SOMATIC GENETIC ANALYSIS OF THE APICAL LAYERS OF CHIMERAL SPORTS IN CHRYSANTHEMUM BY EXPERIMENTAL PRODUCTION OF ADVENTITIOUS SHOOTS

Many commercial chrysanthemum cultivars display unusual somatic variability. The ‘Indianapolis’ family of chrysanthemum sports was analyzed for the genetic potential for color of each of the three layers in the apical meristem of their shoots. Populations of each cultivar were grown and sectors and off-color plants recorded. The location of the pigment within cells and between tissues was determined by microscopic examination of free-hand sections of fresh petals. Adventitious buds were forced from the stems of each cultivar by excising all normal lateral buds. These observations, showed 12 of the 16 ‘Indianapolis’ cultivars to be periclinal chimeras. Adventitious shoots often originated from two or more cells, derived from at least two different apical layers, and thus were themselves periclinal chimeras. While somatic mutation is the ultimate source of the variability in ‘Indianapolis’ chrysanthemums, the most frequent type of sporting resulted from the loss in mitosis of a chromosome carrying a supressor for the formation of yellow chromoplasts, giving a yellow sector or shoot. Sectors resulting from rearrangement of layers in the periclinal chimeras were less frequent than the sectors from chromosome loss.     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

ONTOGENY IN MONOCOTYLEDONS AS REVEALED BY STUDIES OF THE DEVELOPMENTAL ANATOMY OF PERICLINAL CHLOROPLAST CHIMERAS

The developmental anatomy of apically stable periclinal chloroplast chimeras was studied in a number of monocotyledonous genera. Their ontogeny is basically similar to that of dicotyledons. In both there are three independent apical layers (L-I, L-II, and L-III) whose derivatives can be traced in stem, leaf and flower. The bulk of the stem tissue is derived from L-III with only the epidermis and one or two hypodermal cell layers from L-I and L-II. All three layers participate in formation of the leaf with great flexibility in the amount of tissue from each. There is more instability in growth of most monocotyledonous leaves than in dicotyledonous leaves. As a result there is relatively more tissue derived from L-I and L-II and less from L-III. The same is true in floral development so that a significant number of gametes are of L-I origin. As in dicotyledons, there is variation in direction, timing, and frequency of cell division. However, an overriding genetic control results in normal size, shape, and structure. The evidence from genetic and cytochimeras in both monocotyledons and dicotyledons has provided direct experimental proof of the functional reality of the apical layers described by Hanstein and Schmidt.     

CHLOROPHYLL-DEFICIENT CELL LINES WHICH ARE GENETICALLY UNCHARACTERIZED CAN BE INAPPROPRIATE FOR USE AS PHENOTYPIC MARKERS IN DEVELOPMENTAL STUDIES

Periclinal chloroplast chimeras are genetic mosaics which possess shoot apices composed of one or more chlorophyll-deficient histogens and can exist as a series of arrangements of normal and mutant layers (A-B-B, A-B-A, etc.). Three periclinal chimeral cultivars of Sansevieria trifasciata L., each of which possesses normal green cell layer(s) but a genetically different chlorophyll-deficient cell layer(s), were utilized to study the effect of genotype on the ability of the cell layers of leaf cuttings and of cultured leaf tissue to regenerate shoots. The epidermis and LI derivatives were apparently incapable of shoot regeneration via leaf cutting, yet in two cultivars produced some shoots in vitro. In two of the cultivars, the chlorophyll-deficient cells never produced shoots. In the third, the capability of chlorophyll-deficient cell layers to produce shoots was less in vitro than in vivo, indicating that when determining morphogenic potential, direct comparisons between in vitro and in vivo systems may not be valid. Results also demonstrate that because genetically different albino cell layers can differ in their morphogenic response, utilizing a series of periclinal chimeras is useful only if the series is composed of the same two genotypes.     

Nuclear DNA content of ploidy chimeras of Hosta Tratt. (Hostaceae) demonstrate three apical layers in all organs, but not in the adventitious root

