A Histochemical Study of Nectaries of Hibiscus rosa-sinensis

Different histochemical and cytochemical methods were employedon nectaries of Hibiscus rosa-sinensis. Light microscopy revealedthe presence of oil and mucilage cells in the subglandular tissue.Electron microscopy showed intense activity of ATPase in thephloem subtending the nectary. When CaCl₂ or tannic acid areadded to the fixative, electron-dense globular deposits areencountered in close contact with the plasmalemma of the secretorycells. In this case the endoplasmic reticulum appears in alternatingelectron-dense areas. In young nectaries the application oftannic acid results in electron-opaque deposits at the cellplate of dividing cells. The prolonged incubation of nectariesin OsO₄ results in an obvious difference in staining betweennectary hairs and subglandular cells. Structures stained selectivelywith OsO₄ are the endoplasmic reticulum, nuclear envelope, plastids,and mitochondria. The cytochemical experiments support the viewthat in nectaries of Hibiscus rosa-sinensis, the pre-nectaroriginates from the phloem and it is symplastically carriedvia the plasmodesmata to the secretory cells of the hair fromwhere it is secreted. The principal element which is involvedboth in the pre-nectar transport and nectar secretion is theendoplasmic reticulum. Key words: Lipid staining, polysaccharides, tannic acid, calcium binding sites, ATPase activity, osmium impregnation     

Role of ethylene in the senescence of isolated hibiscus petals

Senescence of petals isolated from flowers of Hibiscus rosa-sinensis L. (cv Pink Versicolor) was associated with increased ethylene production. Exposure to ethylene (10 microliters per liter) accelerated the onset of senescence, as indicated by petal in-rolling, and stimulated ethylene production. Senescence was also hastened by basal application of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminooxyacetic acid, an inhibitor of ethylene biosynthesis, effectively inhibited ethylene production by petals and delayed petal in-rolling. In marked contrast to these results with mature petals, immature petals isolated from flowers the day before flower opening did not respond to ethylene in terms of an increase in ethylene production or petal in-rolling. Furthermore, treatment with silver thiosulfate the day before flower opening effectively prevented petal senescence, while silver thiosulfate treatment on the morning of flower opening was ineffective. Application of ACC to both immature and mature petals greatly stimulated ethylene production indicating the presence of an active ethylene-forming enzyme in both tissues. Immature petals contained less free ACC than mature, presenescent petals and appeared to possess a more active system for converting ACC into its conjugated form. Thus, while the nature of the lack of responsiveness of immature petals to ethylene is unknown, ethylene production in hibiscus petals appears to be regulated by the control over ACC availability.     

Endogenous ethylene and abscisic Acid relative to phytogerontology

Endogenous production of ethylene and endogenous levels of abscisic acid were measured from Hibiscus rosa-sinensis L. abscission zone explants at six stages of development: tight bud, open flower, closed flower, petal abscission, calyx abscission, and peduncle abscission.     

The Floral Nectaries of Hibiscus rosa-sinensis: 1. Development of the Secretory Hairs

Floral nectaries of Hibiscus rosa-sinensis occur on the lowerinner side of the fused sepals and each one consists of numerous(50000–55000) secretory hairs, occupying a cylinder-likezone completely lining the inner side of the sepals. Each hairoriginates from a single protodermal mother cell and, at maturity,it is built up of a basal cell, a stalk, 35–40 intermediatecells and a tip secretory cell. Development of protodermal cellsinto secretory hairs is asynchronous, the first cells to initiatedevelopment being those situated in the lowermost part of thecylindrical zone, and development progressing upwards. Volume increase of protodermal mother cells initiating developmentis accompanied by cell polarization manifested by organelledisplacement towards the apical region. Secretory hairs areformed through a sequence of transverse and, later on, anticlinaldivisions. Divisions of apical cells are preceded by well definedpre-prophase microtubule bands, which foreshadow the plane ofthe forthcoming division and predict with accuracy the sitesof parental walls where the new cell plate fuses at cytokinesis. Stalks consist of either one or two cells. Two-celled stalksoccur in 40 per cent of secretory hairs and derive from a transversedivision of one stalk cell; the wall formed is always depositedparallel to the proximal and distal walls, but never to thelateral ones. The significance of this mode of division is discussedin relation to the fact that lateral walls are entirely impregnatedwith a cutin-like material that blocks apoplastic movement ofsolutes. Hibiscus rosa-sinensis, nectaries, development, preprophase microtuble bands, stalk cells     

