Stomata and Structure of Tetraploid Apple Leaves cultured in Vitro

Leaves of anther-derived tetraploid apple (Malus pumila Mill.)shoots were examined by low-temperature scanning electron microscopy(LT-SEM). Leaves were serrate and wide with an undulating adaxialsurface due to convex epidermal cells, apparently without crystallineepicuticular wax. Stomata were absent from the adaxial surface,except for the marginal teeth which exhibited 40-60 stomataper leaf; they probably originated from residual mitotic activity.One third of abaxial stomata was occluded by the residual cuticleof the mother guard cell across the stomatal pore which rupturedwhen the stomata became functional. The stomatal index was 7·2(± 1·6) with 60-75 stomata mm^-2, i.e. abaxialstomata of tetraploid leaves expanded in vitro were less frequentthan those in triploid leaves either cultured in vitro (475-575stomata mm^-2) or grown on the tree (320-390 stomata mm^-2) wherethe stomatal index was 21 (± 4). Freeze-fracture transsectionsshowed that the tetraploid in vitro leaves were composed ofa layer of adaxial epidermal cells, followed by a single layerof palisade cells and four to five layers of spongy mesophyllcells and the abaxial layer of epidermal cells, in contrastto juvenile seedling-grown apple leaves in which the two layersof palisade cells comprised the majority (52-60%) of the leafvolume. The same morphological features, such as wide and lesspointed leaves, reduced stomatal density and stomatal index,and increased stomatal size that were previously reported fortree-grown tetraploid leaves were also expressed in vitro. Thus,causes of the stomatal deformation in tissue-cultured Rosaceaeare interpreted to be in part genetic and not purely environmental.Copyright1994, 1999 Academic Press Malus pumila Mill., apple, biotechnology, breeding, cryo-preservation, CO₂, juvenile, low temperature-scanning electron microscopy (LT-SEM), micropropagation, ploidy, stomata, tissue-culture, transpiration     

The Influence of Prior Water Stress and Abscisic Acid Foliar Spraying on Stomatal Responses to CO2, IAA, ABA, and Calcium in Leaves of Solanum melongena

The influence of a water stress or foliar ABA spraying pretreatmenton stomatal responses to water loss, exogenous ABA, IAA, Ca²⁺,and CO₂ were studied using excised leaves of Solanum melongena.Both pretreatments increased stomatal sensitivity of water loss,in the presence and absence of CO₂, but decreased stomatal sensitivityto exogenous ABA. CO₂ greatly reduced the effect of exogenouslyapplied ABA. IAA decreased leaf diffusion resistance for controland ABA sprayed leaves, but did not influence the LDR of previouslywater-stressed leaves. CA²⁺ did not influence LDR of any leavesof any treatments. Key words: Water stress, stomatal response, pretreatments     

Photosynthetic Development of Micropropagated Strawberry Plantlets following Transplanting

Leaves from plantlets of Fragaria x ananassa Duch. cv. Gentocultured in vitro do not fix sufficient carbon to maintain theplantlets without an added carbon source. Following transplantingto glasshouse conditions these leaves fail to develop significantphotosynthetic ability and degenerate. Those leaves developingsubsequent to transplanting are photosynthetically capable.The possible transitory, nutritive function of leaves producedin vitro is discussed. Fragaria x ananassa Duch., strawberry, in vitro culture, transplanting, photosynthesis     

Response of Cultured Embryos of Phaseolus vulgaris and Hordeum vulgare to Farnesol

Excised embryos of Phaseolus vulgaris incubated in a mediumcontaining 10 mg dm^–3 farnesol showed enhanced root growthwhereas the leaves remained rudimentary At lower concentrationsof exogenous farnesol normal leaf development occurred and rootgrowth was comparable to untreated cultures. Enhanced root growthalso occurred when excised embryos of Hordeum vulgare were treatedwith farnesol but only at 10 mg dm^–3 and this treatmentdid not prevent leaf growth X-ray micro-probe analysis of leavesrevealed an increased phosphorus content in P vulgaris and adecreased sulphur content in H vulgare in comparison to untreatedplants. Hordeum vulgare L., barley, Phaseolus vulgaris, bean, embryo culture, farnesol, X-ray microprobe analysis, root growth     

