Studies on the Expansion of the Leaf Surface: III. THE INFLUENCE OF RADIATION ON CELL DIVISION AND LEAF EXPANSION

The numbers of leaves and the areas and cell numbers of leavesfrom the first five nodes of the cucumber were determined throughouttheir development with three different amounts of daily radiationand two conditions of nutrient supply. The rate of leaf production was found to be constant with timefor any one amount of radiation and to increase with increasedradiation. The transition from the rate in darkness to thatin light was sharp and occurred more quickly the higher theradiation. Provided the nutrient supply was high the ultimate areas ofindividual leaves were greater the higher the radiation; ifmineral nutrients were depleted the maximum area of a leaf occurredwith an intermediate amount of light. This arose because thesefactors exercised a differential effect on the various phasesof growth. Each leaf commenced its life as a mass of dividing cells andthe mean rate of division remained constant until it unfoldedfrom the terminal bud. The mean rate of division was much greaterat high than at low levels of radiation and was interpretedas being regulated by carbohydrate supply. Although expansionof some cells was likely before unfolding, after this stagethere was a marked decrease in the proportion of cells proceedingto division. The duration of division in the leaf after unfoldingwas independent of radiation; although 70–98 per cent,of the final number of cells were formed after unfolding, theultimate number was effectively determined by the rate of divisionprior to unfolding. Low nutrient supply restricted the duration of division in theleaf to a significant extent and the expansion of cells to avery considerable degree. The smaller leaves on plants receivinghigh rather than medium radiation were due to their cells beingmuch smaller.     

LEAF DEVELOPMENT OF PHASEOLUS VULGARIS L. IN LIGHT AND IN DARKNESS

Development of the primary bean leaf in the dark and under continuous white light was studied during 14 days after sowing. The increase in surface area of the blade is the result of a number of sequential processes. Both in the darkness and under illumination, leaf growth is characterized by an initial cell enlargement followed by intensive cell division. Cell division in etiolated leaves continues for one day longer than in illuminated ones, but it proceeds at a slower rate. Mature leaves grown under white light undergo a phase of cell enlargement after cell division has stopped. This increases their surface area up to 800 times when compared with the blade area of the embryo. This enlargement phase is almost absent in dark-grown seedlings. Consequently the blade area of etiolated leaves is only 50 times that of the embryonic state. Thus light appears to have a dual effect on leaf development: it activates cell division and induces cell expansion.     

Studies on the Growth of Spinach Leaves (Spinacea oleracea)

The growth of spinach leaves has been studied from approximately1 cm long to full size. Over-all growth was measured in termsof area and total number of cells. The differential growth ofleaves was measured by the changes in the shape of squares drawnon the leaf surface. Growth differentials in terms of numbersof cells and number displaying mitotic figures were measuredin leaf discs taken from different positions within leaves. It was found that cell division in spinach leaves continueduntil the leaves reach from one-third to one-half full size.Cell division within the lamina of the leaves was not uniformbut ceased at an early stage of development in the leaf tipregion and continued for an extended period at the base.     

Cell Division and Expansion in the Growth of the Leaf

Volumes and numbers of cells were determined at different stagesof development of the fifth leaf of Lupinus albus, and eachof the second pair and the tenth leaf of Helianthus annuus.In the case of the second pair of sunflower leaves the valuescover the whole life of the leaf from initiation to senescence. During both primordial development and the ensuing ‘grandperiod of growth’ division is the determinant of growth.About 10 per cent. of the cells in the fully grown leaf arelaid down before leaf-emergence; the remaining 90 per cent.are formed during unfolding. Division does not cease in thelupin leaf or the second pair of sunflower leaves until theyhave reached half their maximum area. The tenth leaf, on theother hand, is as much as three-quarters fully grown beforedivision ceases. Cell expansion commences soon after leaf initiation and continuesthroughout the life of the leaf. With lupin and the second pairof sunflower leaves there is a fourfold increase in the averagevolume of the cells before emergence from the apical region.During unfolding, there is a further tenfold increase in theaverage volume of the cells of the lupin leaf, and a twentyfoldincrease with the second pair of sunflower leaves. Expansioncontinues after the cessation of division but this further increasein volume is comparatively small. The data are discussed in relation to the ‘two phase’hypothesis of leaf development.     

