IN VITRO DEVELOPMENT OF THE ISOLATED SHOOT APICAL MERISTEM OF ANGIOSPERMS

Development of complete plants was achieved from isolated shoot apical meristems of Nicotiana tabacum L., Daucus carota L., Nicotiana glauca Grah., Tropaeolum majus L., and Coleus blumei Benth. The explants consisted of only meristematic dome tissue with no visible leaf primordia. A simple nutrient medium composed of the Murashige and Skoog salt mixture, 100 mg/liter myo-inositol, 0.4 mg/liter thiamin-HCl, 1-2 mg/liter IAA, 30 g/liter sucrose, and 1% agar was adequate. Histologically there occurred principally tissue enlargement during the first 3-6 days, followed by appearance of bipolar organization in 6-9 days and formation of a well-defined root apex and initiation of first leaf primordium by 12 days.     

香榧营养苗端细胞组织分区的超微结构观察

本文研究了榧树(Torreya grandis)成熟植株在季节生长中营养苗端的超微结构变化。各区域细胞的主要区别特征为:顶端原始细胞与亚顶端细胞相接的细胞壁较厚,液泡多分布于细胞游离面,质体中淀粉粒较小;亚顶端细胞壁较厚,液泡较大,质体中淀粉粒较大而多;周缘区细胞质体多不具淀粉粒,液泡也较小,胞间连丝丰富;肋状区细胞被大量的含淀粉质体及液泡占据了大部分空间,胞间连丝丰富。在季节变化的四个时期中,各区域细胞的亚显微结构特征亦不相同。休眠期各区细胞淀粉质体较发达,细胞壁较厚,液泡大;叶扩展期淀粉质体减少或消失;芽鳞形成期出现大量小液泡;新的顶芽形成期液泡增加,核糖体含量较高。讨论了各区域细胞核形态与其细胞活跃性的关系。     

AN ELECTRON MICROSCOPIC EVALUATION OF CYTOHISTOLOGICAL ZONATION IN THE SHOOT APICAL MERISTEM OF PINUS BANKSIANA

Shoot apical meristems of jack pine (Pinus banksiana) were examined by light and electron microscopy. Cytohistological zonation was evident when meristems were fixed in Craf IV, embedded in paraffin, and stained with Chlorazol Black E. When meristems were fixed for electron microscopy the cytoplasm of the apical initials and central mother cells each contained numerous lipid bodies and their nuclei contained little, if any, heterochromatin. The cytoplasm of the peripheral zone was rich in ribosomes. The nuclei of the peripheral zone and rib meristem were heterochromatic. Thus, the lack of heterochromatin in the nuclei and the dissolution of lipids in the cytoplasm of the apical initials and central mother cells appeared to contribute most to the organization and appearance (cytohistological zonation) of the shoot apex when standard histological techniques are used.     

CELL DIVISION KINETICS IN THE SHOOT APICAL MERISTEM OF CERATOPTERIS THALICTROIDES BRONG. WITH SPECIAL REFERENCE TO THE APICAL CELL

The duration of mitosis and the cell cycle were determined for defined cell populations of the shoot apical meristem of Ceratopteris thalictroides Brong. by using the colchicine-induced metaphase accumulation technique. The results indicate that the apical cell is mitotically active and cycles at an apparently greater frequency than the cells of subjacent populations. Duration of mitosis was similar for all cells of the meristem. These results are correlated with mitotic indices of control apices, the geometry of the apex, and the mean number of cells in the meristem. Shoot apices from adult plants were examined to determine mitotic indices within the meristem; mitotic activity was again noted for the apical cell. These results contradict recent proposals that the pteridophyte apical cell serves as a unicellular quiescent center which lacks histogenic potential and offer experimental support for the classical concept of apical cell function in those fern shoot meristems which terminate in a single apical cell.     

DNA MICROSPECTROPHOTOMETRY OF SHOOT APICAL MERISTEM CELL POPULATIONS IN CERATOPTERIS THALICTROIDES (FILICALES)

A microspectrophotometric analysis of the DNA content of cell populations in the shoot apical meristem of young and adult stages of the filicalean fern Ceratopteris thalictroides was conducted to determine if the previously reported uptake of labeled DNA precursors by the apical cell was a consequence of endomitotic DNA replication or of DNA synthesis preceeding mitosis. Results demonstrate that in both the young and adult plants the estimated DNA content of the apical cell nuclei parallels that of the other cells of the meristem. There was no evidence of an “apical zone” of endopolyploid cells.     

