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.     

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G₂) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.     

CONTROL OF THE INTERNODAL INTERCALARY MERISTEM OF CYPERUS ALTERNIFOLIUS

In the growing culm of C. alternifolius, surgical removal of parts indicated that the stimulus for the prolonged activity of the internodal intercalary meristem (IM) came from the matured leaves and upper internode and that buds were not involved in maintaining internodal growth. Decapitation of the culm resulted in cessation of internodal extension. Various growth regulators were applied to the decapitated internode, and both the total extension and growth rates were analyzed statistically. Gibberellin A₃ (GA) and benzyladenine (BA) substituted for the excised parts in their effect on internodal extension. Indoleacetic acid (IAA) had little effect. (2-chloroethyl) trimethylammonium chloride (CCC) inhibited internodal growth, and its effects were reversed by GA. IAA was antagonistic to BA but not to GA. BA and GA were somewhat antagonistic. The quantitative effects of growth regulators on epidermal and ground parenchyma cell length and number of interstomatal cells were examined. Extension induced by GA was due to both cell division and cell elongation in the IM. Cells were longer, and fewer stomates differentiated than in the control. In internodes induced to extend by GA + BA cell division, cell length, and stomate differentiation were similar to the control. The results indicate that prolonged internodal IM activity is maintained by cytokinins and gibberellins coming from the matured upper portions of the culm. Changes in the levels of these regulators during growth presumably result in the histological gradient in the internode.     

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.     

THE COST OF MERISTEM LIMITATION IN POLYGONUM ARENASTRUM: NEGATIVE GENETIC CORRELATIONS BETWEEN FECUNDITY AND GROWTH

Growth and reproduction in higher plants depend on meristems, which have three developmental fates. A meristem can become reproductive, but doing so terminates its activity, it can differentiate vegetatively, or it can remain quiescent for extended periods. The first two fates are mutually exclusive, and only the second leads to the production of additional meristems for subsequent growth and reproduction. In Polygonum arenastrum (frequently referred to as P. aviculare in North American Floras), an annual species lacking quiescent meristems, a quantitative genetic analysis of inbred full-sibling families revealed genetic variation in the developmental pattern of axillary meristem commitment to vegetative growth versus reproduction. Developmental variation resulted in family differences in the age of first reproduction, in age-specific fecundity and growth, and in final plant size and reproductive output. Furthermore, there were strong negative genetic correlations between age-specific growth and fecundity. Early commitment of meristems to reproduction favors high early fecundity, but reduces the number of meristems available for vegetative differentiation, and leads to lowered growth rates and fecundity later in life, when meristems are limiting. Conversely, meristem commitment to vegetative growth early in life results in low early fecundity but high late fecundity and growth. Meristem limitation, like resource limitation, is a proximate mechanism that generates trade-offs between life history traits. Differences between meristem limitation and resource limitation are discussed. Meristem limitation leads automatically to a senescent life history because of the determinate fate of reproductive meristems. Developmental characters were also found to be genetically correlated with metamer characters (leaf size, internode length) and seed size in this selfing species. The pattern of correlation is suggestive of selection for particular suites of life history and morphological characters.     

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.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

THE RELATIONSHIP BETWEEN THE PRIMARY THICKENING MERISTEM AND THE SECONDARY THICKENING MERISTEM IN YUCCA WHIPPLEI TORR. III. OBSERVATIONS FROM HISTOCHEMISTRY AND AUTORADIOGRAPHY

Observations were made of stem sections stained for RNA and protein of Yucca whipplei ranging from germinated seedlings to 6-month-old plants. One-, two-, and three-month-old plants were labeled with tritiated thymidine, fixed in FAA, sectioned, stained with the Feulgen reaction, and prepared for autoradiography. The serial transverse sections were outlined with a drawing tube recording all labeled nuclei on a computer graphics tablet. Computer-assisted three-dimensional reconstructions were made to observe the locations of labeled nuclei. The two techniques are in agreement: the thickening meristem is broad near the top of the stem, occupies a narrower band at more basipetal levels, and disappears below the level of recent root initiation. There are no gaps in staining or labeling, and there are no changes in staining or labeling that would distinguish between the activities of the primary thickening meristem and the secondary thickening meristem in those plants which possess both. The meristems are continuous at all stages of development in the young vegetative stem. The STM is interpreted to be a developmental continuation of the PTM.     