Some Effects of Temperature and Irradiance on Growth of the First Four Leaves of Wheat, Triticum aestivum

Plants of Heron wheat were grown at 20 and 15 °C and inquantum flux densities of 400 and 200 µmol m^–2 s^–1.At completion of expansion of the first or second leaf, plantswere transferred between temperatures and quantum flux densities.Final size and cell number were measured for each of the firstfour main-stem leaves. Leaf area was affected only slightlyby treatment and effects on leaf length and width were alsosmall. It was concluded that leaf extension rate, which waslower at the lower temperature and in the lower light regime,is inversely related to the duration of leaf expansion. Leafdry wt was higher for plants grown in high light and for plantsgrown at 15 °C; transfer treatments led to readjustmentswhereby dry wts of leaves expanded after transfer resembledthose of leaves on plants kept throughout in the post-transferconditions. Leaf cell number was not affected by treatment but mean drywt per cell was significantly greater in high light, and forthe first two leaves, at 15 °C. There was a major and highlysignificant effect of treatment on the ratio of dry: fresh wtper cell, this being larger for leaves in high light. Transfertreatments between light regimes led to rapid changes in expandingleaves as was found for leaf dry wt. It was concluded that theexpanding grass leaf is much less dependent on older leavesto provide the necessary materials for cell division and expansionthan is the dicotyledon leaf. It is suggested that the increasein cell dry wt in high light is associated with an increasein cell wall material which is under photomorphogenic control. Triticum aestivum, wheat, leaf growth, cell division, cell expansion, cell size     

Bean Leaf Expansion in Relation to Temperature

When dwarf Phaseolus vulgaris plants were grown in a controlledenvironment at 20, 25, 30, and 35° C, expansion of the primaryleaves occurred in two phases with an intermediate lag. Varyingrates and duration of expansion were involved, leading to greatestfinal areas at the two intermediate temperatures. Dry weightsof the leaves and leaf areas were similary influenced by temperature,except that the initial rates of increase continued for a longerperiod for weights than for areas. The rates of cell divisionand final numbers of cells were similar from 25 to 35° C,but both were decreased at 20° C. Final cell sizes were,on the other hand, decreased only at the highest temperature.The time trends of cell expansion varied greatly with temperature. Leaf expansion is discussed as a possible consequence of substratesupply, which may be determined by temperature in a number ofways. Cell division and cell expansion are not considered tobe joint direct determinants of leaf expansion. Temperatureinfluences division, with two consequences; the rate interactswith substrate supply to determine size of cells, and finalcell number affects potential leaf area. Cell size is regardedas being secondary to numbers of cells and total material available,although some factors can vary cell size independently of substrate,e.g. water status. An important control of leaf growth, until the attainment ofabout half the final area, may be exercised by way of the leaf.Subsequently, intra-plant competition is likely to dominate.     

Temporal and Spatial Development of the Cells of the Expanding First Leaf of Arabidopsis thaliana (L.) Heynh

The three-dimensional quantitative leaf anatomy in developingyoung (9–22 d) first leaves of wild type Arabidopsis thalianacv. Landsberg erecta from mitosis through cell and leaf expansionto the cessation of lamina growth has been studied. The domainsof cell division, the relative proportion of the cell typespresent during development and the production of intercellularspace in the developing leaf have been determined by image analysisof entire leaves sectioned in three planes. Mitotic activityoccurs throughout the youngest leaves prior to unfolding andcell expansion is initiated firstly at the leaf tip with a persistentzone of mitotic cells at the leaf base resulting in a gradientof development along the leaf axis, which persists in the olderleaves. Major anatomical changes which occur during the developmentare, a rapid increase in mesophyll volume, an increase in thevein network, and expansion of the intercellular spaces. Thepattern of cell expansion results in a 10-fold variation inmesophyll cell size in mature leaves. In the youngest leavesthe plan area of mesophyll cells varies between 100 µm²and 400 µm² whereas in mature leaves mesophyll cells rangein plan area from 800 µm² to 9500 µm². The volumesof mesophyll tissue and airspace under unit leaf area increase3-fold and 35-fold, respectively, during leaf expansion. Thevolume proportions of tissue types mesophyll:airspace:epiderrnal:vascularin the mature leaf are 61:26:12:1, respectively. This studyprovides comparative information for future identification andanalysis of leaf development mutants of Arabidopsis thaliana. Key words: Arabidopsis, quantitative leaf anatomy, leaf expansion, image analysis     

Devlopmental Physiology of Sugar-Beet: II. EFFECTS OF TEMPERATURE AND NITROGEN SUPPLY ON THE GROWTH, SOLUBLE CARBOHYDRATE CONTENT AND NITROGEN CONTENT OF LEAVES AND ROOTS