A unique combination of chloroplast and ploidy chimeras is found in some Hosta Tratt. (Hostaceae) cultivars. The absence of endopolyploidy in the above–ground parts made it possible to study the fate of the apical or germ layers in all organs. Moreover, we would like to address the question of the number of apical cell layers in Hosta also to explain breeding results. Nuclear DNA content in twelve different organs of Hosta was determined by flow cytometric analysis of four chimeric cultivars with a 4-2-2 or 2-2-4 composition. The margin of the leaf, derived from the L1, can be determined separately and has a 4C amount of DNA in 4-2-2 ploidy chimeras but a 2C amount of DNA in 2-2-4 chimeras. The presence of three layers in Hosta was deduced from comparisons of the percentage of 4C nuclei in (2)-2-4 and (4)-2-2 chimeras. If Hosta organs derive only from two layers the percentage of 4C cells in the different ploidy chimeras should add up to 100%, but they do not. The absence of nuclei with a 2C amount of DNA in the roots of 2-2-4 plants shows that only the L3 participates in adventitious root formation. Gametes and consequently seedlings are mainly derived from the L2. The often found correlation between the color of the centre of the leaf and the color of the seedlings is the consequence of the way these cultivars originate. The presence of three apical layers is visualized in the tricolored leaves of Hosta cultivars like `Striptease'. The nuclear DNA amounts show unequivocally, that in all the above ground parts of Hosta the three apical layers L1, L2 and L3 are present. However, only the L3 participates in adventitious root formation.     

INDEPENDENCE OF TISSUES DERIVED FROM APICAL LAYERS IN ONTOGENY OF THE TOBACCO LEAF AND OVARY

Six different homoplastidic periclinal chimeras of tobacco carrying the plastogene DP₁ were selected after somatic segregation in heteroplastidic seedlings. Direct observation of the plane of division in epidermal cells of young leaves, and the number and size of sub-epidermal green spots on leaves with the Green-White-White (G-W-W) pattern of variegation, indicated that the ratio of periclinal to anticlinal divisions in L-I during development of the lamina was 1:3100. The number of green and white seedlings obtained from the different chimeral branches indicated a similar frequency of periclinal divisions in development of the ovary. The arrangement of green and white tissue in mature leaves of the various chimeral types indicated the extent of participation by the three apical layers in the initiation of the buttress, development of the axis, and formation of the lamina. During development of the lamina there must be three independent initial-groups present. L-I and L-II initials remain marginal, but early in the growth of the lamina the leading edge of tissue derived from L-III becomes separated from the submarginal (L-II) initials by the products of frequent periclinal divisions of the L-II initials.     

STRUCTURAL INVESTIGATIONS OF ASEXUAL REPRODUCTION IN NEPHROLEPIS EXALTATA AND PLATYCERIUM BIFURCATUM

Nephrolepis exaltata cv. Bostoniensis, the Boston fern, exhibits extreme stem dimorphism. The plant has orthotropic, dictyostelic shoots which bear pinnatifid leaves and plagiotropic, protostelic stolons which are aphyllous. Vegetative reproduction occurs by budding from primary and secondary stolons. Secondary stolons arise exogenously from derivatives of the apical cell of the primary stolon, whereas root primordia develop endogenously. Shoots develop in vivo when a creeping stolon makes contact with the substrate via extensive root proliferation. When stolon segments are excised and grown in vitro, secondary stolon primordia expand and initiate leaf primordia, forming new leafy shoots. In Platycerium bifurcatum, the staghorn fern, asexual propagation occurs on ageotropic roots ramifying among the basal nest fronds. Root bud initiation is marked by root tip hypertrophy following cortical parenchyma expansion. Root apical cell derivatives produce the bud apex; the root apical cell remains separate from the developing root bud. Superficially, vegetative reproduction in Nephrolepis and Platycerium appears to involve unusual organs. However, both ferns exhibit leafy bud development from distinct sites of origin, not from undetermined primordia or from direct transformation of root to shoot. Thus, distinctness of organ types is maintained in these two ferns and no evidence for interconvertibility of organ types has been found.     

Arrangement of cell layers in the shoot apical meristems of periclinal chimeras influences cell fate

Utilizing a complete set of six periclinal graft chimeras composed of Nicotiana tabacum and Nicotiana glauca (TGG, GTT, TTG, GGT, TGT, and GTG), the fate of the three apical cell layers in both vegetative and reproductive organs has been traced. An analysis of leaf phenotype indicated that only rarely did deviations from expected cell lineage occur and in only TTG did such deviations originate in the shoot apical meristem rather than during leaf development. In most plants that possess a stratified shoot apical meristem, gametes are derived from the second apical layer (L2). A phenotypic and/or DNA analysis of seed progeny following reciprocal crosses between all chimeras and their component species indicated that pollen and eggs were sometimes derived from non-L2 lineage in all but one periclinal chimera. There was no evidence for non-L2-derived gametes in 95 crosses where GTT was a parent whereas 40 of 104 crosses with TTG as a parent yielded some offspring that resulted from non-L2-derived gametes. Of these 40 cases, non-L2-derived pollen grains were responsible 39 times while non-L2-derived eggs were responsible just once. Therefore, the occurrence of non-L2-derived gametes was not random. The disruption of ‘normal’ lineage patterns was dependent on the specific arrangement of genetically dissimilar tissue layers in the shoot apices of the chimeras and was different for different organs.     