HORMONAL REQUIREMENTS OF EXCISED DIANTHUS CARYOPHYLLUS L. SHOOT APICAL MERISTEM IN VITRO

Whereas a medium containing kinetin alone enabled a few Dianthus caryophyllus L. apical meristem dome explants to develop into rooted plants, the highest frequency of plants was obtained in one containing supplements of both IAA and kinetin. In an unsupplemented medium, continued development required that explants have 2 pairs of primordial and a pair of expanding leaves. Kinetin alone caused production of many new leaves, but the development was significantly less than when it was furnished in combination with IAA. IAA given alone caused meristem explants to develop primarily callus, roots, and a few leaves. Gibberellin and abscisic acid were without promotive effects on leaf and shoot formation. A balance of hormonal substances, synthesized in young leaf structures and relocated to the meristem, is proposed as the fundamental mechanism that regulates new leaf initiation in the shoot apex.     

APICAL DOMINANCE: THE EFFECTS OF GROWTH REGULATORS ON THE GAMETOPHYTE OF ANEMIA PHYLLITIDIS

Gametophytes of the fern, Anemia phyllitidis (L.) Sw. were aseptically cultured until nearly mature, then collected and surgically divided into meristematic and nonmeristematic halves. Indoleacetic acid, benzyladenine, abscisic acid, and the auxin inhibitor, triiodobenzoic acid, were employed to elucidate the role of growth regulators during regeneration of adventitious gametophytes. Explants possessing actively growing meristems failed to produce adventitious thalli regardless of treatment. Untreated nonmeristematic halves initiated numerous filamentous outgrowths within two weeks of excision. These outgrowths eventually developed into the cordate prothalli typical of this species. Nontoxic levels of IAA (10⁻⁴ g/1 and 10⁻⁵ g/1) promoted early regeneration by basal explants. TIBA, at 10⁻⁵ m, completely suppressed regeneration. Benzyladenine, at 10⁻² g/1, exerted its most striking effect by greatly increasing the number of adventitious outgrowths formed by treated basal explants. ABA at all concentrations delayed regeneration and reduced the number of basal halves that displayed renewed mitotic activity. This evidence suggests that auxin acts as an essential promoter of regeneration, and that the maintenance of endogenous hormonal interactions is necessary to preserve the form of an intact gametophyte.     

HISTOCHEMISTRY OF THE RHIZOMORPH MERISTEM OF ARMILLARIA MELLEA

Histochemical studies disclose the presence of relatively large amounts of protein and nucleic acids in the apical region of the rhizomorph of Armillaria mellea. No specific center for the production of protein was noted, but nucleic acid studies suggest that RNA synthesis may be confined to a limited region of the rhizomorph tip. Intranuclear, crystalline protein bodies are present while selective fluorescent staining of DNA indicates that the relative amounts of that substance, per nucleus, may be quite small and may account for some of the difficulty in selective nuclear staining by more commonly employed methods. Large stores of glycogen in the area adjacent to the apical center and its apparent rapid turnover in that region implicates this polysaccharide as a participant in the differentiation of the primary medulla.     

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.     

ONTOGENETIC REORGANIZATION OF THE ROOT MERISTEM IN THE COMPOSITAE

The organization of the root meristem in selected Compositae was investigated to determine whether changes in the pattern of cell arrangement occurred during root growth in species other than Helianthus annuus. Embryonic, short, and long primary roots of one species of each of twelve genera were prepared for microscopic examination. Additional intermediate growth stages were prepared for Echinacea pallida. The meristem of embryonic roots showed layers of initials typical for dicotyledons. The meristem in many of the short roots of eight species was reorganized by the development of a secondary columella. The long roots showed patterns similar to the embryonic roots. In three species which maintained closed meristems, two layers of cortical initials were common in the embryonic root, and as a general trend, a single layer of cortical initials became more common during root elongation. The cellular changes that resulted in the initiation of a secondary columella are characterized by the conversion of cortical initials to secondary columella initials by a shift in their plane of cell division. It is proposed that the size and shape of the quiescent center changes as the conversion takes place. No intermediate stages were observed which could account for the reduction of two layers of cortical initials to one layer.     