Plants were grown at temperatures of 15 and 25 ?C with two ratesof nitrogen supply. The changes in dry weight, leaf area, cellnumber, mean cell volume, soluble carbohydrate, and total nitrogenconcentration of the cotyledons, the first and second pair oftrue leaves, and the storage root were measured. Changes incell number and cell volume of the first pair of true leavesand storage root of plants were also measured at 11, 18, 25,and 32 ?C. Leaf growth before unfolding was chiefly by increase in cellnumber and after unfolding by increase in mean cell volume,while the growth of the storage root was almost entirely byincrease in cell number. The rates of cell division and cellexpansion were fastest at 25 ?C, but the initially high ratesof cell division in the terminal bud and in individual leavesdecreased rapidly and greater rates were maintained at the sub-optimaltemperatures, i.e. 15 and 18 ?C. After an initial period ofslow growth, the first-formed leaves grew faster and becamelarger at 15 than at 25 ?C. Leaves were produced, unfolded,grew faster, and became larger with increase in the externalconcentration of nitrogen, because cells divided and expandedfaster, so that nitrogen increased the number and size of cells. Sugar concentration was greater at 15 than at 25 ?C in leavesbut not in the storage root. Sugar concentration in the petiolesof the first and second pair of true leaves increased to 1.2and 2.0 per cent fresh weight respectively. Decreased nitrogensupply temporarily increased the sugar concentration of cotyledonpetioles and the seedling hypocotyl, but later decreased itin the leaves and storage root. Nitrogen concentration was greaterin the leaves and storage root at 15 than at 25 ?C with thelarger nitrogen supply. Nitrogen concentrations were similarin young leaves of all treatments but as the size of leavesincreased nitrogen concentrations decreased most rapidly at25 ?C with the smaller nitrogen supply. It is suggested that when increased leaf production and storage-rootgrowth occurs at temperatures below the growth optimum (25 ?C),they may be due to an effect of increased carbohydrate supplyon cell division and sugar storage.     

Effects of cell number and cell size on petiole length variation in a stoloniferous herb

In stoloniferous species, the length of petioles is of pivotal importance because it determines the position of leaf blades within the canopy. From a mechanistic perspective, two developmental processes, cell division and cell elongation, are responsible for the length of a given petiole. This study aimed at quantifying the relative contributions of cell division and cell elongation to genotypic and plastic variation in petiole length of the stoloniferous herb Trifolium repens. Thirty-four genotypes of T. repens were grown under high light conditions and simulated canopy shade. Cells were counted and their lengths measured on epidermal prints from fully grown petioles of leaves that had been initiated in the experimental light conditions. Cell number was the main trait explaining petiole length differences among genotypes grown under high light, while both cell number and length changed in response to shading. Our study revealed a strong negative correlation between shade-induced changes in cell number and cell length: genotypes that responded to shading by increasing cell numbers hardly changed in cell length, and vice versa. Our results suggest that genotypic and phenotypic variation in petiole length results from a complex interplay between the developmental processes of cell elongation and cell division.     

Ultraviolet-B radiation reduces the rates of cell division and elongation in the primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman)

The primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman) was used as a model system to examine how elevated ultraviolet‐B (UV‐B; λ= 280–320 nm) radiation affected growth. A reduction in the rate and duration of growth of the primary leaf, in response to UV‐B, was the result of changes in both the rate and extent of cell division and elongation. UV‐B reduced the proportion of mitotically active cells (mitotic index) and increased the time taken for cell division (cell doubling time). Thus the supply of cells into the elongation zone was reduced, and this, coupled to a reduction in the rate of elongation, resulted in reduced leaf growth. This analysis of the spatial distribution of growth provided a means of calculating the age of cells within the leaves. Cells of UV‐B‐treated leaves were found to age more quickly than those of the controls. This analysis will enable future studies to take account of age‐related changes when interpreting the response of plants to any number of environmental stresses that affect leaf development.     

Co-Ordination of Cell Division and Tissue Expansion in Sunflower, Tobacco, and Pea Leaves: Dependence or Independence of Both Processes?