A HISTOCHEMICAL STUDY OF THE SEEDLING SHOOT APICAL MERISTEM OF PINUS LAMBERTIANA

Vernalized seeds of Pinus lambertiana were scarified and planted in perlite. At 5, 8, 10, 13 and 16 days after planting, seedlings were selected for morphological examination and histochemical study. The shoot apical meristem consisted of a relatively homogeneous population of cells at 5 days. Cytohistological zonation was observed in the meristem by the eighth day and needle primordia initiation began at this time. Acid phosphatase (AP) activity was high in the extreme tip of the apex at 5 days. At 8 days AP activity was intense in the peripheral zone but weak in the apical initial and central mother cell zones. The apical meristem of the 10–16-day-old seedlings exhibited high AP activity in the peripheral zone only during the early stages of needle primordia initiation. The distribution of cytoplasmic and nuclear protein-bound SH was correlated with cytohistological zonation. Protein-bound SH was distributed relatively uniformly at 5 days, but by the eighth day the 4 cytohistological zones contained differential quantities. Succinic dehydrogenase (SD) activity was observed throughout the apex at 5 days, but by the eighth day the apical initial and central mother cell zones exhibited differentially greater levels of SD activity. Irradiation with 500 R of X-rays at 7 days after planting completely inhibited needle primordia initiation and disrupted the cytohistological zonation of the apex. Correlated with the inhibition of needle primordia initiation was the loss of SD activity in the apical initial and central mother cell zones. Irradiation also resulted in the gradual loss of protein-bound SH from the cytoplasm of the apical initial, central mother cell and peripheral zone.     

香石竹原生质体再生的研究

香石竹(Dianthus caryophyllus L.)是一种受到世界性欢迎的观赏植物。香石竹的快速繁殖已有不少研究报告。为了寻找新颖的育种手段,培育新的品种,我们进行了该植物的原生质体培养。米益(Mii, et al., 1682)已对香石竹的原生质体培养作过报道,但他们所用的材料为叶肉原生质体。为期望得到更多变异的再生植株,我们选用愈伤组织做为游离原生质体的材料。     

STEREOLOGICAL STUDY OF ULTRASTRUCTURAL CHANGES IN THE SHOOT APICAL MERISTEM OF NICOTIANA TABACUM DURING FLORAL INDUCTION

Shoot apices of a short-day sensitive line of Nicotiana tabacum L. cv. NCTG-22 have been examined by electron microscopy for ultrastructural changes which occurred in the central zone over a 17-day period during the transition from vegetative to reproductive growth. Plants were grown in controlled-environment chambers of the NCSU Phytotron and exposed to an inductive photoperiod after a 6-wk juvenile phase. Ultrastructural changes were investigated from photomicrographs using a semi-automatic stereological procedure and a microcomputer. After exposure to only one inductive cycle cell and nuclear cross sectional areas in short-day plants were significantly larger than in long-day controls. Subsequently, under short days cross sectional areas of cells, nuclei, vacuoles and proplastids decreased, while mitochondrial cross sectional area and relative volume increased during the course of the induction period. In induced apices as cross sectional areas and relative volumes of vacuoles and proplastids decreased, their profile numbers increased. The reduction in cross sectional areas of cells and most organelles was associated with an increase in rate of leaf initiation and size of the apical dome. The demand for sufficient energy input to maintain the surge in growth and activity preceding floral initiation was reflected by the significant increases in cross sectional area, profile numbers and density of the mitochondria population. Even though the transition period is quite long for Nicotiana, cells and organelles in the central zone were observed to progress through similar changes in morphology that are known to occur in Sinapis and Xanthium which exhibit a more rapid and absolute response to photo-induction.     

THE PICEA ABIES SHOOT APICAL MERISTEM IN CULTURE. I. AGAR AND AUTOCLAVING EFFECTS

Shoot apical meristems of Picea abies seedlings can be cultured on a relatively simple, defined, basal medium. Dome-like explants initially about 200 μ tall, without externally obvious primordia, and having dry weights of about 3 μg, usually initiate 5–10 new primordia within a week. They typically show 10- to 30-fold dry weight increases in three weeks. None of the 5,000 meristems cultured has produced any basal callus. Growth is strongly influenced by both the type and concentration of agar used to gel the medium. Dry weight yield increases as agar concentration decreases. This is probably partly due to increased diffusion rates of enzymes or other large molecules through more dilute agar gels but possibly also partly ascribable to unknown agarborne inhibitors. About half of the agar concentration effect can be eliminated by substituting glucose and fructose for sucrose in the medium. This suggests that diffusion of invertase through the agar gel in this medium may be a growth limiting factor. Growth of cultures is also promoted by autoclaving sucrose in the presence of the agar. The basis of this effect is not yet understood.     

