Growth of Carnation Meristems in vitro: Anatomical Structure of Abnormal Plantlets and the Effect of Agar Concentration in the Medium on their Formation

Carnation meristems cultured in vitro grow into shoots of threetypes: normal, translucent and succulent. The apical meristemof succulent shoots was of the mantle-core type and it lackedpro-vascular tissue. The leaf had large vacuolated mesophyllcells, fewer stomata (often plugged), and no plate meristem.A higher agar concentration in the medium increased the percentageof normal shoots developing. This supports other indicationsthat the water potential of the medium affects morphogenesis. carnation, Dianthus caryophyllus L, meristem culture, abnormal plantlets, shoot meristem     

Surface Changes on Fresh Carnation Shoot Apices During Scanning Electron Microscopy

The general morphology of fresh intact shoot apices of carnation,Dianthus caryophyllus L. cv. Improved White Sim was studiedwith a scanning microscope. The changes in the surface withtime under the electron beam were: (1) individual cells beingvisible, (2) a smooth apex and (3) wrinkling until the specimenbecame unsatisfactory for study. As indicated by time coursestudies, the initial individual cell observations representthe true features of the intact apex. The observation of a smoothapex was related to a ballooning of the surface layer resultingfrom water vaporization. Incisions in the apex to prevent pressurebuild-up under the surface layer revealed the smooth characteristicof the apex surface was due to lifting of the cuticle from underlyingcells. Areas adjacent to a micropuncture in the apical domedid not exhibit ballooning.     

The Carnation Succulent Plantlet -- A Stable Teratological Growth

Axillary buds of carnation (cv. Cerise Royalette) cultured invitro, frequently became ‘succulent’ plantlets,which proved to be a teratalogical stable type of growth. Agarconcentration (0.8–1.2 per cent) in the medium influencedthe type of development, and 0.05, 1 or 2 mg l^–1 of NAAin the medium did not change the results. The succulent plantletsdid not revert to normal growth when transferred to medium containingmore agar, which favoured normal plantlet development. Succulentexcised meristems developed mainly into succulent plantlets. A hypothesis is made that a rearrangement of the meristem occursin the first days of growth, the consequence of which is thesucculent plantlet, which is no longer influenced by agar concentrationin the medium. Carnation, Dianthus caryophyllus L. cv. Cerise Royalette, vegetative shoot meristem, agar effect, meristem organization     

The Effect of Cytokinins on Vitrification in Melon and Carnation

The influence of exogenous cytokinin on vitrification of melonand carnation buds grown in vitro was assessed. In melon, theanatomical traits of vitrification were found to be similarto those previously described for carnation grown on a highconcentration of auxin. In carnation, the plantlets treatedwith high cytokinin concentrations became bushy but the anatomicalstructure remained normal. The relation of cytokinin and vitrificationis discussed. Cucumis melo L., Dianthus caryophyllus L., vitrified plantlets, habituation, cytokinin     

'Vitrified' Dianthus--Teratomata in vitro due to Growth Factor Imbalance

Cultured shoot tips of Dianthus caryophyllus often develop asabnormal, ‘vitrified’ plants that have the characteristicsof teratomata: stable, unlimited development with fasciatedshoot apices, a bush habit, reduced stem elongation and succulentleaves. High concentrations of NAA in the culture medium increasedand benzylaminopurine decreased the proportion of shoot-tipsthat developed as abnormal plants. The requirements of abnormaland normal plants for continued development differed. Duringthe initial stages of development abnormal plants did not requireauxin and they were able to continue their growth in the absenceof roots. It is suggested that the abnormal plants are teratomata,similar to those induced by Crown Gall bacteria, and their inductionand abnormal traits could be due to imbalance of auxins andcytokinins. Dianthus caryophyllus L., carnation, abnormal plant growth, habituation, vitrification, teratoma (plant)     

Normal and Abnormal Developments of the Stem Apex in Carnation

Normal and abnormal development of the stem apex is describedfor the carnation Dianthus caryophyllus, cultivated varieties.Abnormalities described include adventitious buds within theflower, carpel-like structures in the stamen whorl, excessivedevelopment of ovules, and axile secondary flower formations.Some comments are made on their occurrence and they are discussedin the light of present hypotheses concerning flower initiationand development.     

Development of suitable protocol to overcome hyperhydricity in carnation during micropropagation

Hyperhydricity during micropropagation of carnation (Dianthus caryophyllusL.) was reduced by media modifications. Three commercial varieties (White sim, Exquisite and Scania) tested, varied in optimal growth without hyperhydricity. Increased concentration of iron and/or magnesium reduced hyperhydricity with 0.7–0.8% agar. At some concentrations, hyperhydricity was reduced to 0% and shoot multiplication was increased. All nonhyperhydrified micropropagated plantlets survived in a glasshouse during acclimatization.     

Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

A Cytochemical Study of Differentiation and Breakdown of Vessel End Walls

A cytochemical approach was used to study the architecture ofthe end wall and its modifications during vessel differentiationin Populus italica and Dianthus caryophyllus. The combinationof cytochemical techniques with selective extractions to removeend wall subunits provides information on the different componentsof the end wall and on the method of perforation. The end wallappears to be formed mainly from pectins and hemicelluloses.No cellulosic components nor lignin were found though the 3,3'-diaminobenzidine assay revealed the presence of peroxidases.That the method of perforation may depend on the species isdiscussed. Dianthus caryophyllus L., carnation, Populus italica, poplar, xylem vessel, cell wall, cytochemistry, vessel differentiation     

Increased Survival and Differentiation of Frozen Herbaceous Plant Organ Cultures through Cold Treatment

Cold treatment of donor carnation plants (Dianthus caryophyllus L.) at 4 C for 3 days or more resulted in a doubling in the percentage of excised, frozen shoot apices which survived freezing and a 6- to 7-fold increase in the percentage which formed leaf primordia or shoots. The optimal freezing parameters for both survival and differentiation were as follows: size of the shoot apex-two to three sets of leaf primordia; dimethylsulfoxide concentration in the freezing solution-5%; time in dimethylsulfoxide prior to freezing->30 minutes; average cooling rate-≥50 C/minute; initial warming rate-about 1450 C/minute. In general, the cells in the meristematic region of the shoot apex remained viable after freezing while those in the leaf primordia did not. Viability of the meristematic cells appears to be maintained by preventing the growth of intracellular ice crystals formed during rapid cooling by rapidly passing the tissue through the temperature zone in which lethal crystal growth occurs (mechanism of Luyet). Applications of this technique are discussed.     

Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots

Carnation plantlets (Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm⁻³), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2 to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 μm²). However, the number of regenerated shoots also decreased (17.7 in 2 g dm⁻³ agar to 4.3 in 12 g dm⁻³). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis.     

Some Aspects of Ontogeny of the Shoot Apices of Solanum melongena L. and Capsicum annuum L.

The ontogeny of the vegetative shoot apices of two importantvegetable plants, Solanum melongena L. and Capsicum annuum L.is described. Each shoot apex is studied at different stagesof seed germination. The relationship between time of germinationand (i) area of vacuoles in the cell, (ii) total area of thecell, (iii) area of the nucleus in the cell, and (iv) ratioof area of vacuoles in the cell to the total area of the cellin each apex is examined. The differentiation of cytohistologicalzonation in both apices is distinct only after one or two leavesare initiated and developed. At a certain stage heterogeneityin staining in the peripheral region of the shoot apex of S.melongena is found. The zonation in both plants differentiatesgradually in histological and cytological features. Vacuolationincreases or decreases in all the cells of the shoot apex orin the cells of a particular region of the shoot apex at differentstages of its ontogeny.     

Shoot Regeneration from Fragmented Flower Buds of Carnation (Dianthus caryophyllus)

Adventitious shoots were regenerated from fragmented flowerbuds, individual petals and receptacles in a number of differentcarnation cultivars. The major site of shoot formation was thesubepidermal cells at the proximal end of the petals. The yieldof shoots from a single flower bud was high, ranging between70 and 275, for the 11 cultivars tested. The regeneration mediumcontained Murashige and Skoog basal medium supplemented with4–8 µm -naphthaleneacetic acid and 4–8 µmbenzyladenine. The preferred regeneration protocol appears highlysuited to the development of gene transfer systems. Adventitious shoots, Dianthus caryophyllus L., tissue culture, explant, auxin, cytokinin, cut flowers, floriculture, organogenesis     

Chalcone Synthase Localization in Shoot Apices of Fagopyrum, Brassica and Pisum

Chalcone synthase (CHS), a key enzyme of flavonoid synthesis,was localized in shoot apices of Fagopyrum, Brassica and Pisum.The enzyme was detected in initial cells of the shoot apex,which gives rise to the whole plant body. In Fagopyrum and BrassicaCHS was located in the rib and flank meristems, whereas in theArgenteum mutant of Pisum this enzyme was localized at an earlierstage in the ontogenesis of the shoot. It occurs in a few cellsof the tunica, which gives rise to the protoderm, and then tothe epidermis which contains anthocyanins in these plants. Chalcone synthase, immunogold labelling, promeristem, shoot apex, Brassica, Fagopyrum, Pisum     

Micropropagation of Dianthus caryophyllus L. — Control of Vitrification

Vitrification is a morphological and physiological disorder affecting in vitro regenerated plants. Vitrified shoots of carnation (Dianthus caryophyllus L.) regenerated from cultured cotyledons were abnormally glassy, thick and bushy with wider translucent leaves. Such vitrified shoots were recovered by culturing them on a medium supplemented with GA₃. Differentiation of shoot buds from the cultured cotyledons of D. caryophyllus occurred on MS medium supplemented with BAP (2.0 mg/l). Shoot buds subcultured on the same medium resulted in further prolific development of shoot buds and bushy shoot growths. Key words: carnation, shoot morphogenesis, micropropagation, cotyledons vitrification.     

