Improvement of Ex Vitro Desiccation through Elevation of CO2 Concentration in the Atmosphere of Culture Vessels during In Vitro Growth

in HortScience

Acclimatization of in vitro plantlets is one of the key steps in successful tissue culture propagation. Gaseous atmosphere during in vitro culture can influence the rate of ex vitro acclimation of the plantlets produced. In the current study, effects of elevated CO2 concentration on the leaf water loss dynamic responses of in vitro–produced walnut leaves during ex vitro desiccation were investigated. Elevated CO2 concentration in the headspace of culture vessels caused a considerable decrease in stomatal aperture. Although the traits related to stomatal size were not influenced by CO2 elevation, the number of small stomata was increased, and the number of large stomata was decreased at elevated CO2 concentration. Higher CO2 concentration resulted in a lower transpiration rate and a higher relative water content (RWC) during ex vitro desiccation. This improvement was due to decreased stomatal aperture during the first phase of water loss. Osmotic potential (ψs) was decreased under an elevated CO2 concentration, but no influence was observed on the concentration of compatible solutes. In conclusion, increasing the CO2 concentration of culture vessel headspace can be an efficient tool for improving acclimation of in vitro–grown walnuts without negative effects on plantlet growth.

Contributor Notes

We would like to thank Iran National Science Foundation (INSF), Center of Excellence of Walnut Improvement and Technology of Iran, and University of Tehran for their supports.

Corresponding authors. E-mail: kvahdati@ut.ac.ir or aliniaeifard@ut.ac.ir.

Article Sections

Article Figures

  • View in gallery

    Work stages for CO2 elevation in the atmosphere of the vessel headspaces. (A) Microshoots were cultured in the culture vessels. (B) Vials containing 3 mL of mixed NaHCO3 and Na2CO3 solution (3 M) in the ratio of 73/27 (v/v), respectively, were placed in the vessels. (C) The samples were incubated and grown at 25 ± 2 °C under 16/8 h light/dark cycles for 30 d.

  • View in gallery

    Stomata on abaxial surface of in vitro–grown walnut (cv. Chandler) leaves developed under ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars (A) without CO2-releasing solution as control plantlets or (B) with CO2-releasing solution in the headspace of culture vessels. The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

  • View in gallery

    Variation in stomatal size on leaves of in vitro–grown walnut (cv. Chandler) microshoots exposed to ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

  • View in gallery

    (A) Leaflet transpiration rate, (B) relative water content and (C) transpiration rate as a function of RWC during 1.5 h of ex vitro desiccation of leaves excised from tissue cultured walnut microshoots (cv. Chandler). In vitro–formed leaves were grown under either ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, the leaves of plantlets were detached and placed with their abaxial side up on a balance in an environment with 50% relative humidity, 21 °C, and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

Article References

  • AliniaeifardS.HajilouJ.TabatabaeiS.J.Sifi-KalhorM.2016Effects of ascorbic acid and reduced glutathione on the alleviation of salinity stress in olive plantsIntl. J. Fruit Sci.16395409

    • Search Google Scholar
    • Export Citation
  • AliniaeifardS.Malcolm MatamorosP.van MeeterenU.2014Stomatal malfunctioning under low VPD conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?Physiol. Plant.152688699

    • Search Google Scholar
    • Export Citation
  • AliniaeifardS.van MeeterenU.2013Can prolonged exposure to low VPD disturb the ABA signalling in stomatal guard cells?J. Exp. Bot.6435513566

    • Search Google Scholar
    • Export Citation
  • AliniaeifardS.van MeeterenU.2014Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognise the mechanism of disturbed stomatal functioningJ. Exp. Bot.6565296542

    • Search Google Scholar
    • Export Citation
  • AliniaeifardS.van MeeterenU.2016Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humiditySci. Hort.2058489

    • Search Google Scholar
    • Export Citation
  • AllawayW.G.MansfieldT.A.1967Stomatal responses to changes in carbon dioxide concentration in leaves treated with 3-(4-chlorophenyl)-1, 1-dimethylureaNew Phytol.6615763

    • Search Google Scholar
    • Export Citation
  • ArveL.E.TerfaM.T.GisleordH.R.OlsenJ.E.TorreS.2013High relative air humidity and continuous light reduce stomata functionality by affecting the ABA regulation in rose leavesPlant Cell Environ.36382392

    • Search Google Scholar
    • Export Citation
  • AvilesF.RiosD.GonzalezR.Sanchez-OlateM.2010Effect of culture medium in callogenesis from adult walnut leaves (Juglans regia L.)Chil. J. Agr. Res.69460467