A Humidity-induced Convective Throughflow Ventilation System Benefits Annona squamosa L. Explants and Coconut Calloid

A simple apparatus is described for generating pressurized throughflowventilation in plant tissue culture vessels. No pumps or gas-cylindersare required and the flow is driven by humidity-induced diffusionacross microporous membranes. In the experiments described,pressurized flows of sterile humidified air were supplied atrates of up to 1 ml min^-1and these had beneficial effects onleaf survival and production inAnnona cuttings and on calloidform in coconut. Ethylene (ethene) was removed more quicklyfrom the pressure-flow ventilated culture vessels (t ₅₀, 0.4–0.7h) than from those aerated by diffusion through conventionalpolypropylene membranes (t ₅₀, 1.6–2.4 h). InAnnona cuttings leaf production was greatly increased andethylene-induced leaf fall considerably delayed when culturedwith the forced as opposed to diffusion-based ventilation ofthe vessels. With throughflow ventilation, coconut calloid was more convolutedthan under wholly diffusive aeration and had a smooth distinctepidermal surface and clearly defined sub-epidermal meristematicnodules. It resembled freshly initiated calloid from which regenerationof plantlets via somatic embryogenesis can be obtained. Underwholly diffusive aeration, calloid developed a rough, relativelyundifferentiated surface, more haustorial (i.e. cotyledonary)in appearance, and characteristic of cultures where regenerationpotential has been lost. It is suggested that other benefits of the pressurized throughflowventilation may be the removal of volatiles such as ethanoland acetaldehyde, the removal of excess carbon dioxide at nightand its improved supply during the day, and sustained oxygenconcentrations at levels close to atmospheric both night andday. Abscission; callus; ethylene; explants; tissue-culture; growth; ventilation     

Rapid Ethylene Production in Soybean in Response to the Cupric Ion

Very rapid accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC), followed by stimulation of ethylene production wereinduced by a Cu²⁺ in soybean cuttings. The accumulation mustbe attributed to an increase in ACC synthesis, because: (1)it was completely inhibited by aminoethoxyvinylglycine (AVG);and (2) the ethylene stimulation was inhibited by AVG, indicatingthat free ACC cannot be released from its conjugated form. Theactivity of the ethylene-forming enzyme slightly decreased followingthe Cu²⁺ pulse, and this event was accompanied by a slight increasein electrolyte leakage from the treated soybean tissues. Glycine max L., soybean, ethylene, cupric ion     

Initiation of Rapid Ethylene Synthesis by Apple and Pear Fruits in Relation to Storage Temperature

The influence of storage temperature on the onset of rapid ethyleneproduction was investigated for fruits of Conference pear (Pyruscommunis L.) and five cultivars of apple (Malus domestica Borkh.).The time taken from harvest to rapid ethylene production wasshorter and more uniform at 3 ?C than at 18–20 ?C forConference pears and Golden Delicious apples. Increases in internalethylene concentration, 1-amino cyclopropane-1-carboxylic acidconcentration and ethylene production were simultaneous in GoldenDelicious apples at 3 ?C. When Golden Delicious apples wereheld at 3 ?C for 48 h and then kept at 20 ?C the mean time ofonset of ethylene production was similar to that for applesheld continuously at 20 ?C. However, two periods of 48 h at3 ?C caused earlier ethylene production. Conversely, ethyleneproduction at 3 ?C was delayed by transfer to 20 ?C for twoperiods of 48 h. Cox's Orange Pippin and other apple cultivarstended to show more synchronous ethylene production at 3 ?Cthan at higher temperatures but the mean time of onset was eitherunaffected by temperature or slighdy delayed at lower temperature.Acceleration of the onset of ethylene production by low temperaturewas never observed in Cox's Orange Pippin apples harvested atweekly intervals from 10 August to 17 September. Key words: Ethylene, Storage temperature, Pyrus communis, Malus domestica     

