Stomatal Morphology and Desiccation Response of Persian Walnut Tissue Culture Plantlets Influenced by the Gelling Agent of In Vitro Culture Medium

Document Type : Research Article


1 MSc student, Department of Horticulture, College of Aburaihan, University of Tehran, Tehran, Iran

2 Assistant Professor, Department of Horticulture, College of Aburaihan, University of Tehran, Tehran, Iran

3 Professor, Department of Horticulture, College of Aburaihan, University of Tehran, Tehran, Iran


There are diverse types of gelling agents that are used in media cultures. Agar and Gelrite are among the gelling agents used in DKW culture medium, as the common culture medium for the micropropagation of walnut plants. The effects of these gelling agents have not been investigated on the successful production of in vitro explants and the response of the explants to ex vitro evaporative demand is overlooked so far. Stomata and water relations of tissue culture medium determine the successful production of in vitro plants, therefore this experiment was conducted to investigate the effect of two types of gelling agents (Agar and Gelrite) on the stomatal characteristics, transpiration rate (E), and desiccation responses of in vitro walnut explants. Stomatal morphology, transpiration rate, RWC, and some morpho-physiological traits such as shoot length, chlorophyll content, osmotic potential (ψs), proline, and glycine betaine content were evaluated in micropropagated walnut explants cultured on Agar or Gelrite. Analysis of results indicated no considerable changes in the morpho-physiological characteristics of explants grown in DKW medium containing Agar or Gelrite gelling agents. Compared with the medium containing Agar, adding Gelrite to the DKW medium caused a decrease in E and an increase in relative leaf water content (RWC) of the walnut explant's leaves during desiccation. Gelrite induced generation of more closed stomata leading to a reduction in E and increase in RWC during desiccation. This resulted in improvement of walnut plantlet's capacity to conserve their water content and as the consequence promoted ability to prevent ex vitro wilting.