Induction and origin of adventitious shoots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The chimeras between tuber mustard (Brassica juncea) and red cabbage (B. oleracea) were artificially synthesized in our previous study. Adventitious shoots were induced from nodal segments and leaf discs of TCC (LI-LII-LIII, LI -the outmost layer of shoot apical meristem; LII -the middle layer; LIII -the innermost layer. T = Tuber mustard, C = Red cabbage) chimeras. The origin of the shoots was analyzed by histology and molecular biology. As a result, the frequency of adventitious shoot induction rose with the increase of BA in MS medium in the area of the nodes. However, there was no different induction frequency of adventitious shoots from nodal segment bases in media with different BA concentrations. Most adventitious shoots (clustered shoots) arising from the node area were TTT (Tuber mustard- Tuber mustard- Tuber mustard) and only 4 shoots were chimeras, which indicated that more shoots originated from LI than from LII and LIII. All shoots from nodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicating that the shoots originated from LII or LII and LIII. There were significant differences in the regeneration rate in the margin of the leaf discs among the three combinations of BA and NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 out of 70 were chimeras, which indicated that more shoots originated from LII or LII and LIII than from LI. All chimeras obtained by regeneration were different from the original explant donor in type in the present study. The origin of the adventitious shoots varied with the site of origin on the donor plant, and could be multicellular and multihistogenic.     

The Effect of 2,3.5 Triiodobenzoic Acid on Caulogenesis in Callus Cultures of Tomato and Pelargonium

Stem explants from winter grown tomatoes cultured on a cytokinin, auxin-free medium, developed one or two adventitious shoots at the top end of the explant. Addition of the auxin transport inhibitor. 2,3,5-triiodobenzoic acid (TIBA) to the medium stimulated caulogenesis with loss of polarity. Callus, initiation in pelargonium and ‘geranium’ petiole explants requires both auxin and cytokinin. On transfer, after callus induction to an auxin-free medium, rhizogenesis occurs in pelargonium cultures followed by caulogenesis. Few shoots develop and unless these are removed, further caulogenesis is suppressed. Bud-like structures were formed in the callus. Subculture on auxin-free medium containing cytokinin and TIBA resulted in shoot formation from these bud-like organs. An analogy with apical dominance is suggested. In ‘geranium’ callus, shoots developed with a low frequency (c. in 2% of the cultures): caulogenesis was increased to 80% when calli were subcultured from auxin-free, cytokinin medium after green nodule formation to cytokinin-TIBA medium. Histological studies of green nodules in ‘geranium’ callus indicated a variation in morphological development within and between nodules. It is suggested that auxin synthesis may occur at some microscopic stage in morphogenesis in ‘geranium’ cultures which suppresses further caulogenesis. This may be overcome by the addition of TIBA to the medium at the appropriate stage in morphogenesis. The possible interaction of endogenous auxin in morphogenesis is discussed.     

RELATIONSHIPS BETWEEN SHOOT GROWTH AND CHANGING PHYLLOTAXY OF RANUNCULUS

Growth of Ranunculus shoots through ontogeny is quantified by techniques utilizing scanning electron microscopy and studies on living plant material. The order of the contact parastichy phyllotaxy in the apical system is related to the relative plastochron rates of growth of the shoot. There is a change in the (2, 3) contact parastichy pattern of vegetative phyllotaxy to a transitional (3, 5) contact pattern which is maintained through sepal production. Formation of a 5(1, 1) whorl of petal primordia establishes a (5, 8) contact pattern with the sepal primordia. Subsequent initiation of stamen primordia, in spiral sequence, results in (5, 8, 13) triple contacts between petal and stamen primordia. The stamen primordia and carpel primordia arrangement is characterized by a (8, 13) contact parastichy pattern of phyllotaxy. Through ontogeny the volume of the shoot apex progressively increases but the shape of the apex, described by a second degree polynomial, remains constant. The plastochron and the relative plastochron rates of radial and vertical displacement of primordia progressively decrease during transition but there is no alteration of the chronological rate of apical expansion. The change in contact parastichy phyllotaxy through ontogeny is interpreted as a change in the relative positions of primordia insertion on the apex resulting from an increase in apical volume and an increased rate of primordia initiation.     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