Effect of Photoperiodic Induction on the Transpiration Rate and Stomatal Behaviour of Debudded Xanthium Plants

In controlled-environment studies with debudded Xanthium plants,appreciable changes in stomatal activity and attendant ratesof transpiration were found to be associated with photoperiodicinduction. Leaves of plants kept on a 10-h inductive photoperiodafter removal of the apical and axillary buds grew at ratescomparable to those of similarly debudded plants kept on a 10-h-interruptednon-inductive photoperiod (consisting of an 8-h photoperiodand a 2-h interruption of the dark period). There was a large effect of leaf age on stomatal behaviour:the minimum stomatal resistance of leaves of non-induced plantsdecreased from about 5 s cm^–1 when the leaf was 25 percent of its final size to 1.6 s cm^–1 when almost fullyexpanded. Thereafter, it slowly increased with time. Superimposedon this age response was a marked effect of photoperiodic induction.The stomata on induced leaves opened more widely than thoseon non-induced leaves, the response being greatest with theleaves which were youngest at the time induction commenced.However, this ‘opening’ tendency was maintainedfor only a few weeks; thereafter, the stomata failed to openas widely. This later ‘closing’ tendency of stomataon leaves of induced plants progressed rapidly and in a basipetalsequence and presaged a necrotic form of leaf senescence whichdeveloped in the same sequence. The closing tendency on leavesof non-induced plants progressed slowly in an acropetal direction;leaves senesced in the same sequence with the familiar yellowingsymptoms. It is suggested that flower induction sets in train a sequenceof events which influence stomatal movement (and other processes)and inevitably leads to the death of the induced axis. Transpiration rates calculated from measurements of the physicalenvironments and stomatal resistances agreed well with thosemeasured.     

Stomatal Functioning of In Vitro and Greenhouse Apple Leaves in Darkness, Mannitol, ABA, and CO2

Leaves from in vitro and greenhouse cultured plants of Malusdomestica (Borkh.) cv. Mark were subjected to 4 h of darkness;4 h of 1 M mannitol induced water stress; 1 h of 10^–4M to 10^–7 M cis-trans abscisic acid (ABA) treatment; 1h of 0.12% atmospheric CO₂. Stomatal closure was determinedby microscopic examination of leaf imprints. In all treatments,less than 5% of the stomata from leaves of in vitro culturedplants were closed. The diameter of open stomata on leaves fromin vitro culture remained at 8 µm. In contrast, an averageof 96% of the stomata on leaves of greenhouse grown plants wereclosed after 4 h in darkness; 56% after 4 h of mannitol inducedwater stress; 90% after 1 h of 10^–4 M ABA treatment; 61%after 1 h in an atmosphere of 0.12% CO₂. Stomata of in vitroapple leaves did not seem to have a closure mechanism, but acquiredone during acclimatization to the greenhouse environment. Thelack of stomatal closure in in vitro plants was the main causeof rapid water loss during transfer to low relative humidity.     

江西铅山红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的建立

以江西铅山红芽芋脱毒苗为试材,研究不同因素对红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的影响,以期对红芽芋脱毒苗的再生体系进行优化。结果表明,红芽芋脱毒苗球茎愈伤组织诱导的最佳培养基是MS+TDZ 2 mg·L^-1+2,4-D 1 mg·L^-1。红芽芋脱毒苗球茎愈伤组织分化的最佳培养基是MS+TDZ 2 mg·L^-1+NAA 1 mg·L^-1。红芽芋脱毒苗不定芽生根的最佳培养基是1/2MS+NAA 0.5 mg·L^-1+PP₃₃₃ 0.5 mg·L^-1。红芽芋再生苗最好的移栽基质为发酵后的腐锯木屑。红芽芋脱毒苗球茎愈伤组织再生苗移栽时最佳的PP₃₃₃浓度为20～50 mg·L^-1。本试验成功建立了红芽芋脱毒苗球茎愈伤组织的再生体系,为红芽芋脱毒苗转基因的研究和种质创新奠定了基础。     