Comparative Requirement for Endogenous Ethylene during Seed Germination

Requirement for endogenous ethylene during seed germinationof the following ten species was determined: Lycopersicon esculentumMill, (tomato), Allium cepa L. (onion), Avena fatua L., dormantpure line AN-51 (wild oats), Cucumis sativus L. (cucumber),Sinapis arvensis L. (wild mustard), Tagetes erecta L. (marigold),Raphanus sativus L. (radish), Triticum aeslivum L. (wheat),Catharanthus roseus L. (periwinkle), and Phaseolus aureus L(mung bean). Experiments were done under controlled conditionssuited for the germination of each species. Criteria used todetermine the need for endogenous ethylene were: (i) temporalrelationship between ethylene production and seed germination;(ii) parallel inhibition of ethylene synthesis and seed germinationby L-     

Role of Aminocyclopropane-1-carboxylic Acid (ACC) in Control of Ethylene Production in Fresh and Cold-stored Rose Flowers

The relationships between ethylene production, aminocyclopropane-1-carboxylicacid (ACC) content and ethylene-forming-enzyme (EFE) activityduring ageing and cold storage of rose flower petals (Rose hybridaL. cv. Gabriella) were investigated. During flower ageing at20 °C there was a climacteric rise in petal ethylene production,a parallel increase in ACC content, but a continuous decreasein EFE activity. Applied ACC increased petal ethylene productionc. 200-fold. During cold storage of flowers at 1 °C therewere parallel increases in petal ethylene production and ACCcontent, to levels greater than those reached in fresh flowersheld at 20 °C. EFE activity decreased during storage. Immediatelyafter cold-stored flowers were transferred to 20 °C ethyleneproduction and ACC levels were c. four times greater than infreshly cut flowers. These levels increased to maximum valuesof two to four times the maximum values reached during ageingof fresh, unstored, flowers. It was concluded that in rose petalsethylene synthesis is probably regulated by ACC levels and thatcold storage stimulates ethylene synthesis because it increasesthe levels of ACC in the petals. Key words: Rose flower, senescence, ethylene     

Factors Controlling the Basipetal Pattern of Tuberization in Induced Potato (Solanum tuberosum L.) Cuttings

Potato plants (Solanum tuberosum L. cvs Chippewa and Katahdin)were grown in a glasshouse under continuous light. Various numbersof long (16 h) nights were given to these plants and stem cuttingswere taken. Treatments were applied to the cuttings, which werethen placed in a mist bench under continuous light and examinedfor tuberization after 12 days. The general tendency for the strongest tuberization to occurat the most basipetal nodes, which is commonly seen with intactpotato plants, was also found on stem cuttings. This patterncould not be attributed primarily to orientation with respectto gravity, proximity to the mother tuber, or age of buriedbuds. Buried buds farthest from active leaves tended to tuberizethe most strongly. However, distance of the buried bud fromstem exposed to light may have been of equal or greater importance. potato, Solanum tuberosum L., stem cuttings, tuberization     

In vitro culture of <Emphasis Type="Italic">Hibiscus rosa-sinensis</Emphasis> L.: Influence of iron,calcium and BAP on establishment and multiplication