DETERMINATION OF NUMBER AND MITOTIC ACTIVITY OF SHOOT APICAL INITIAL CELLS BY ANALYSIS OF MERICLINAL CHIMERAS

The dimensions of mericlinal sectors in periclinal chimeras resulting from replacement-displacement phenomena have been used to determine the number of shoot apical initial cells and their mitotic activity. Narrow sectors were always short, extending an average of less than three nodes. All long sectors were wide, involving 1/3 or 1/2 of the circumference of the stem. These observations define the origin of all primary growth as from 1–3 apical initial cells in each of the apical layers. The sectors reveal a surprising stability of cellular position at the center of growth, with a specific initial cell maintaining its position during formation of over 100 nodes. During vigorous vegetative growth of Ligustrum ovalifolium the initials themselves divide only about once in 12 days during the formation of three nodes. The mitotic index of the initials in privet shoot apices is 1.4, and this rate of division is sufficient for them to be the ultimate source of all cells composing the shoot.     

THE SHOOT APICAL ONTOGENY OF THE PICEA ABIES SEEDLING. IV. PROTOXYLEM INITIATION AND AGE OF INTERNODES

In Picea abies seedlings the distance below the base of the shoot apical dome to the first protoxylem (px) to be differentiated in the internodes beneath is a linear function of apical dome basal diameter. By using mathematical relations derived in earlier papers of this series, we computed the morphogenic age in plastochrons and the chronometric age in days of the internode in which px is first differentiated (n_px). As the seedlings age from 30 to 140 days, the distance from the base of the apical dome to px increases from 186 to 295 μm, the n of n_px increases from 16 to 53, but the chronometric age of n_px remains within the range of 10 to 12 days. Protoxylem differentiation in young internodes is, therefore, more closely related to chronometrie age than it is to morphogenic age or to distance from the base of the shoot apical dome.     

DEVELOPMENTAL STUDIES IN SMILAX (LILIACEAE). I. ORGANOGRAPHY AND THE SHOOT APEX

The ontogeny of the shoot and the tendril-bearing leaf was studied in four species of Smilax which differ considerably in growth habit. Growth is indeterminate in S. bona-nox, S. rotundifolia, S. laurifolia and determinate in S. pumila. Shoot apical organization in all four species is consistent with the tunica-corpus concept. The number of tunica layers varies from 2 in S. bona-nox, S. pumila, and S. rotundifolia to 3 (2–4) in S. laurifolia. A primary thickening meristem was observed in 5. laurifolia. Shoot tip abortion involving a cessation of vegetative growth resembling that found in many woody plants occurs in S. pumila. Even though there are distinct differences in growth habit, vigor, and anatomy, the four species share many developmental similarities. Hence, subtle changes in apical ontogeny may be associated with marked differences in habit.     

FLEXIBILITY IN ONTOGENY AS SHOWN BY THE CONTRIBUTION OF THE SHOOT APICAL LAYERS TO LEAVES OF PERICLINAL CHIMERAS

The development of leaves on apically stable, periclinal chimeras was studied in a number of dicot genera. The mutant cell layers of the shoot apex and the tissues derived from them were as active developmentally as the normal layers. Ontogeny was the same in these chimeras as in nonchimeras, and growth of their leaves can be outlined as follows. Formation of the buttress, the axis, and the lamina of simple dicot leaves were independent events. In each the first growth included derivatives of the apical layers, usually three in number, found in the apex of the shoot and the lateral buds. Most cell divisions in the outer layers (L-I and L-II) were anticlinal relative to the new structures. Therefore, in the proximal regions of the buttress, axis (petiole and midrib), and lamina, the derivative cells of L-I and L-II were usually present in single layers. The rest of the internal tissue was from L-III. As formation of the axis and the lamina proceeded, derivatives of L-II replaced L-III internally in the distal and marginal regions leaving cells of L-III behind. Both the determinate growth of leaves and the pattern of cell divisions at and near the leading edges of growth meant that no cells in the leaf were comparable to the initial cells of the shoot apex. As the lamina extended, there were extensive intercalary cell divisions, both anticlinal and periclinal, so that in any given region of a leaf the layers of internal cells were from either L-II or L-III. At any point along the axis, L-III participated or did not participate in laminar extension. At any given stage in laminar growth either of two sister cells in any internal layer divided either a few times or extensively. The extreme variability in direction and frequency of cell division during leaf development was under an overriding genetic control, which resulted in the normal or typical size, shape and thickness of leaves.     