The Effect of Shade During Leaf Expansion on Photosynthesis by White Clover Leaves

In three experiments measurements of photosynthesis were madeon single leaves of white clover (Trifolium repens L.) on threecultivars grown in a controlled environment. Plants which had grown under an irradiance of 30 J m^–2s^–1, or in shade within a simulated mixed sward, producedleaves with photosynthetic capacities some 30 per cent lowerthan did plants grown at 120 J m^–2 s^–1 without shade.There were no differences between treatments either in photosynthesismeasured at 30 J m^–2 s^–1, or in respiration ratesper unit leaf dry weight. Respiration per unit leaf area washigher in the plants grown at 120 J m^–2 s^–1, reflectingthe lower specific leaf area of these leaves. There were nodifferences between the three cultivars examined. Leaves which were removed from the shade of a simulated swardshortly after becoming half expanded achieved photosyntheticcapacities as high as those which were in full light throughouttheir development. It is suggested that it is this characteristicwhich enables clover plants growing in an increasingly densemixed sward to produce a succession of leaves of high photosyntheticcapacity, even though each lamina only reaches the top of thesward at a relatively late stage in its development. Trifolium repens L., white clover, photosynthesis, leaf expansion, shade, specific leaf area, stomatal conductance     

Potassium Involvement in Stomatal Movements of Paphiopedilum

There are conflicting reports about whether guard cells of Paphiopedilumspp. accumulate K during stomatal opening. In this study X-raymicroprobe analysis and histochemical staining for K indicatedthat K accumulated in guard cells in leaves of Paphiopedilumspp. when stomata opened. Additionally, stomatal opening inepidermal strips of P. harrisseanum could only be induced inan MES buffered KC1 medium. Element analysis ofP. harrisseanumepidermis also indicated substantial levels of K, Na and Cawithin the tissue. We conclude that K is involved in the stomatal movements ofPaphiopedilum. Key words: K⁺ transport, Paphiopedilum, Stomatal movements     

Effects of Different CO2 Environments on the Photosynthesis-Yield Relationship and the Carbon and Water Balance of a White Clover (Trifolium repens L. cv. Blanca) Sward

Effects of atmospheric CO₂ enrichment to a level above 600 parts10^–6 on leaf and canopy gas exchange characteristics wereinvestigated in Trifolium repens, using an open system for gasexchange measurement. The cuvettes of the system served as growthchambers, allowing continuous measurement in a semi-controlledenvironment of ±350 and ±600 parts 10^–6CO₂, respectively. Carbon balance data were compared with cropyield and effects on the canopy level were compared with measuredleaf responses of photosynthesis and stomatal behaviour. Photosyntheticstimulation by high CO₂ was stronger at the canopy level (103%on average) than for leaves (90% in full light), as a consequenceof accelerated foliage area development. The latter increasedabsolute water consumption by 16%, despite strong stomatal closure.The overall result was a 63% improvement in canopy water useefficiency (WUE), while leaf WVE increased almost 3-fold insaturating light. The stomatal response was such that, whilethe internal CO₂ concentration in the leaf, ch increased withrising atmospherical CO₂ concentration, c_a, ci/c_a was somewhatdecreased. Total canopy resistance, R_c, was generally lowerat high CO₂ levels, despite higher leaf resistance. Higher canopyCO₂ loss at night and faster light extinction in a larger-sizedhigh CO₂ canopy were major drawbacks which prevented a furtherincrease in dry matter production (the harvest index was increasedby a factor 1.83). Key words: CO₂ enrichment, canopy CO₂ exchange, carbon balance, water use efficiency, leaf and canopy resistance     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Flag Leaf Photosynthesis of Triticum aestivum and Related Diploid and Tetraploid Species