Growth Substances in the Roots of Vicia faba II. THE EFFECTS OF AGEING AND EXCISION OF THE MAIN TAP-ROOT MERISTEM

A quantitative chromatographic study has been made of the changesin the activity and distribution of the main ether-soluble acidauxins of Vicia faba seedling root systems during developmentand resulting from excision of the main tap-root meristem. AnIAA-like auxin (AP (ii)) is apparently synthesized predominantlyin apical and at a lower rate in lateral meristems. Productionseems to stop when meristematic growth stops. Its concentrationin mature extended cells is much lower and may fall to zeroin old cells, suggesting active degradation by an auxin-oxidase.Excision of the main tap-root tip gradually results in a greatlyaugmented production of AP (ii) in lateral meristems, conceivablythe result of correlative growth promotion. A second auxin and root-growth inhibitor (AP (iii)) is presentat higher activity levels than AP (ii) in tap-root meristemsand at the same level in lateral meristems. In mature cellsits activity is much lower than that of AP (ii). In contrastto AP (ii) it accumulates in both tap-root meristems and maturetissue as the root system ages. It could also be produced duringmeristematic growth but is not subsequently degraded. As withAP (ii), excision of the tap-root tip brings about a great increasein its concentration in lateral tips. A third auxin and root-growth accelerator (AP (i)) (accelerator?) is present in lower concentrations in both meristem and maturetissue. Its concentration tends to decrease with ageing and,in lateral meristems, is not affected by tap-root tip excision. It is suggested that AP (ii) produced by the meristem is normallyat suboptimal levels in the extending cells and may be the principalhormone controlling extension growth. AP (iii) accumulationmay account for growth deceleration on ageing. The role of AP(i) remains obscure. It is unlikely that correlative effectsof the taproot tip on lateral root growth are exercised directlyvia these auxins.     

A MORPHOMETRY STUDY OF THE ULTRASTRUCTURE OF ECHINOCEREUS ENGELMANNII (CACTACEAE). II. THE MATURE,ZONATE SHOOT APICAL MERISTEM

The shoot apical meristems of adult Echinocereus engelmannii plants are zonate and have a tunica, central mother cells, a peripheral zone, and a pith-rib meristem. An ultrastructural, stereological study showed that each zone has its own distinct ultrastructure, but that the differences between the zones are quite small, both on a protoplasmic basis and on a cytoplasmic basis. Furthermore, the ultrastructure present in the adult apices differed only slightly from that which had been found in seedling apices, demonstrating a long-term stability of structure. The standard deviations found in the sample were small, indicating little variability from one plant to the next and suggesting that there are little or no cyclic changes during the plastochron or a 24-hr photoperiod. The ultrastructures found in the shoot apical meristems differed significantly and markedly from mature tissues of the same plants.     

HISTOCHEMISTRY OF THE PRIMARY THICKENING MERISTEM IN THE VEGETATIVE STEM OF ALLIUM CEPA L.

Stems of Allium cepa L., 1, 2, 5, and 6 months old respond similarly when stained for protein and RNA. The primary thickening meristem (PTM) stains more intensely than surrounding stem tissues. The acropetal region of the PTM is a broadly staining band which narrows basipetally to the level of the initiation of shoot-borne roots in the stem and disappears more basipetally. These staining patterns are consistent with the hypothesis that the PTM functions in stem thickening and root production, and also indicate that the meristem functions before histological evidence of the cambial-like zone exists in the onion stem. Histochemical staining may be an accurate method of locating the PTM.     

Effect of growth regulators on dormant lenticels ofCedrela toona ROXB.

Exogenously applied IAA, GA₃ and Niagara (ethyl-hydrogen-1-propyl-phosphonate) activated dormant lenticel meristems of stem cuttings ofCedrela toona Roxb. Maximum activation was achieved by IAA at 0.5 mg ml⁻¹ concentration.     

SELECTIVE INHIBITION OF CELL CYCLE STAGES IN THE ALLIUM ROOT MERISTEM BY COLCHICINE AND GROWTH REGULATORS

The treatment of root tips of Allium carinatum, Allium cepa, and Allium flavum with colchicine, abscisic acid, kinetin, and indole-3-acetic acid, applied in appropriate concentrations, combinations, and durations, makes possible the selective blockade of the cell cycle in G₁, G₂, any mitotic stage, and between karyokinesis and cytokinesis. Moreover, treatment with abscisic acid followed by a recovery period stimulates polyploid nuclei in mature tissues to divide. Colchicine, kinetin, and indole-3-acetic acid applied together cause end-to-end association of metaphase chromosomes. These results together with earlier findings suggest that any step of the cell cycle is independently controlled both by specific balance of the growth regulators and by specific synthesis of the nucleic acids.     

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.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.