Control of relative humidity and root-zone water content for acclimation of in vitro-propagated M9 apple rootstock plantlets

  • Sang-Min Ko
  • Jin-Hui Lee
  • Myung-Min Oh
Research Report Cultivation Physiology


The present study aimed to evaluate the effects of controlling the relative humidity (RH) and water content of the root-zone on the survival rate and growth of in vitro-propagated virus-free M9 apple plantlets in closed-type plant production systems. In the first experiment, three RH regimes were applied to pre-acclimated (PA) and non-PA apple plantlets for 6 weeks after transplantation. In the second experiment, the apple plantlets were transplanted into several growth media, including a mixture of peat moss and perlite (PP), rock wool (RW), and urethane sponge (SP), and in a deep flow technique (DFT) system for controlled root zone water content under controlled RH. In the first experiment, pre-acclimation improved the survival rate by preventing the loss of leaf water potential and promoting antioxidant capacity during the acclimation period. However, no clear difference was found among the three RH regimes. The antioxidant capacity was increased at 2 weeks after transplantation, followed by root initiation. The leaf water potential, which decreased continuously until 3 weeks after transplanting, tended to remain constant after root initiation. These results suggested that pre-acclimation is necessary for the survival of in vitro-propagated apple plantlets, and that the underdeveloped roots of apple plantlets have restricted water absorption under controlled RH. In the second experiment, the survival rate of plantlets grown in PP at 6 weeks after transplantation was only 70% accompanied by an increase in antioxidant capacity, whereas the survival rates of plantlets grown in RW, SP, DFT, and DFT-PP (replanted to PP from DFT 4 weeks after transplantation) were 98, 96, 93.8, and 93.8%, respectively. Most of the growth parameters of the plantlets grown in DFT were the highest among the growth media at 6 weeks after transplantation. The results of the second experiment implied that the application of DFT for in vitro-propagated apple plantlets can reduce the problems caused by poor root architecture during acclimation.


Antioxidant capacity Closed-type plant production systems Deep flow technique Leaf water potential Pre-acclimation Virus-free plants 



This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (315003051SB020).