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.     

Observations on the Growth of Carnation (Dianthus caryophyllus L.) in Natural and Long Days

Observations were made on the growth and development of carnationsgrown in containers under natural or 24 h days. The number ofleaf pairs produced before flower initiation and the final lengthof each flowering stem were affected by the date at which theshoot appeared and its position on the plant. Dusk-to-dawn lighting reduced the number of axillary shootsin each generation but increased their rate of development.This resulted in similar numbers of flowers being produced inboth treatments. Dianthus caryophyllus L., Carnation, growth, flowering, day length     

The Natural Philosophy of Plant Form: Cellular Autoreproduction as a Component of a Structural Explanation of Plant Form

A map-L-system is described which simulates the developmentof the two-dimensional patterns of cell walls displayed at thesurfaces of shoot apices of Psilotum nudum. The simulation ofthese cellular patterns commences with the division of a triangularcell and continues until a complete set of ten different cells,including new triangular cells, is formed amongst the descendantsof each merophyte. The triangular cells generated by means ofthis division pathway, P1, are, in their three-dimensional aspect,four-sided apical cells. In the plant, they have the potentialityto support the development of a shoot apex. The generation ofnew triangular cells by pathway P1 therefore seems to be a preconditionfor the branching of the shoot. Observed variations upon thecellular pattern developed by pathway P1 have also been analysed.Two of these variant pathways, P2 and P3, suggest the typesof controls which are required to bring about all three (P1–P3)patterns of cells. These controls may involve the participationof the plant cytoskeleton and may also require an influencefrom the apical cell itself. The triangular shoot apical cellsof Psilotum are autoreproductive cells: that is, at each division,one of the daughters is a new triangular cell, the other daughterhas some other shape. This example of triangular cell autoreproductionand self-maintenance and its relation to organogenesis is discussedin light of the views on reproduction and self-maintenance expressedby Agnes Arber (1950) in her book The natural philosophy ofplant form(Cambridge: Cambridge University Press). Copyright2001 Annals of Botany Company Agnes Arber, apical cell, cell division patterns, computer simulation, cytoskeleton, L-systems, Living Systems Theory, meristems, Psilotum, shoot apex, stem cell     

Relative water content and water potential of tissue 1

Relative water content (RWC) and water potential as measuredwith the pressure chamber were evaluated as indicators of waterstatus of tissue-cultured apple shoots and plantlets (shootswith roots). During the hydration required for RWC measurement,both water content and water potential exhibited the same hydrationkinetics, indicating that 10 h were required for full hydration.Once full hydration was reached, shoot mass remained relativelyconstant. Moisture release characteristics were also constructedand the associated shoot and plantlet water relations parameterswere estimated. Underin vitroconditions, both shoot and plantletwater potential were similar to the water potential of the culturemedium in which they were grown. The moisture release characteristicof shoots and plantlets was consistent with that expected fortypical plant tissues, and gave estimates of maximum modulusof elasticity (6.201.14 MPa), osmotic potential at saturation(–0.85 0.10 MPa), osmotic potential at zero turgor (–1.16 0.14 MPa) and RWC at zero turgor (78 2%) which were similarto values in the literature. Higher values of leaf conductanceand RWC were found in shoots and plantlets placed at 95% RH(21 C) compared to those at 90% RH. Plantlets had higher valuesof both conductance and RWC compared to shoots, suggesting thatinvitroroots are functional in water uptake. Relative water contentwas related to measures of physiological activity such as leafconductance, and it was also easier to measure than water potential.Relative water content is suggested as a sound index of waterstatus in tissue culture plants. Key words: Conductance, microculture, water status, water stress.     

Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets

The ability of shoot tips from carnation (Dianthus caryophyllus L., var. Eolo) cultured in vitro to develop resistance to freezing in liquid nitrogen depends on the physiological state of the cell material and the pretreatment conditions. Regrowth rates close to 100% have been obtained with apical shoot tips isolated from 2 month-old stems, precultured on medium supplemented with sucrose (0.75M) and treated with dimethylsulfoxide (5% or more). Resistance of axillary shoot tips decreased progressively as a funtion of their distance from the apical shoot tip. During the development of the stem from axillary buds (obtained by cutting), progressive increases in the regrowth rate of frozen apices were noted, from 30% before cutting (axillary buds) to 98% after 3 weeks of culture.Abbreviations DMSO dimethylsulfoxide - LN liquid nitrogen