Some factors influencing in vitro cultures of potted Hibiscus rosa-sinensis L. using nodal cuttings were investigated. A protocol using a modified MS medium helped to overcome chlorosis, shoot tip necrosis (STN) and leaf drop. These disorders have been caused by mineral imbalance associated with calcium and iron deficiency. STN and leaf drop were overcome by increasing calcium level from 3 mM (MS standard concentration) to 9 mM, and this increase, in addition, enhanced shoot dry weight and shoot extension. The chlorophyll content and leaf area increased by increasing the iron concentration 3-fold from 98 μM to 295 μM. Furthermore, substituting Fe-EDTA with Fe-EDDHA resulted in an increase in chlorophyll content, leaf area and shoot extension. The most suitable multiplication medium for H. rosa-sinensis L. was demonstrated to be a modified MS medium containing 2.2 μM BAP and increased concentrations of calcium at 9 mM and iron at 295 μM provided as Fe-EDDHA. The shoots were rooted in half-strength modified MS medium containing 2.7 μM NAA. Acclimatization was successful with all shoots with or without roots.     

Effect of Host Plants on Honeydew Production of an Invasive Mealybug, Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Honeydew production plays a key role in mutualism between the mealybugs and ants. However, no studies have focused on the amount and circadian rules of honeydew excreted by Phenacoccus solenopsis Tinsley, a new invasive species which has conditional mutualism with Solenopsis invicta Buren in China. To address this problem, we measured the weight and estimated honeydew production in all stages of development of the invasive mealybug, P. solenopsis, as well as its honeydew production on tomato (Solanum lycopersicun), Hibiscus rosa-sinensis, and cotton (Gossypium sp.) for 24 h. The honeydew excreted by each instar of the mealybug in H. rosa-sinensis was measured for 2 weeks. Our results revealed that the weight of mealybugs significantly varied at different development stages. Host plants had no significant effect on the weight of nymphs, although the weight of a single adult reared on S. lycopersicun was significantly heavier than those reared on H. rosa-sinensis and G. sp. The amount of honeydew excreted by the 1st instar nymphs in S. lycopersicum was significantly greater than that on H. rosa-sinensis and G. sp. Each instar mealybug produced more honeydew when fed with S. lycopersicum compared with H. rosa-sinensis and G. sp. The amount of honeydew excreted by mealybugs when provisioned with H. rosa-sinensis was no different from mealybugs provisioned with G. spp. while in the same instar. The amount of honeydew excreted by the 1st and 2nd instar nymphs was not significantly different on the same host plant. However, there was a significant difference between the 3rd instar nymph and the adult. The amount of honeydew excreted by a single adult when provisioned with H. rosa-sinensis decreased from 3085.3 μg to 572.0 μg in 2 weeks. The 2nd instar nymph, 3rd instar nymph, and adult excreted honeydew more frequently during the day than at night, while the frequency of honeydew excretion of the 1st instar nymph had no significant difference between daytime and night.     

Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings

This study investigated the role of ethylene action in ethylene production and in poststorage performance of pelargonium cuttings. Cuttings of zonal pelargonium (Pelargonium x hortorum L.H. Bailey) of the cultivars Isabell and Mitzou were treated with ethylene and with the ethylene action inhibitors 1-methylcyclopropene (MCP), silver-thiosulfate (STS) and silver nitrate (SN) and were stored in the dark at different temperatures (5, 12, and 20 °C) for 48 h. Ethylene concentrations in the storage boxes were monitored and poststorage leaf senescence, survival and root formation of cuttings were determined. Applications of MCP resulted in a significant increase of ethylene evolution by cuttings of both cultivars which was more pronounced with increasing storage temperature. After 48 h of storage at 20 °C, ethylene concentrations were more than 20-fold higher for the MCP-treated cuttings as compared to those of the untreated controls. Also preharvest applications of STS and SN increased ethylene evolution by cuttings, even though these effects were less pronounced. However, application of these inhibitors caused severe poststorage leaf injury. Application of ethylene during storage had no effect on subsequent leaf damage. Leaf senescence during rooting and decay of cuttings, which raised with increasing storage temperature, could only partially been reduced by MCP. The results strongly support the conclusion, that in zonal pelargonium cuttings, ethylene production is controlled by autoinhibition, and clearly indicate, that temperature dependent processes other than ethylene action are substantially involved in storage-induced leaf senescence and decay.     