    • Search Google Scholar
    • Export Citation
  • BadrA.AngersP.DesjardinsY.2011Metabolic profiling of photoautotrophic and photomixotrophic potato plantlets (Solanum tuberosum) provides new insights into acclimatizationPlant Cell Tissue Organ Cult.1071324

    • Search Google Scholar
    • Export Citation
  • BatesL.WaldrenR.TeareI.1973Rapid determination of free proline for water-stress studiesPlant Soil39205207

  • BrainerdK.FuchigamiL.1982Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA, and CO2J. Exp. Bot.33388392

    • Search Google Scholar
    • Export Citation
  • Buddendorf-JoostenJ.WolteringE.1994Components of the gaseous environment and their effects on plant growth and development in vitroJ. Plant Growth Regul.15116

    • Search Google Scholar
    • Export Citation
  • CarvalhoL.SantosP.AmancioS.2002Effect of light intensity and CO2 concentration on growth and the acquisition of in vivo characteristics during acclimatization of grapevine regenerated in vitroVitis41116

    • Search Google Scholar
    • Export Citation
  • Cha-umS.ChanseetisC.ChintakovidW.PichakumA.SupaibulwatanaK.2011Promoting root induction and growth of in vitro macadamia (Macadamia tetraphylla L. ‘Keaau’) plantlets using CO2-enriched photoautotrophic conditionsPlant Cell Tissue Organ Cult.106435444

    • Search Google Scholar
    • Export Citation
  • Cha-umS.MosaleeyanonK.KirdmaneeC.SupaibulwatanaK.2003A more efficient transplanting system for Thai neem (Azadirachta siamensis Val.) by reducing relative humiditySci. Asia29189196

    • Search Google Scholar
    • Export Citation
  • Cha-umS.UlziibatB.KirdmaneeC.2010Effects of temperature and relative humidity during in vitro acclimatization, on physiological changes and growth characters of Phalaenopsis adapted to in vivoAustral. J. Crop. Sci.4750756

    • Search Google Scholar
    • Export Citation
  • Chaari-RkhisA.MaalejM.DriraN.StandardiA.2011Micropropagation of olive tree (Olea europaea L.) cv. ‘Oueslati’Turk. J. Agr. For.35403412

    • Search Google Scholar
    • Export Citation
  • CraneJ.HughesH.1990Medium overlays for improved hardening of micropropagated potatoesHortScience25794795

  • CuiY.Y.HahnE.J.KozaiT.PaekK.Y.2000Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitroPlant Cell Tissue Organ Cult.62219226

    • Search Google Scholar
    • Export Citation
  • de Paiva NetoV.B.OtoniW.C.2003Carbon sources and their osmotic potential in plant tissue culture: Does it matter?Sci. Hort.97193202

  • DesjardinsY.GosselinA.YelleS.1987Acclimatization of ex vitro strawberry plantlets in CO2 enriched environments and supplementary lightingJ. Amer. Soc. Hort. Sci.112846851

    • Search Google Scholar
    • Export Citation
  • DonnellyD.J.VidaverW.E.1984Pigment content and gas exchange of red raspberry in vitro and ex vitroJ. Amer. Soc. Hort. Sci.109177181

  • DriverJ.A.KuniyukiA.H.1984In vitro propagation of Paradox walnut rootstockHort. Sci.19507509

  • DuringH.StollM.1996Stomatal patchiness of grapevine leaves. I. Estimation of non-uniform stomatal apertures by a new infiltration techniqueVitis356568

    • Search Google Scholar
    • Export Citation
  • FanourakisD.CarvalhoS.M.AlmeidaD.P.HeuvelinkE.2011Avoiding high relative air humidity during critical stages of leaf ontogeny is decisive for stomatal functioningPhysiol. Plant.142274286

    • Search Google Scholar
    • Export Citation
  • FanourakisD.HeuvelinkE.CarvalhoS.M.2013A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidityJ. Plant Physiol.170890898

    • Search Google Scholar
    • Export Citation
  • FuchigamiL.ChengT.SoeldnerA.1981Abaxial transpiration and water loss in aseptically cultured plumJ. Amer. Soc. Hort. Sci.106519522

  • FujiwaraK.KozaiT.WatanabeI.1987Measurements of carbon dioxide gas concentration in closed vessels containing tissue cultured plantlets and estimates of net photosynthetic rates of the plantletsJ. Agr. Met.432130

    • Search Google Scholar
    • Export Citation
  • GalzyR.CompanD.1992Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro. Plant Cell TissueOrgan Cult.31239244

    • Search Google Scholar
    • Export Citation
  • GhashghaieJ.BrenckmannF.SaugierB.1992Water relations and growth of rose plants cultured in vitro under various relative humidities. Plant Cell TissueOrgan Cult.305157

