will allow better design of future grazing studies, and can be used directly by range managers. Flowering culm production, location of growing point, and clump basal area are frequently used by range managers to document growth. Since pinegrass is a rhizomatous and infrequently flowering species, this study was limited to vegetative growth characteristics. Authors are research scientist and technican, Agriculture Canada, Range Research Station, 3015 Ord Road, Kamloops, B.C. V2B 8A9, Canada. Manuscript accepted September 27, 1984. 312 Materials and Methods The study was conducted during 1978, 1979, and 1982. The site was adjacent to the Poison Creek site described earlier (Stout et al. 1980), 18 km north of Kamloops airport at 1,189 m elevation. Limited weather data for this site and a nearby site (Pass Lake) have been published (McLean et al. 1969, Stout et al. 1980). The soil is a Gray Luvisol (A.L. van Ryswyk personal communication) (Canadian Soil Survey Committee 1978) on glacial till material, with a high percentage of parent rock fragments in the profile (Tisdale and McLean 1957). The forest cover was a moderately open stand of trembling aspen (Populus tremuloides Doug].), lodgepole pine (pinus contorta Dougl.) and douglasfir. The major shrubs were birch-leaved spirea (Spirea betulifoliu Pall.), rose (Rosa sp. L.), twinflower (Linnueu borealis L.), and creeping oregongrape (Berberis repens Lindl.). Pinegrass was the predominant graminoid and the major ground cover constituent. Forbs included broad-leaved lupine (Lupinus lutifofius Agardh), heart-leaf arnica (Arnicucordz~oliu Hook.), cream-flowered peavine (Larhyrusochroleucus Hook.) and wild strawberry (Fruguriu virginiunu Duchesne). In 1978,20 tillers within a 3 X6-m area were randomly selected. On each collection date, height above the forest floor of these 20 tillers was measured. Twenty tillers of similar height were then selected from within the 3 X 6-m area, harvested, placed in plastic bags containing a little water, and transported to the laboratory on ice for leaf area and weight measurements. In 1979 individual tillers were taken at l-m intervals from a different starting point on a permanent transect on each date. Each tiller selected was the first one intercepted by a meter stick laid at right angles to the line at the predetermined point. The height of a selected tiller was measured and then the selected tiller was harvested and transported to the laboratory as in 1978. In 1982 six collection points chosen visually to represent the site were staked. Based on visual observations, a representative group of tiller tufts was taken from within a 0.5-m radius. Twenty tillers were collected from each location on each date. All tillers in a tuft were taken before moving on to another tuft to get the total of 20 tillers. A tuft typically contained 6 tillers. The height of each tiller was recorded and then the tiller was harvested and transported to the laboratory. Leaf blade area, a measure of photosynthetic area, was measured in 1978 and 1979. At the laboratory, a tiller was removed from the plastic bag, number of leaves were counted, and the blades were immediately removed from the tiller and mounted on paper. Pinegrass has 4 or 5 small rudimentary leaves that were excluded from our measurements. The lowest 3 or 4 rudimentary leaves do not extend above the litter layer and they become dry brown scales as the tiller matures. The upper 1 or 2 rudimentary leaves extend above the litter and may turn green. Leaves were numbered, starting with the lowest on the stem (the oldest) having a blade length greater than 1 cm. Only the leaf blade area exerted from the sheath (of the preceding leaf) was measured. This included some immature leaves that were still rolled, so they were unrolled before being mounted. Leaves that were not at all exposed were discarded. In mounting, a 1.25 cm wide length of Magic (3M) transparent tape was laid on the working surface, adhesive side up, anchored at both ends. The pinegrass blade was placed on the tape, JOURNAL OF RANGE MANAGEMENT 38(4), July 1985 abaxial side down, then flattened out so that its entire surface was in contact with the tape. The tape bearing the leaf was then affixed to a labelled sheet of paper. As soon as they were prepared, the pages of mounted leaves were photocopied since leaves were observed to shrink and curl on drying. Therefore, the photocopies were considered to be a more reliable record of leaf blade area. A dot planimeter (Bruning areagraph chart no. 4850) was used for leaf area determination. Means of duplicate counts for each of 3 planimeter placements on each blade were converted to square centimeters. Total and senesced blade areas were determined for each leaf blade. For blade weight determination, the mounted blades were peeled off the tape, dried 24 h in an 80°C oven, and weighed. This measurement was done on the whole (exerted) blade and so included both live and senesced tissues. Distribution of dry weight and percent dry matter within a tiller were determined in 1982. In the field the tiller was divided into 3 components. These were: “blade”, all completely exerted blades over 1 cm in length, and the exerted portion of immature blades; “sheath”, the foliage standing above the litter layer, excluding exerted blades and comprising sheaths, and blades 1 cm or less in length and all immature tissue enveloped in the sheaths;“subterranean tiller base”, the base of the tiller, lying beneath the surface of the litter layer, comprising (the vegetative) stem, rudimentary basal leaves and leaf sheaths of aerial leaves. The length of the tiller base ranged from I to 3 cm depending upon its point of origin on the previous year’s stem or on the rhizome, and on the depth of the litter layer. A bulk 20-tiller sample for each component was prepared for each of the 6 plots, weighed, oven-dried at 80’ C, and reweighed. Total tiller weight was calculated from the component weights. Growth analysis was done as described by Radford (1967). In 1978 and 1979, a relationship between leaf blade area and tiller height was established. In 1982, above ground level tiller dry weight (W) was related to growth time (t) by a second degree polynomial, W = a + bt + ct*. Leaf blade area (A) was estimated from tiller height and then related to growth time by a second degree polynomial, A q a’ + b’t + c’tz. April 31 was arbitrarily chosen as day 0 since observations over several years indicate that snow melt typically occurs during the period April 24 to May 7. Net assimilation rate (NAR) at a particular time was then estimated using NAR = l/A dW/dt and relative growth rate (RGR) at a particular time was estimated using RGR q I/W dW/dt. Leaf area ratio (LAR) is defined by LAR = RGR/NAR. Average NAR was calculated using NAR q [(Wz-Wl)/(Az-At)] [(In Az-ln Ar)/(tz-tt)], and average RGR was calculated using RGR = (In Wrln Wl)/(tz-tl). Each experiment reported here had a completely random design. Statistical analysis involved calculating_? f SE for a sample size of n. Curve fitting was done using a computer program th