Structures and synthesis of seed-germination inhibitors from hibiscus rosa-sinensis

8-Nonynoic, 9-decynoic acids and their methyl esters were isolated for the first time as natural products from Hibiscus rosa-sinensis. They were inhibitors of the germination of lettuce seeds and were synthesized via o-nitrophenyl selenides.     

Chloroplast genome of Hibiscus rosa-sinensis (Malvaceae): Comparative analyses and identification of mutational hotspots

Previous studies to resolve phylogenetic and taxonomic discrepancies of Hibiscus remained inconclusive. Here, we report chloroplast genome sequence of Hibiscus rosa-sinensis. Hibiscus rosa-sinensis chloroplast genome was 160,951 bp, comprising of large single copy (89,509 bp) and small single copy (20,246 bp) regions, separated by IRa and IRb (25,598 bp each). The genome contained 130 genes including 85 protein-coding genes, 37 transfer RNAs and 8 ribosomal RNAs. Comparative analyses of chloroplast genomes revealed similar structure among 12 species within family Malvaceae. Evolutionary rates of 77 protein-coding genes showed 95% similarities. Analyses of codon usage, amino acid frequency, putative RNA editing sites, and repeats showed a great extent of similarities between Hibiscus rosa-sinensis and Hibiscus syriacus. We identified 30 mutational hotpots including psbZ-trnG, trnK-rps16, trnD-trnY, trnW-trnP, rpl33-rps18, petG-trnW, trnS-trnG, trnH-psbA, atpB-rbcL, and rpl32-trnL that might be used as polymorphic and robust markers to resolve phylogenetic discrepancies in genus Hibiscus.     

The Role of 1-Aminocyclopropane-1-carboxylic acid in the Control of Low Temperature Induced Ethylene Production in Leaf Tissue of Phaseolus vulgaris L .

Ethylene production from leaf discs of dwarf bean (Phaseolausvulgaris L.) was less than 02 nl g^–1 h^–1 at 5 Cbut rapidly increased tenfold on transfer to 25 C. The lowethylene production at 5 C and the potential for overshootproduction on transfer to 25C were not associated with accumulationof the ethylene synthesis intermediate 1-aminocyclopropane-1-carboxylicacid (ACC). Addition of exogenous ACC to leaf discs incubatedat 5C increased ethylene production, while similarly incubatedleaf discs did not synthesize increasing amounts of endogenousACC until they were transferred to 25 C. The basis for theovershoot in ethylene production when leafdiscs were transferredfrom 5 to 25 C appears to reside in changes to the pathwayleading to the synthesis of ACC or an earlier intermediate inthe pathway of ethylene biosynthesis. Ethylene, 1-aminocyclopropane-l-carboxylic acid, Phuseolru vulgaris L., dwarf bean, temperature     

Micropropagation of Potato: Evaluation of Closed, Diffusive and Forced Ventilation on Growth and Tuberization

Different types of ventilation of the culture vessel headspace,each with and without the ethylene inhibitor AgNO₃(3.0 µM)or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid(ACC) (2.0 µM) in the culture medium, were investigatedin terms of their effects on the growth of potato cuttings (Solanumtuberosum L. ‘cara’). Concentrations of CO₂ , O₂andethylene in the culture vessel headspaces were monitored duringthe study. Growth was substantially enhanced and vitrification(stunting and epinasty of leaves and hooking of stem apices)was reduced by increasing the efficiency of ventilation, theeffects being greatest with forced ventilation. In the conventionaldiffusive treatment (via a polypropylene membrane), leaf epinastyoccurred but the leaves were not stunted unless ACC had beenadded. AgNO₃prevented vitrification in the latter case and reducedit in the sealed treatment. On the other hand, with all forcedventilation treatments, even with the addition of ACC, the plantletsgrew well and some of the growth parameters exceeded those inthe diffusive + AgNO₃treatment. Ethylene removal was clearlyan important factor contributing to the better growth foundwith diffusive and especially with the forced ventilation treatment;with the latter, ethylene concentrations in the culture vesselswere virtually zero. In addition, enhanced CO₂supply probablycontributed to the better performance under forced ventilationcompared to diffusive ventilation. Callus developed on the stembases in all sealed (airtight) and diffusive treatments exceptwhere AgNO₃was used. No callus was observed in any treatmentwhere forced ventilation was applied and in vitro tuberization(tuber size) was considerably improved by this treatment. Copyright2001 Annals of Botany Company Callus, ethylene, potato, tuberization, vitrification     