THE PICEA ABIES SHOOT APICAL MERISTEM IN CULTURE. II. DEPOSITION OF POLYSACCHARIDES AND LIGNIN-LIKE SUBSTANCES BENEATH CULTURES

Excised shoot apical meristems of Picea abies seedlings grow and develop primordial leaves when cultured on Millipore (mixed esters of cellulose) filter membranes lying on a simple, defined medium gelled with agarose. When the cultures are removed from the membranes, each leaves a spot of altered light transmission, spectral characteristics, hygroscopicity, and chemical reactivity. These spots are the manifestation of deposition in the membrane pore space of polysaccharides, lignin-like components, and probably other substances. Deposition of water-insoluble, Schiff's reagent-positive substances can be detected in the filter membranes after only 3–6 hr exposure to a meristem and continues for 10–15 days or longer. Precursors of the insoluble deposition materials can diffuse through at least nine layers of Millipore membrane before deposition at a site remote from living cells. Placement of a dialysis membrane between the meristem and the Millipore membrane prevents detectable deposition in the latter. The observations are consistent with the hypothesis that apical meristems can synthesize and export mobile precursors of cell wall components as well as any substances necessary to promote their condensation or polymerization into insoluble materials at remote sites. The system may be useful in studying synthesis of cell wall components and investigating the functional role of growth regulators in shoot apical development.     

PRIMORDIAL LEAF AND PHYTOHORMONE EFFECTS ON EXCISED SHOOT APICAL MERISTEMS OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. apical meristems and apical meristems +1, +2, +3 primordial leaf pairs were cultured to examine phytohormone influences on development and correlative effects of developing primordial leaves on in vitro responses. The meristem with no phytohormones or low levels of IAA could not develop in vitro. At least 0.1 mg/l IAA and optimumly 1-2 mg/l IAA were required for development into complete plants. IAA from 0.1 to 3 mg/l also resulted in root development with no apparent leaf or shoot formation. Levels of IAA higher than 3 mg/l were inhibitory to development. Kinetin, as a substitute for naturally occurring cytokinins, alone (0.0003 to 3 mg/l) resulted in development of rosettes of leaves. In the presence of IAA (***1 mg/l) and kinetin (0.003 mg/l) plants, rosettes, individual leaves with roots, and roots developed from isolated meristems. Glutamine and adenine sulfate both appeared inhibitory to meristem development. With +1, +2, +3 developing primordial leaf pairs left attached to the apical dome, three pairs were required for plant formation in the absence of phytohormones. In the presence of IAA, two pairs of primordial leaves resulted in plant formation; whereas, with IAA and low levels of kinetin one pair of primordial leaves was enough. Higher levels of kinetin were inhibitory to plant development with primordial leaves present. ABA appeared to be inhibitory to development of meristems and meristems +1, +3 primordial leaves at low concentrations and resulted in death at ***1 mg/l. Developing primordial leaves appear to supply the apical meristem with a balance of phytohormones during growth. Meristem development into a plant first involved formation of leaf primordia. Establishment of a bipolar axis with root formation followed.     

HISTOCHEMICAL STUDY OF ENZYME ACTIVITY IN THE SHOOT APICAL MERISTEM OF BRASSICA CAMPESTRIS DURING TRANSITION TO FLOWERING. IV. GLUCOSE-6-PHOSPHATASE

Glucose-6-phosphatase (G6P) activity was determined in fresh-frozen, cryostat sections in the shoot apical meristem of Brassica campestris L. Enzymatic activity was differentially distributed in a zonate pattern in the vegetative meristem, but not in the transition and floral meristem. Vegetative apices showed a heterogenous localization with the highest activity in the central zone and the pith-rib meristem zone. At the early transition stage of development, G6P activity in the peripheral zone increased slightly. At the late transitional (prefloral) stage, G6P activity was not localized within the peripheral zone in island-like areas of activity. This is the first demonstration of G6P in shoot apical meristem at the vegetative, transition, and floral stage. The results indicate that G6P activity 1) is an accompanying event of evocation, but 2) does not mark incipient floral primordia. G6P may play an important role in the maintenance of glucose-6-phosphate homeostasis in an evoked shoot apical meristem.     

RELATIONSHIPS AMONG SHOOT APICAL MERISTEM ONTOGENIC FEATURES IN TRICHOCEREUS PACHANOI AND MELOCACTUS MATANZANUS (CACTACEAE)

Various morphometric parameters of shoot apical meristems were measured during the ontogeny of Melocactus matanzanus and Trichocereus pachanoi. While some ontogenic features varied dramatically (i.e., apical surface curvature, dK/dl, varied by 0.15–0.17, while the apical volume increased from 10⁵ μm³ to 10⁷ or 10⁸ μm³), the percent volume of the central-mother-cell zone (CMCZ), pith-rib meristem (PRM), and the peripheral zone (PZ) showed remarkable intraspecific constancy: CMCZ = 9.2%, PRM = 10.4%, PZ = 80% for M. matanzanus, and CMCZ = 10%, PRM = 24.5%, PZ = 65% for T. pachanoi. Seedlings (2 plastochrons old) showed distinctly different percent volumes from older forms: CMCZ = 7.8%, PRM = 5.9%, PZ = 86% for M. matanzanus, and CMCZ = 8.9%, PRM = 13%, PZ = 77% for T. pachanoi. Geometric expressions of cell patterns within zones of the apex remained relatively constant throughout development. On the basis of intra- and interspecific comparisons of the percent volumes among 23 taxa and the chronology in the appearance of zones within the apex, it is concluded that while there are no apparent biological reasons for the actual percent volume values computed from our sample set, the constancy in zones reflects a high degree of homeostasis which is achieved early in ontogeny.     