Abstract Temporal analyses of cell division and tissue expansion in pea, tobacco, and sunflower leaves reveal that both processes follow similar patterns during leaf development. Relative cell division and relative tissue expansion rates are maximal and constant during early leaf development, but they decline later. In contrast, relative cell expansion rate follows a bell-shaped curve during leaf growth. Cell division and tissue expansion have common responses to temperature, intercepted radiation, and water deficit. As a consequence, final leaf area and cell number remain highly correlated throughout a large range of environmental conditions for these different plant species, indicating that cell division and tissue expansion are co-ordinated during leaf development. This co-ordination between processes has long been explained by dependence between both processes. Most studies on dicotyledonous leaf development indicate that leaf expansion rate depends on the number of cells in the leaf. We tested this hypothesis with a large range of environmental conditions and different plant species. Accordingly, we found a strong correlation between both absolute leaf expansion rate and leaf cell number. However, we showed that this relationship is not necessarily causal because it can be simulated by the hypothesis of independence between cell division and tissue expansion according to Green's theory of growth (1976). Received 23 February 2000; accepted 3 March 2000     

草甘膦和百草枯对毛桃幼苗根系形态及地上部生长的影响

以砧木毛桃幼苗为研究对象,通过土施草甘膦和百草枯研究2种桃园常用除草剂对毛桃营养生长、根系结构、根尖细胞分裂、叶片光合特性等的影响,为除草剂在桃生产中的安全使用提供科学依据。结果表明: 草甘膦处理显著抑制毛桃地上部和根生长,与对照相比,株高降低31.5%,总根系长度、总根表面积、总根体积和总根尖数分别降低了39.5%、39.5%、49.8%和44.6%,而百草枯处理以上指标与对照差异均不显著;草甘膦和百草枯处理后毛桃根尖细胞有丝分裂指数分别降低38.0%和35.9%,且草甘膦处理分裂中期细胞数占分裂细胞总数的比例显著低于对照和百草枯处理;毛桃根尖细胞对2种除草剂响应迅速,从处理第2天开始根尖细胞电解质渗漏率始终显著高于对照。叶片细胞电解质渗漏率则从处理5 d后开始显著升高,且草甘膦处理出现幼叶基部变黄并向叶尖蔓延,同时部分叶尖逐渐焦枯的现象;2种除草剂处理导致毛桃叶片净光合速率、气孔导度、蒸腾速率有不同程度的降低,其中草甘膦处理下降更明显。综上,使用草甘膦和百草枯均会降低毛桃幼苗根尖细胞分裂指数,提高根尖细胞电解质渗透率,总体降低叶片净光合速率。草甘膦对毛桃营养生长、叶片光合作用影响更大,而且会造成幼叶变黄、叶尖焦枯等现象。     

Leaf dynamics,self-shading and carbon gain in seedlings of a tropical pioneer tree

We examined leaf dynamics and leaf age gradients of photosynthetic capacity and nitrogen concentration in seedlings of the tropical pioneer tree, Heliocarpus appendiculatus, grown in a factorial design under controlled conditions with two levels each of nutrients, ambient light (light levels incident above the canopy), and self-shading (the gradient of light levels from upper to lower leaves on the shoot). Correlations among these parameters were examined in order to determine the influence of self-shading, and the regulation of standing leaf numbers, on leaf longevity and its association with leaf photosynthetic capacity. Leaf longevity and the number of leaves on the main shoot were both reduced in high light, while in the low light environment, they were reduced in the steeper self-shading gradient. In high nutrients, leaf longevity was reduced whereas leaf number increased. Leaf initiation rates were higher in the high nutrient treatment but were not influenced by either light treatment. Maximum-light saturated photosynthetic rate, on an area basis, was greater in the high light and nutrient treatments, while the decline in photosynthetic capacity in realtion to leaf position on the shoot was more rapid in high light and in low nutrients. Leaf longevity was negatively correlated among treatments with initial photosynthetic capacity. The leaf position at which photosynthetic capacity was predicted to reach zero was positively correlated with the number of leaves on the shoot, supporting the hypothesis that leaf numbers are regulated by patterns of self-shading. The negative association of longevity and initial photosynthetic capacity apparently arises from different associations among gradients of photosynthetic capacity, leaf numbers and leaf initiation rates in relation to light and nutrient availability. The simultaneous consideration of age and position of leaves illuminates the role of self-shading as an important factor influencing leaf senescence and canopy structure and dynamics.     

Can meristematic activity determine variation in leaf size and elongation rate among four Poa species? A kinematic study

We studied inherent variation in final leaf size among four Poa spp. that live at different elevations. The average final length of leaf 7 of the main stem of the smallest species (Poa alpina) was only one-half that of the largest species (Poa trivialis); it was correlated with leaf elongation rate, but not with the duration of leaf elongation. A faster rate of leaf elongation rate was associated with (a) larger size of the zone of cell expansion, and (b) faster rates of cell production (per cell file) in the meristem, which in turn were due to greater numbers of dividing cells, whereas average cell division rates were very similar for all species (except Poa annua). Also we found that the proliferative fraction equaled 1 throughout the meristem in all species. It was remarkable that rates of cell expansion tended to be somewhat higher in the species with slower growing leaves. We discuss the results by comparing the spatial and material viewpoints, which lead to different interpretations of the role of cell division. Although the presented data do not strictly prove it, they strongly suggest a regulatory role for cell division in determining differences in growth rate among the present four Poa spp.     