Aguilar M, Espadas F, Coello J, Maust B, Trejo C, Robert M, Santamaria J (2000) The role of abscisic acid in controlling leaf water loss, survival and growth of micropropagated Tagetes erecta plants when transferred directly to the field. Journal of Experimental Botany. 51, 1861-1866.
Aliniaeifard S, Asayesh Z.M, Driver J, Vahdati K (2020) Stomatal features and desiccation responses of Persian walnut leaf as caused by in vitro stimuli aimed at stomatal closure. Trees. 34,1219–1232.
Aliniaeifard S, Malcolm Matamoros P, van Meeteren, U (2014) Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Physiologia Plantarum. 152, 688-699.
Aliniaeifard S, van Meeteren U (2013) Can prolonged exposure to low VPD disturb the ABA signalling in stomatal guard cells? Journal of Experimental Botany. 64,3551-3566.
Aliniaeifard S, van Meeteren U (2014) Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning. Journal of Experimental Botany. 65, 6529-6542.
Aliniaeifard S, van Meeteren U (2016) Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humidity. Scientia Horticulturae. 205, 84-89.
Apostolo N.M, Llorente B.E (2000) Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (Link) Schn. In Vitro Cellular & Developmental Biology-Plant. 36, 243-249.
Asayesh M.Z, Vahdati K, Aliniaeifard, S (2017a) Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants. Scientia Horticulturae. 224,1-7.
Asayesh Z.M, Vahdati K, Aliniaeifard S, Askari, N (2017b) Enhancement of ex vitro acclimation of walnut plantlets through modification of stomatal characteristics in vitro. Scientia Horticulturae. 220, 114-121. scienta.2017.03.045
Bates L, Waldren R, Teare, I (1973) Rapid determination of free proline for water-stress studies. Plant and soil. 39, 205-207.
Cha-um S, Ulziibat B, Kirdmanee C (2010) Effects of temperature and relative humidity during in vitro acclimatization, on physiological changes and growth characters of'Phalaenopsis' adapted to in vivo. Australian Journal of Crop Science 4:750.
Debergh P (1983) Effects of agar brand and concentration on the tissue culture medium. Physiologia plantarum. 59, 270-276.
Driver J.A, Kuniyuki A.H (1984) In vitro propagation of Paradox walnut rootstock. HortScience. 19, 507-509.
Eshghi Khas M, Abbasifar A, ValizadehKaji B (2020) Optimization of in vitro Propagation of Purple Passion Fruit (Passiflora edulis), an Important Medicinal and Ornamental Plant. International Journal of Horticultural Science and Technology. 7(3), 305-314. doi: 10.22059/ijhst.2020.297194.342
Fanourakis D, Aliniaeifard S, Sellin A, Giday H, Korner O, Nejad AR, Delis C, Bouranis D, Koubouris G, Kambourakis E (2020) Stomatal behavior following mid-or long-term exposure to high relative air humidity: A review. Plant Physiology and Biochemistry.153, 92–105.
Fanourakis D, Heuvelink E, Carvalho S.M (2013) A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidity. Journal of Plant Physiology. 170, 890-898.
Farsi M, Fatahi Moghadam M, Zamani Z, Hassani D (2018) Effects of Scion Cultivar, Rootstock Age and Hormonal Treatment on Minigrafting of Persian Walnut. International Journal of Horticultural Science and Technology. 5(2), 185-197. doi: 10.22059/ijhst.2018.255460.233
Franck T, Kevers C, Gaspar T, Dommes J, Deby C, Greimers R, Serteyn D, Deby-Dupont G (2004) Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response. Plant Physiology and Biochemistry. 42, 519-527.
Gandev S (2014) State-of-the-art and Problems of Walnut Propagation Methods. Agroznanje. 15, 95-110.
Grieve C, Grattan S (1983) Rapid assay for determination of water soluble quaternary ammonium compounds. Plant and soil. 70, 303-307.
Grouh M.S.H, Vahdati K, Lotfi M, Hassani D, Biranvand N.P (2011) Production of haploids in Persian walnut through parthenogenesis induced by gamma-irradiated pollen. Journal of the American Society for Horticultural Science. 136, 198-204.
Hassankhah A, Vahdati K, Lotfi M, Mirmasoumi M, Preece J, Assareh M (2014) Effects of Ventilation and Sucrose Concentrations on the Growth and Plantlet Anatomy of Micropropagated Persian Walnut Plants. International Journal of Horticultural Science and Technology, 1(2), 111-120. doi: 10.22059/ijhst.2014.52781
Hazarika B (2006) Morpho-physiological disorders in in vitro culture of plants. Scientia horticulturae. 108, 105-120.
Ivanova M, Van Staden J (2011) Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell, Tissue and Organ Culture (PCTOC). 104, 13-21.