Rates of net photosynthesis of the flag leaves of 15 genotypesof wheat and related species were measured throughout theirlife, using intact leaves on plants grown in the field. At thestage when rates were maximal, they were in general highestfor the diploid species, intermediate for the tetraploidspeciesand lowest for Triticum aestivum (means of 38, 32 and 28 mgCO₂ dm^–2 h^–1 respectively). Rates were stronglynegatively correlated with leaf area, leaf width and the meanplan area per mesophyll cell and positvely correlated with stomatalfrequency and number of veins per mm of leaf width. The differencesamong species in these attributes were mainly related to ploidylevel. It was not possible to determine the relative importanceof each anatomical feature, though the changes in stomatal frequencyhad only slight effects on stomatal conductance and the observeddifferences in rates of photosynthesis were much greater thanwould be expected from those in stomatal conductance alone. There was genetic variation in rates of light dependent oxygenevolution of isolated protoplasts and intact chloroplasts butno difference attributable to ploidy. The mean rate, 91 µmolO₂ mg^–1 chlorophyll h^–1, equivalent to 3.9 mg CO₂mg^-1chlorophyll h^-1 was considerably less than the rate of photosynthesisin comparable intact leaves, which was 7.2 mg CO₂ mg^–1chlorophyll h^–1. The total above-ground dry matter yields were least for thewild diploids T. urartu and T. thauodar and the wild tetraploidT. dicoccoides, but the other wild diploids produced as muchdry matter as the hexaploids. The prospects of exploiting differences in photosynthetic ratein the breeding of higher yielding varieties are discussed. Triticum aestivum L., wheat, Aegilops spp, photosynthesis, stomatal conductance, stomatal frequency, polyploidy     

Ionic Relations of Garden Orache, A triplex hortensis L.: Growth and Ion Distribution at Moderate Salinity and the Function of Bladder Hairs

The growth of garden orache, A triplex hortensis was studiedunder conditions of mild NaCl or Na₂SO₄ salinity. Growth, drymatter production and leaf size were substantially stimulatedat 10 mM and 50 mM Na⁺ salts. Increased growth, however, appearedto be due to a K⁺-sparing effect of Na⁺ rather than to salinityper se. The distribution of K⁺ and Na⁺ in the plant revealeda remarkable preference for K+ in the roots and the hypocotyl.In the shoot the K/Na ratio decreased strongly with leaf age.However, the inverse changes in K⁺ and Na⁺ content with leafage were dependent on the presence of bladder hairs, which removedalmost all of the Na⁺ from the young leaf lamina. Measurementsof net fluxes of K⁺ and Na⁺ into roots and shoots of growingAtriplex plants showed a higher K/Na selectivity of the netion flux to the root compared to the shoot. With increasingsalinity the selectivity ratio S_{K, Na}^* of net ion fluxes tothe roots and to the shoots was increased. The data suggestthat recirculation of K⁺ from leaves to roots is an importantlink in establishing the K/Na selectivity in A. hortensis plants.The importance of K⁺ recirculation and phloem transport forsalt tolerance is discussed. Key words: Atriplex hortensis, Salinity, Potassium, Sodium, K⁺ retranslocation, Bladder hairs, Growth stimulation     

Comparative Leaf Surface Morphology and the Glossy Characteristic of Sorghum, Maize, and Pearl Millet