CONSTANCY OF RELATIVE VOLUMES OF ZONES IN SHOOT APICAL MERISTEMS IN CACTACEAE: IMPLICATIONS CONCERNING MERISTEM SIZE,SHAPE, AND METABOLISM

The volumes and dimensions of shoot apical meristem zones were determined for 21 species in 10 genera of the Cactaceae. If the volumes of the central-mother-cell zone (CMCZ), the peripheral zone (PZ), and the pith-rib meristem (PRM) are expressed as percentages of the total volume of the corpus, then they are remarkably constant (CMCZ = 11.1% of the volume of the corpus, PZ = 61.2% and PRM = 27.1%) regardless of the relative size of the apical meristems. The relative volume of the tunica decreases, however, as the whole apex increases. The zones have variable shapes, and whereas the PZ and PRM are always trapezoidal in longitudinal section, in apices with large volumes, these zones have a flatter, more flaring shape than they have in smaller apices. Thus large apices are flatter and less hemispherical than are small apices. The CMCZ, however, maintains a constant shape and is always relatively hemispherical regardless of its volume. A hypothesis that relates all of these shape and volume relationships as an integrated growth sequence is proposed.     

EXPERIMENTAL STUDIES OF THE SHOOT APICAL MERISTEM OF SEED PLANTS. I. MORPHOLOGICAL AND CYTOCHEMICAL EFFECTS OF IAA APPLIED TO THE EXPOSED MERISTEM OF LUPINUS ALBUS

Application of 1.5 μg indoleacetic acid (IAA) in a lanolin droplet to the exposed apical meristem of Lupinus albus seedlings caused: (1) axillary buds to form closer to the apex than normal, (2) displacement of primordia formed during the first two plastochrons following treatment, and (3) significant increases in concentration of RNA, protein, and unsaturated lipids in the meristems. Primordial displacement tended to be random relative to the site of the treatment, which may be a feature common to dicotyledonous plants exhibiting spiral phyllotaxis. That IAA conferred initiation site capabilities to all of the peripheral zone for a short time was indicated by (2) and (3) above, and by decreases in concentrations of the observed compounds toward control levels after the second plastochron following treatment. Effects of IAA on RNA suggest that nucleic acid metabolism, and possibly gene action, was involved in the response. Kinetin or gibberellic acid had no apparent morphogenetic effect on Lupinus meristems.     

THE HORMONAL CONTROL OF ORGAN FORMATION IN CALLUS OF MEDICAGO SATIVA L. CULTURED IN VITRO

Organogenesis in alfalfa callus (Medicago sativa L. cv. ‘Regen S’) has been obtained by the transfer of callus from an induction medium containing growth regulators to a regeneration medium lacking growth regulators. The transfer of callus from induction medium containing high levels of 2,4-D and low levels of kinetin to regeneration medium resulted in the formation of shoots. Conversely, the transfer of callus from induction medium containing low levels of 2,4-D and high levels of kinetin resulted in the formation of roots. The pattern of organogenesis on regeneration medium was modified by the nutritional composition of that medium. When Blaydes medium supplemented with inositol and yeast extract was employed as regeneration medium, root organogenesis was inhibited. Root organogenesis was not inhibited by either Shenk and Hildebrandt medium or Gamborg's B5 medium. Shoot formation occurred on all of these media. A survey of the in vitro organ-forming capacity of 14 genotypic clones from the cv. ‘Regen S’ was conducted. The capacity to form organs differed quantitatively among the clones analyzed. A more detailed analysis of a highly responsive clone (RA3) and a poorly responsive clone (RA5) revealed no significant qualitative difference in their organogenic responses.     

THE ROOT APICAL MERISTEM OF OSMUNDA REGALIS

Roots of the Osmundaceae differ from most ferns in having more than one apical cell. The size of the apical initial group, which includes cells that are considered to be apical cells, varies directly with root diameter in Osmunda regalis L. Mitotic indices were 6.63% for apical cells, 7.45% for the entire apical group, 6.25% for distal derivatives, and 7.15% for developing cortical cells. Cytophotometric measurements of Fuelgen-stained nuclei indicated no endopolyploidy in the populations of cells studied. These findings demonstrate that there is no quiescent center in the roots of O. regalis.