Kacar Y, Bicen B, Varol I, Mendi Y, Serce S, Cetiner S (2010) Gelling agents and culture vessels affect in vitro multiplication of banana plantlets. Genetics and Molecular Research. 9, 416-424.
Klimaszewska K, Cyr D, Sutton B (2000) Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L. In Vitro Cellular & Developmental Biology-Plant. 36, 279-286.
Koubouris G, Vasilakakis M (2006). Improvement of in vitro propagation of apricot cultivar ‘Bebecou’. Plant cell, tissue and organ culture 85, 173-180. DOI 10.1007/s11240-005-9066-y Lebedev V, Arkaev M, Dremova M, Pozdniakov I, Shestibratov K (2019) Effects of growth regulators and gelling agents on ex vitro rooting of raspberry. Plants. 8(1), 3.
Martıinez J.P, Lutts S, Schanck A, Bajji M, Kinet J.M (2004) Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L? Journal of Plant Physiology. 161,1041-1051.
Masondo N.A, Aremu A.O, Finnie J.F, Van Staden J (2015) Growth and phytochemical levels in micropropagated Eucomis autumnalis subspecies autumnalis using different gelling agents, explant source, and plant growth regulators. In Vitro Cellular & Developmental Biology-Plant. 51, 102-110.
Payghamzadeh K, Kazemitabar S.K (2011) In vitro propagation of walnut-A review. African Journal of Biotechnology. 10, 290-311.
Pospisilova J, Synkova H, Haisel D, Semoradova S (2007) Acclimation of plantlets to ex vitro conditions: Effects of air humidity, irradiance, CO2 concentration and abscisic acid (a Review). Acta Horticulturae 748(1), 29-38.
Pourkhaloee A, Khosh-Khui M (2015) Pupae are excellent explants with low microbial contamination and high regeneration frequency for micropropagation of Freesia × hybrida Bailey 'Argenta'. International Journal of Horticultural Science and Technology. 2(1), 97-109. doi: 10.22059/ijhst.2015.54268
Rezaei Nejad A, U Van Meeteren (2005) Stomatal response characteristics of Tradescantia virginiana grown at high relative airhumidity. Physiologia Plantarum. 125, 324–332.
Saadat Y, Hennerty M (2002) Factors affecting the shoot multiplication of Persian walnut (Juglans regia L.). Scientia Horticulturae. 95, 251-260.
Santamaria J, Davies W, Atkinson C (1993) Stomata of micropropagated Delphinium plants respond to ABA, CO2, light and water potential, but fail to close fully. Journal of Experimental Botany. 44, 99-107.
Seifeldin A.M, Ozzambak M.E (2016) Effect of cultivar and gelling agent on in Vitro multiplication of Gerbera (Gerbera jamesonii Bolus) shoots. Journal of Agricultural Sciences. 24(1),
Shim S-W, Hahn E-J, Paek KY (2003). In vitro and ex vitro growth of grapevine rootstock5BB'as influenced by number of air exchanges and the presence or absence of sucrose in culture media. Plant Cell, Tissue and Organ Culture 75,57-62.
Slavik B (1974) Methods of studying plant water relations . London: Chapman and Hall. 121–156.
Thapa R, Thapa P, Ahamad K, Vahdati K (2021) Effect of grafting methods and dates on the graft take rate of Persian Walnut in open field condition. International Journal of Horticultural Science and Technology. 8(2), 133-147. doi: 10.22059/ijhst.2020.311553.401
Te-chato S, Petsut P, Nuchum P (2005) Effect of gelling agents on shoot growth and multiple shoot formation of mangosteen. Songklanakarin Journal of Science and Technology. 27, 637-643.
Tsay H.S, Lee C.Y, Agrawal DC, Basker S (2006) Influence of ventilation closure, gelling agent and explant type on shoot bud proliferation and hyperhydricity in Scrophularia yoshimurae—a medicinal plant. In Vitro Cellular & Developmental Biology-Plant. 42, 445-449.
Vahdati K, Asayesh Z.M, Aliniaeifard S, Leslie C (2017) Improvement of ex vitro desiccation through elevation of CO2 concentration in the atmosphere of culture vessels during in vitro growth. HortScience. 52, 1006-1012. doi: 10.21273/HORTSCI11922-17
Vahdati K, Jariteh M, Niknam V, Mirmasoumi M and Ebrahimzadeh H (2006) Somatic embryogenesis and embryo maturation in Persian walnut. Acta Horticulturae. 705, 199-205.
Vahdati K, Leslie C, Zamani Z, McGranahan G (2004) Rooting and acclimatization of in vitro-grown shoots from mature trees of three Persian walnut cultivars. HortScience. 39, 324-327.
Van den Dries N, Gianni S, Czerednik A, Krens F.A, de Klerk G.J.M (2013). Flooding of the apoplast is a key factor in the development of hyperhydricity. Journal of experimental botany. 64, 5221-5230.
Ziv M (1991) Quality of micropropagated plants—vitrification. In Vitro Cellular & Developmental Biology-Plant. 27, 64-69.