Leaf surfaces of seven genotypes of Sorghum bicolor, two ofmaize, Zea mays, and two pearl millet, Pennisetum americanum,were examined by scanning electron microscopy for possible morphologicaldifferences. Leaves 1, 3, 5 and 7 were photographed and printswere used to estimate waxiness, hairiness or pubescence andstomatal density. Glossiness was determined by spraying water,which adhered to the glossy leaves. Cuticular transpirationof detached third and fifth leaves was estimated from the rateof water loss after abscisic acid induced stomatal closure.Sorghum lines SC283, CSM63, CSM90, and pearl millets Souna andTiotioni (all from Mali), were non-glossy, well covered withwax, and exhibited variable hairiness. Older leaves of sorghumvarieties Martin and Redlan were glossy and, like older leavesof the other glossy lines SC1096 and SC90, had little or nowax deposits on their cuticles. The two maize cultivars, NB611and N7A, were non-glossy with dense wax covering; no trichomeswere observed until the 5 to 7 leaf stage. Thus, the glossycharacter was correlated with the reduction or absence of waxdeposits on the leaf surfaces, while hairiness might occur ineither glossy or non-glossy genotypes. Unlike sorghum and maize,in which all leaves after the fifth or seventh were glossy,pearl millet showed no glossiness through the ninth leaf. Measurementsshowed that cuticular transpiration of glossy leaves was oftenmore than double that of non-glossy leaves. Comparisons amongsorghums showed that non-glossy lines had higher stomatal densitiesthan glossy lines. Epicuticular wax, trichome, glossy mutant, stomata, cuticular transpiration, Sorghum bicolor, (L.) Moench, Zea mays L., Pennisetum americanum, (L.) Leeke     

The Use of 14C-Ethane Diol as a Quantitative Tracer for the Transpirational Volume Flow of Water and an Investigation of the Effects of Salinity upon Transpiration, Net Sodium Accumulation and Endogenous ABA in Individual Leaves of Oryza sativa L.

Yeo, A. R., Yeo, M. E., Caporn, S. J. M., Lachno, D. R. andFlowers, T. J. 1985. The use of ¹⁴C-ethane diol as a quantitativetracer for the transpirational volume flow of water and an investigationof the effects of salinity upon transpiration, net sodium accumulationand endogenous ABA in individual leaves of Oryza sativa L.—J.exp. Bot. 36: 1099–1109. Oryza sativa L. (rice) seedlings growing in saline conditionsexhibit pronounced gradients in leaf sodium concentration whichis always higher in the older leaves than the younger ones.Individual leaf transpiration rates have been investigated todiscover whether movement of sodium in the transpiration streamis able to explain these profiles from leaf to leaf. The useof ¹⁴C labelled ethane diol to estimate transpiration was evaluatedby direct comparison with values obtained by gas exchange measurements.Ethane diol uptake was linearly related to the transpirationalvolume flow and accurately predicted leaf to leaf gradientsin transpiration rate in saline and non-saline conditions. ¹⁴C-ethanediol and ²²NaCl were used to compare the fluxes of water andsodium into different leaves. The youngest leaf showed the highesttranspiration rate but the lowest Na accumulation in salineconditions; conversely, the older leaves showed the lower transpirationrates but the greater accumulation of Na. The apparent concentrationof Na in the xylem stream was 44 times lower into the youngerleaf 4 than into the older leaf 1. Exposure to NaCl (50 molm^–3) for 24 h elicited an increase in endogenous ABA inthe oldest leaf only, but no significant changes occurred inthe younger leaves. Key words: —Salinity, rice, Oryza sativa L., transpiration, volume flow, abscisic acid     

The Tropical Fig Ficus microcarpa L. f. cv. Golden Leaves Lacks Heat-Stable Dehydroascorbate Reductase Activity