STRUCTURE AND DEVELOPMENT OF THE PERIANTH IN DOWNINGIA BACIGALUPII

The structure and ontogeny of the calyx and corolla of Downingia bacigalupii Weiler (Campanulaceae; Lobelioideae) were investigated for the purpose of comparing perianth development with previous observations on the floral bract, as well as elucidating the mechanism of development of the zygomorphic, sympetalous corolla. Sepals are uni-traced with a palmate, reticulate venation. They have basal and apical hydathodes, as well as storage tracheids. Sepals show a reduction in size, venation and hydathode number when compared to the bract. The pentamerous, zygomorphic corolla is bilabiate, consisting of a three-lobed adaxial lip and a two-lobed abaxial lip connected by a short tubular region. The constituent petal lobes are also uni-traced and have a reticulate venation, resembling that of the sepal and bract, but lack storage tracheids and hydathodes. Sepals arise in an adaxial to abaxial succession and are initiated in the outer corpus layer of the floral apex. Expansion of the floral apex follows and is accompanied by the establishment of a second tunica layer. Sepals undergo apical, marginal, and intercalary growth accompanied by acropetal differentiation of procambium. The petals arise simultaneously and are initiated in the second tunica layer and the outer corpus cells. After initiation, the petals exhibit a period of apical and marginal growth followed by intercalary growth. Apical growth in petals is less protracted than in sepals, but plate meristem activity is more extensive. The free petal lobes become temporarily fused by an interlocking of marginal epidermal layers, but they separate at anthesis. Zonal growth beneath the originally free lobes forms the tube and lip regions of the sympetalous corolla. Zygomorphy is evident from the time of initiation of petals and is accentuated by later differential growth. Comparative observations of corolla ontogeny in autogamous species of Doumingia indicate that the reduced corollas in these taxa are derived by a simple process of neoteny.     

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

DETERMINATE (det) MUTANT OF PISUM SATIVUM (LEGUMINOSAE: PAPILIONOIDEAE) EXHIBITS AN INDETERMINATE GROWTH PATTERN

Inflorescence ontogeny and morphology of the det mutant of Pisum sativum L. were investigated using scanning electron microscopy. This mutation causes the production of a limited number of axillary flowers followed by the formation of an apparent terminal flower slightly offset from the vertical. Our study indicates that the apparent terminal flower arises from an axillary meristem. The terminal meristem senesces and differentiates hairs, forming a rudimentary stub in the same manner as axillary meristems of conventional (Det) and det plants. Thus the dramatic effect of the det gene on inflorescence architecture results from early apical arrest rather than conversion of the terminal meristem to a flower as implied by the symbol det. This mutant will be valuable in elucidating regulation of apical arrest.     

Water relations of Picea abies. II. Determination of the apoplastic water content and other water relations parameters of needles by means of capillary microcryoscopy and the pressure-volume

Using the pressure volume analysis (PV analysis) on the shoots of Norway spruce (Picea abies [L.] Karst.) and the here presented capillary microcryoscopy of the needle press sap of the same shoots, it was possible to determine the amount of apoplastic water in the needles (W_an) as well as in the defoliated shoots (W_as). Additionally, the bulk osmotic pressure at full water saturation in the symplast of the needles and defoliated shoots (π_on and π_os) was determined. The dependence of the bulk-averaged turgor pressure (P_t) on the water content and the relationship between the bulk modulus of elasticity of the needles (?_n) and the bulk-averaged needle turgor pressure (P_tn) was shown with help of the PV analysis on the whole shoots and defoliated shoots. The study was conducted at the end of the vegetation period in 1987 and during winter 1988. The proportion of W_an in the total needle water content (W_tn) was 14% in September 1987 and 12.5% in March 1988. The respective percentage of W_as in W_ts were 27% and 25%. The amount of apoplastic water depended on the ratio of the dry weight of defoliated shoots to the dry weight of the whole shoots. A standard mean value for the amount of W_an in the total water content of the shoots (W_t) was therefore not possible. The bulk osmotic pressure at full water saturation in the needle symplasts was –1.9 MPa in September 1987 and –2.2 MPa in winter 1988. The respective values of the bulk osmotic pressures in the symplast of the defoliated shoots (π_os) were –1.5 MPa and –1.7 MPa. Thus π_on was 0.1 MPa lower and π_os 0.3–0.4 MPa higher than the average osmotic pressure during full water saturation in the symplast of the whole shoots (π_o). The relation between bulk-averaged turgor pressure and water content showed that during water loss P_tn dropped more rapidly than the turgor pressure of defoliated shoots (P_ts). Consequently the needles were less elastic than the defoliated shoots. The turgor values of whole shoots followed an intemediate course between P_tn and P_ts. The flat course of P_ts seems to be the main reason for the often observed “plateau” of ψ-isotherms of whole shoots near full turgor.