Temperature Affects Expansion Rate of Maize Leaves without Change in Spatial Distribution of Cell Length (Analysis of the Coordination between Cell Division and Cell Expansion)

We have analyzed the way in which temperature affects leaf elongation rate of maize (Zea mays L.) leaves, while spatial distributions (observed at a given time) of cell length and of proportion of cells in DNA replication are unaffected. We have evaluated, in six growth chamber experiments with constant temperatures (from 13 to 34[deg]C) and two field experiments with fluctuating temperatures, (a) the spatial distributions of cell length and of leaf elongation rate, and (b) the distribution of cell division, either by using the continuity equation or by flow cytometry. Leaf elongation rate was closely related to meristem temperature, with a common relationship in the field and in the growth chamber. Cell division and cell elongation occurred in the first 20 and 60 mm after the ligule, respectively, at all temperatures. Similar quantitative responses to temperature were observed for local cell division and local tissue expansion rates (common x intercept and normalized slope), and both responses were spatially uniform over the whole expanding zone (common time courses in thermal time). As a consequence, faster cell elongation matched faster cell division rate and faster elongation was compensated for by faster cell displacement, resulting in temperature-invariant profiles of cell length and of proportion of dividing cells. Cell-to-cell communication, therefore, was not necessary to account for coordination.     

MITOTIC INDUCTION AND SYNCHRONIZED CELL DIVISION IN OEDOGONIUM CARDIACUM (HASS.) WITTR.

Waves of mitosis are induced in Oedogonium cardiacum grown under a 15 hr light/9 hr dark cycle. Mitosis starts 4 to 5 hr after the start of the dark period. Each mitotic stage has a high initial rate which plateaus at a lower rate for several additional hours. Partial synchronization of mitotic stages results from this induction of cell division. Mitotic divisions last 9 to 10 hr after induction. During the remainder of the 24-hr light/dark cycle, cells are in interphase. Along a filament, several dividing cells tend to be adjacent, with the most advanced stage in the cap cell. Progressively earlier mitotic stages are basal to the dividing cap cell. This pattern of mitotic division differs from the state in nature where only the cap cell usually divides. Chromosomes probably maintain a telophase arrangement during interphase. The suitability and advantages of Oedogonium, a haploid alga with sexual reproduction, as an experimental plant for cytological, developmental, biochemical, and genetic studies is pointed out.     

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.     

Changes in chloroplast number per cell during leaf development in spinach

Summary The amounts of chlorophyll and nitrogen and the numbers of cells per unit area change as the green leaves of spinach plants grow and increase in size in the light. The changes in the numbers of chloroplasts per cell were measured by a new method. A 5-fold increase in the numbers of chloroplasts per cell took place in both palisade and mesophyll cells over a growing period of 10 days during which time the area of the leaves increased from 1 to 50 cm². Proplastids were not present in the young green leaves but electron-microscope and phase-contrast observations showed the presence of grana-containing chloroplasts, many of which appeared to be undergoing division by constriction. It is suggested that the large increase in chloroplast numbers as leaf cells grow and expand in the light is from the division of differentiated chloroplasts containing grana.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Chromosome constitution and cell division in in vitro cultures ofZea mays L. endosperm

Summary Cultures of maize (Zea mays L.) endosperm grown in vitro for over 3 years were examined cytologically. Conditions of aneuploidy and polyploidy were noted. Chromosome numbers ranged from 21 to over 200, with 30 to 60 being observed most often. Although a few extra large cells with polyploid nuclei were scattered throughout the smear preparation, a large proportion of the interphase nuclei appeared similar in volume and probably contained a near normal complement of chromosomes. Anaphase bridges were the most commonly observed chromosome aberration. No cell divisions were observed the first 24 hr after transfer. From 2 to 8 days after transfer the proportion of cells in division was relatively constant with a mitotic index of approximately 5.5%. The proportion of cells in division began to decline 8 days after transfer and in the final sample taken after 13 days only 2.6% of the cells were in division. Examples of localized synchrony were observed and mitotic indices for individual cell clumps ranged from 0 to 17%. Authorized for publication on October 16, 1973 as paper number 4552 in the Journal Series of the Pennsylvania State Agricultural Experiment Station.