Dehydroascorbate reductase (DHAR) is an enzyme which mediatesa regeneration of ascorbate from dehydroascorbate using GSHas the electron donor. Here we report a tropical fig lackinga heat-stable DHAR activity in leaves. The leaves of Ficus microcarpaL. f. cv. Golden Leaves are normally green under dim light butare yellow under full sunlight in the field. We compared DHARactivity from the leaves of Golden Leaves to that of the wildtype using two distinct assay methods. Total leaf DHAR activityof Golden Leaves, determined in the presence of 2-mercaptoethanoland EDTA, was 0.3 µmol min^–1 (mg protein)^–1which was comparable to that of the wild type or spinach leaves.This activity was completely eliminated by heat treatment at80°C, whereas 40% of the activity was resistant to thistreatment in the wild type leaves. Similar results were obtainedwith another assay method employing dithiothreitol and gel filtration.We conclude that the absence of heat-stable DHAR activity isa significant biochemical marker that distinguishes Golden Leavesfrom the wild type. A physiological role for DHAR in adaptationto high light stress is proposed. (Received December 4, 1998; Accepted April 15, 1999)     

Ammonium Nutrition Enhances Chlorophyll and Glaucousness in Kohlrabi

Kohlrabi (Brassica oleracea var.gongylodes) plants were grownin the greenhouse under autumn conditions and fertilized eitherwith pellets containing nitrogen as 40% ammonium sulphate and60% urea or with nutrient solution containing nitrogen predominantlyas nitrate. Plants given nitrogen as ammonium ions developedglaucous leaves compared to those supplied with nitrate whichformed glossy leaves. Ammonium-induced glaucousness was theresult of a two-fold increase in the amount of epicuticularwax and a markedly altered fine structure. Leaves from ammoniumfertilized kohlrabi plants also showed a 21% increase in chlorophyllcontent together with a reduction in the chlorophyll a:b ratioand decreased ground state fluorescence compared to plants suppliedwith nitrate. Photosynthesis and stomatal transpiration wereunaffected by the form of supplied nitrogen. Brassica oleracea ; chlorophyll; chlorophyll fluorescence; epicuticular wax; glaucousness; photosynthesis; transpiration     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration     

Salt Tolerance in the Triticeae: K/Na Discrimination in Barley

Concentrations of ions were measured in the youngest fully-expandedleaves of Triticum aestivum, T. durum, Hordeum vulgare, H. spontaneum,Secale cereale, and Aegilops squarrosa accessions grown in hydroponicculture in the presence of salt (NaCl+CaCl₂). Triticum aestivum,Secale cereale, and Ae. squarrosa had the low leaf Na and highleaf K concentrations typical of plants which contain the enhancedK/Na discrimination character originally found in Ae. squarrosa.T. durum and the Hordeum species did not have this character.The better growth of H. vulgare than of T. durum with similarsalt concentrations in the youngest fully-expanded leaves maybe a result of better compartmentation of Na, Cl, and K betweendifferent tisssues or between different compartments withincells. The enhanced K/Na discrimination character was expressedin disomic addition lines of H. vulgare chromosomes in Triticumaestivum. The H. vulgare variety Herta and its slender mutantboth had similar leaf cation concentrations, although they differedin growth rate when grown at 60 mol m^–3 NaCl. H. vulgareand T. durum seedlings grown in the absence of monovalent cationsaccumulated more ²²Na in their shoots than seedlings of otherspecies when incubated in 1.0 mol m^–3 NaCl labelled with²²Na. Key words: Salt, ion transport, I genome, barley, wheat     

Measurement of the Apoplastic Activity of K+ and Cl- in the Leaf Epidermis of Commelina communis in Relation to Stomatal Activity

Bowling, D. J. F. 1987. Measurement of the apoplastic activityof K⁺ and Cl^– in the leaf epidermis of Commelina communisin relation to stomatal activity.–J. exp. Bot. 38: 1351–1355. Ionic activity of K⁺ and Cl^– in the apoplast of the lowerepidermis of the leaf of Commelina communis was measured usingion selective micro-electrodes. Large gradients across the stomatalcomplex were observed which were related to stomatal aperture.On stomatal closure the activity of K⁺ and Cl^– in theapoplast of the guard cell rose from 3·0 mol m^–3to 100 mol m^–3 and 33 mol m^–3 respectively. It wasconcluded that the apoplast is an important pathway for iontransport between the cells. Key words: Stomata, ionic activity, leaves, apoplast