Inherited chilling tolerance in somatic hybrids of transgenic Hibiscus rosa-sinensis x transgenic Lavatera thuringiaca selected by double-antibiotic resistance

Summary Improvement of Hibiscus rosa-sinensis for increased frost tolerance has been attempted through somatic hybridization with the frost tolerant Lavatera thuringiaca. Cell suspensions from Hibiscus and Lavatera were transformed with A. tumefaciens harboring plasmids containing selectable genes coding for kanamycin and hygromycin resistance, respectively. We provided evidence that H. rosa-sinensis and L. thuringiaca were transformed by strong selection of transformed calluses in medium containing antibiotics, by GUS activity determination in protein extracts and by molecular confirmation of chromosomal integration and expression of the selectable genes. Protoplasts isolated from a kanamycinresistant Hibiscus callus and from a hygromycin-resistant Lavatera callus were fused and selected in medium containing both antibiotics. We determined unambiguously that the regenerated double-antibiotic resistant clones obtained are indeed somatic hybrids through analysis of acid phosphatase zymograms and nuclear DNA content. Plant regeneration through somatic embryogenesis was accomplished from both isolated protoplasts and transgenic calluses of L. thuringiaca. However, regeneration from the double-antibiotic resistant fusant calluses was unsuccessful. Analysis of the somatic hybrids at the callus level showed that chilling and freezing tolerance are governed by independent genetic components. The somatic hybrids displayed significant improvement for chilling tolerance at conditions lethal to H. rosa-sinensis, although frost tolerance was not expressed.     

One-Leaf Cuttings as a Model to Study Second Growth in the Potato (Solanum tuberosum) Plant

Second growth is an important physiological disorder of thepotato (Solanum tuberosum L.) plant. A model system to studysecond growth was developed using one-leaf cuttings. Photoperiod,temperature, decapitation and leaf removal treatments were carriedout on the plants from which the cuttings were taken and onthe cuttings themselves. Tuberized, one-leaf cuttings takenfrom moderately-induced plants and exposed to 35 °C afterleaf removal showed 95% second growth within 10 d after treatmentinitiation. Conditions that promoted second growth also reducedstarch and dry-matter content, even in tubers that did not developsecond growth. Cuttings, second growth, potato, Solanum tuberosum L, cv, Bintje, Solanum tuberosum L. cv., Désirée, Solanum tuberosum L. cv., Russet Burbank, tuberization, starch content, dry-matter, heat, photoperiod, decapitation, leaf removal     

Stimulation of ethylene or ethane production by malformin

Malformin stimulated ethylene production of Phaseolus vulgarisL. seedlings and explants. However, when malformin was vacuum-infiltratedinto apical bud sections, the production of ethylene was inhibited,ethane production was stimulated and the sections became softand pliable; in pure oxygen, ethylene production was- stimulatedand the sections remained firm. Prolonged stimulation of ethaneproduction by malformin-treated sections required oxygen. Indoleaceticacid (IAA) had no effect on the stimulation of ethane productionby malformin-infiltrated tissues; malformin and IAA stimulatedethylene production synergistically at the same time that malformininducedethane production had increased markedly. ¹This work was supported by grant GB-7158 from the NationalScience Foundation. ²Journal Paper No. 3560 of the Purdue Agricultural ExperimentStation. (Received July 23, 1969; )