    • Search Google Scholar
    • Export Citation
  • GrieveC.GrattanS.1983Rapid assay for determination of water soluble quaternary ammonium compoundsPlant Soil70303307

  • GroutB.AstonM.1977Transplanting of cauliflower plants regenerated from meristem culture. I. Water loss and water transfer related to changes in leaf wax and to xylem regenerationHort. Res.1717

    • Search Google Scholar
    • Export Citation
  • HazarikaB.N.2006Morpho-physiological disorders in in vitro culture of plantsSci. Hort.108105120

  • HazarikaB.N.ParthasarathyV.A.NagarajuV.2004Influence of in vitro preconditioning of Citrus sp. microshoots with sucrose on their ex vitro establishmentIndian J. Hort.612931

    • Search Google Scholar
    • Export Citation
  • HetheringtonA.M.WoodwardF.I.2003The role of stomata in sensing and driving environmental changeNature424901908

  • JoshiN.DaveA.VyasS.PurohitS.2009Growth and shoot proliferation in Chlorophytum borivilianum Sant. et Fernand. in vitro under different carbon dioxide environmentIndian J. Biotechnol.8323327

    • Search Google Scholar
    • Export Citation
  • KilbB.WietoskaH.GoddeD.1996Changes in the expression of photosynthetic genes precede loss of photosynthetic activities and chlorophyll when glucose is supplied to mature spinach leavesPlant Sci.115225235

    • Search Google Scholar
    • Export Citation
  • KozaiT.1991Photoautotrophic micropropagationIn Vitro Cell. Dev. Biol. Plant274751

  • KozaiT.2010Photoautotrophic micropropagation environmental control for promoting photosynthesisProp. Ornam. Plants10188204

  • KozaiT.KubotaC.JeongB.R.1997Environmental control for the large-scale production of plants through in vitro techniquesPlant Cell Tiss. Org. Cult.514956

    • Search Google Scholar
    • Export Citation
  • KubotaC.2002Photoautotrophic micropropagation: Importance of controlled environment in plant tissue cultureProc. Intl. Plant Prop. Soc.52906913

    • Search Google Scholar
    • Export Citation
  • KwaS.H.WeeY.C.LimT.M.KumarP.P.1995Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmentJ. Exp. Bot.4615351542

    • Search Google Scholar
    • Export Citation
  • LamhamediM.S.ChamberlandH.TremblayF.M.2003Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimatizationPhysiol. Plant.118554561

    • Search Google Scholar
    • Export Citation
  • MajadaJ.P.FalM.A.Sanchez-TamesR.1997The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus LIn Vitro Cell. Dev. Biol. Plant336269

    • Search Google Scholar
    • Export Citation
  • MartinezJ.P.LuttsS.SchanckA.BajjiM.KinetJ.M.2004Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L?J. Plant Physiol.16110411051

    • Search Google Scholar
    • Export Citation
  • MeloniA.D.GulottaM.R.MartinezC.A.OlivaM.A.2004The effects of salt stress on growth, nitrate reduction and proline and glycinebetaine accumulation in Prosopis albaBraz. J. Plant Physiol.1613946

    • Search Google Scholar
    • Export Citation
  • MitraA.DeyS.SawarkarS.1998Photoautotrophic in vitro multiplication of the orchid Dendrobiom under CO2 enrichmentBiol. Plant.411145148

    • Search Google Scholar
    • Export Citation
  • NoguesS.AllenD.J.MorisonJ.I.BakerN.R.1998Ultraviolet-B radiation effects on water relations, leaf development, and photosynthesis in droughted pea plantsPlant Physiol.117173181

    • Search Google Scholar
    • Export Citation
  • PayghamzadehK.KazemitabarS.K.2011In vitro propagation of walnut—A reviewAfr. J. Biotechnol.10290311

  • PospisilovaJ.HaiselD.SynkovaH.CatskyJ.WilhelmovaN.PlzakovaS.ProchazkovaD.2000Photosynthetic pigments and gas exchange of in vitro grown tobacco plants during ex vitro acclimationPlant Cell Tissue Organ Cult.61125133

    • Search Google Scholar
    • Export Citation
  • PospisilovaJ.SynkovaH.HaiselD.SemoradovaS.2007Acclimation of plantlets to ex vitro conditions: Effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review)Acta Hort.7482938

    • Search Google Scholar
    • Export Citation
  • PosposilovaJ.TichaI.KadlecekP.HaiselD.PlzakovaS.1999Acclimatization of micropropagated plants to ex vitro conditionsBiol. Plant.42481497

    • Search Google Scholar
    • Export Citation
  • ReddyA.RasineniG.RaghavendraA.2010The impact of global elevated CO2 concentration on photosynthesis and plant productivityCurr. Sci.994657

    • Search Google Scholar
    • Export Citation
  • Rezaei NejadA.Van MeeterenU.2005Stomatal response characteristics of Tradescantia virginiana grown at high relative air humidityPhysiol. Plant.125324332

    • Search Google Scholar
    • Export Citation
  • Rezaei NejadA.Van MeeterenU.2007The role of abscisic acid in disturbed stomatal response characteristics of Tradescantia virginiana during growth at high relative air humidityJ. Exp. Bot.58627636

    • Search Google Scholar
    • Export Citation
  • RybczynskiJ.J.BorkowskaB.FiukA.GawronskaH.SliwinskaE.MikułaA.2007Effect of sucrose concentration on photosynthetic activity of in vitro cultures Gentiana kurroo (Royle) germlingsActa Physiol. Plant.29445453

    • Search Google Scholar
    • Export Citation
  • SaldanhaC.W.OtoniC.G.NotiniM.M.KukiK.N.da CruzA.C.F.NetoA.R.DiasL.L.C.OtoniW.C.2013A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]In Vitro Cell. Dev. Biol. Plant49433444

    • Search Google Scholar
    • Export Citation
  • SallanonH.MaziereY.1992Influence of growth room and vessel humidity on the in vitro development of rose plantsPlant Cell Tissue Organ Cult.30121125

    • Search Google Scholar
    • Export Citation
  • SantamariaJ.DaviesW.AtkinsonC.1993Stomata of micropropagated Delphinium plants respond to ABA, CO2, light and water potential, but fail to close fullyJ. Exp. Bot.4499107

    • Search Google Scholar
    • Export Citation
  • ShackelK.NovelloV.SutterE.1990Stomatal function and cuticular conductance in whole tissue-cultured apple shootsJ. Amer. Soc. Hort. Sci.115468472

    • Search Google Scholar
    • Export Citation
  • ShimS.W.HahnE.J.PaekK.Y.2003In vitro and ex vitro growth of grapevine rootstock 5BB’as influenced by number of air exchanges and the presence or absence of sucrose in culture mediaPlant Cell Tissue Organ Cult.755762

    • Search Google Scholar
    • Export Citation
  • SlavikB.1974Methods of studying plant water relations. Chapman and Hall London

  • SolarovaJ.PospisilovaJ.1997Effect of carbon dioxide enrichment during in vitro cultivation and acclimation to ex vitro conditionsBiol. Plant.392330

    • Search Google Scholar
    • Export Citation
  • SutharR.RathoreP.PurohitS.2009In vitro growth and shoot multiplication in Terminalia bellerica Roxb. under controlled carbon dioxide environmentIndian J. Exp. Biol.47204209

    • Search Google Scholar
    • Export Citation
  • SutterE.1988Stomatal and cuticular water loss from apple, cherry, and sweetgum plants after removal from in vitro cultureJ. Amer. Soc. Hort. Sci.113234238

    • Search Google Scholar
    • Export Citation
  • TanakaK.FujiwaraK.KozaiT.1992Effects of relative humidity in the culture vessel on the transpiration and net photosynthetic rates of potato plantlets in vitroActa Hort.31935964

    • Search Google Scholar
    • Export Citation
  • TaylorC.B.1996Proline and water deficit: Ups, downs, ins, and outsPlant Cell812211224

  • ThomasP.1999Relationship between tissue growth, CO2 level and tendril formation during in vitro culture of grape (Vitis vinifera L.)Vitis382529

    • Search Google Scholar
    • Export Citation
  • UehleinN.LovisoloC.SiefritzF.KaldenhoffR.2003The tobacco aquaporin NtAQP1 is a membrance CO2 pore with physiological functionsNature4256959734737

    • Search Google Scholar
    • Export Citation
  • XiaoY.NiuG.KozaiT.2011Development and application of photoautotrophic micropropagation plant systemPlant Cell Tissue Organ Cult.105149158

    • Search Google Scholar
    • Export Citation
  • ZobayedS.Afreen-ZobayedF.KubotaC.KozaiT.1999Stomatal characteristics and leaf anatomy of potato plantlets cultured in vitro under photoautotrophic and photomixotrophic conditionsIn Vitro Cell. Dev. Biol. Plant35183188

    • Search Google Scholar
    • Export Citation

Article Information

Google Scholar

Related Content

Article Metrics

All Time Past Year Past 30 Days
Abstract Views 89 89 6
Full Text Views 66 66 0
PDF Downloads 7 7 0