  1. Amiri EM, Elahinia A (2011) Optimization of medium composition for apple rootstocks. Afr J Biotechnol 10:3594–3601Google Scholar
  2. Bhatti S, Jha G (2010) Current trends and future prospects of biotechnological interventions through tissue culture in apple. Plant Cell Rep 29:1215–1225CrossRefPubMedGoogle Scholar
  3. Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–194CrossRefPubMedPubMedCentralGoogle Scholar
  4. Carvalho LC, Osório ML, Chaves MM, Amâncio S (2001) Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex-vitro acclimation. Plant Cell Tissue Organ Cult 67:271–280CrossRefGoogle Scholar
  5. Cembali T, Folwell RJ, Wandschneider P, Eastwell KC, Howell WE (2003) Economic implications of a virus prevention program in deciduous tree fruits in the US. Crop Prot 22:1149–1156CrossRefGoogle Scholar
  6. Chatenet M, Delage C, Ripolles M, Irey M, Lockhart BEL, Rott P (2001) Detection of Sugarcane yellow leaf virus in quarantine and production of virus-free sugarcane by apical meristem culture. Plant Dis 85:1177–1180CrossRefGoogle Scholar
  7. Dewir YH, Chakrabarty D, Ali MB, Hahn EJ, Paek KY (2005) Effects of hydroponic solution EC, substrates, PPF and nutrient scheduling on growth and photosynthetic competence during acclimation of micropropagated Spathiphyllum plantlets. Plant Growth Regul 46:241–251CrossRefGoogle Scholar
  8. Díaz-Pérez JC, Sutter EG, Shackel KA (1995) Acclimation and subsequent gas exchange, water relations, survival and growth of microcultured apple plantlets after transplanting them in soil. Physiol Plant 95:225–232CrossRefGoogle Scholar
  9. Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fila G, Ghashghaie J, Hoarau J, Cornic G (1998) Phothosynthesis, leaf conductance and water relation of in vitro cultured grapevine rootstock in relation to acclimation. Physiol Plant 102:411–418CrossRefGoogle Scholar
  11. Geng F, Moran R, Day M, Halteman W, Zhang D (2015) In-vitro shoot proliferation of apple rootstocks ‘B.9’, ‘G.30’, and ‘G.41’ grown under red and blue light. Hortic Sci 50:430–433Google Scholar
  12. Grout BWW, Aston MT (1977) Transplanting of cauliflower plants regenerated from meristem culture. I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration. Hortic Res 17:1–7Google Scholar
  13. Hayashi M, Nakayama M, Kozai T (1988) An application of the acclimation unit for growth of carnation explants, and for rooting and acclimation of the plantlets. Acta Hortic 230:189–194CrossRefGoogle Scholar
  14. Hazarika BN (2003) Acclimation of tissue-cultured plants. Curr Sci 85:1705–1712Google Scholar
  15. Hazarika BN (2006) Morpho-physiological disorders in in vitro culture of plants. Sci Hortic 108:105–120CrossRefGoogle Scholar
  16. Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Stn 347:4–32Google Scholar
  17. Hu G, Dong Y, Zhang Z, Fan X, Ren F, Zhou J (2015) Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell Tissue Organ Cult 121:135–443Google Scholar
  18. Huylenbroeck JMV, Piqueras A, Debergh PC (1998) Photosynthesis and carbon metabolism in leaves formed prior and during ex-vitro acclimation of micropropagated plants. Plant Sci 134:21–30CrossRefGoogle Scholar
  19. Huylenbroeck JMV, Piqueras A, Debergh PC (2000) The evolution of photosynthetic capacity and the antioxidant enzymatic system during acclimation of micropropagated Calathea plants. Plant Sci 155:50–66Google Scholar
  20. James DJ, Thurbon IJ (1979) Rapid in vitro rooting of the apple rootstock M.9. J Hortic Sci 54:309–311CrossRefGoogle Scholar
  21. Jeon MW, Ali MB, Hahn EJ, Paek KY (2006) Photosynthetic pigments, morphology and leaf gas exchange during ex-vitro acclimation of micropropagated CAM Doritaenopsis plantlets under relative humidity and air temperature. Environ Exp Bot 55:183–194CrossRefGoogle Scholar
  22. Kadleček P, Tichá I, Haisel D, Čapková V, Schäfer C (2001) Importance of in vitro pretreatment for ex-vitro acclimation and growth. Plant Sci 161:695–701CrossRefGoogle Scholar
  23. Kepenek K, Karoğlu Z (2011) The effects of paclobutrazol and daminozide on in vitro micropropagation of some apple (Malus domestica) cultivars and M9-rootstock. Afr J Biotechnol 10:4851–4859Google Scholar
  24. Kim JH, Kim CC, Ko KC, Kim KR, Lee JC (1998) The particular of pomology, 4th edn. Hyangmunsa, Seoul, pp 41–45Google Scholar
  25. Kozai T (1991) Acclimation of micropropagated plant. In: Bajaj YPS (ed) High-tech and micropropagation I. Springer, Berlin, pp 127–141CrossRefGoogle Scholar
  26. Lamhamedi MS, Chamberland H, Tremblay FM (2003) Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimation. Physiol Plant 118:554–561CrossRefGoogle Scholar
  27. Lim YJ, Kwak TY, Kim DI, Kim MJ, Kim JK, Kim TC, No KM, Park YM, Park JK et al (2015) Apple luxury strategy. Se-Myeong Munhwasa, Seoul, pp 119–125Google Scholar
  28. Meijneke CAR, van Oosten HJ, Peerboom H (1973) Growth, yield, and fruit quality of virus-infected and virus-free golden delicious apple trees. Acta Hortic 44:209–212Google Scholar
  29. Miller NJ, Rice-Evans CA (1996) Spectrophotometric determination of antioxidant activity. Redox Rep 2:161–171CrossRefPubMedGoogle Scholar
  30. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410CrossRefPubMedGoogle Scholar
  31. Nhut DT, Huong NTD, Khiem DV (2004) Direct microtuber formation and enhanced growth in the acclimation of in vitro plantlets of taro (Colocasia esculenta spp.) using hydroponics. Sci Hortic 101:207–212CrossRefGoogle Scholar
  32. Oh MM, Trick HN, Rajashekar CB (2009) Secondary metabolism and antioxidants are involved in environmental adaptation and stress tolerance in lettuce. J Plant Physiol 166:180–191CrossRefPubMedGoogle Scholar
  33. O’Toole JC, Cruz RT (1980) Response of leaf water potential, stomatal resistance, and leaf rolling to water stress. Plant Physiol 65:428–432CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pospíšilová J, Tichá I, Kadleček P, Haisel D, Plzáková Š (1999) Acclimation of micropropagated plants to ex-vitro conditions. Biol Plant 42:481–497CrossRefGoogle Scholar
  35. Pospíšilová J, Synková H, Haisel D, Semorádová Š (2007) Acclimation of plantlets to ex-vitro condition: effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review). Acta Hortic 748:29–38CrossRefGoogle Scholar
  36. Saliendra NZ, Sperry JS, Comstock JP (1995) Influence of leaf water status on stomatal response to humidity, hydraulic conductance, and soil drought in Betula occidentalis. Planta 196:357–366CrossRefGoogle Scholar
  37. Sgherri CLM, Navari-Izzo F (1995) Sunflower seedlings subjected to increasing water deficit stress: oxidative stress and defence mechanisms. Physiol Plant 93:25–30CrossRefGoogle Scholar
  38. Shackel KA, Novello V, Sutter EG (1990) Stomatal function and cuticular conductance in whole tissue-cultured apple shoots. J Am Soc Hortic Sci 115:468–472Google Scholar
  39. Theiler-Hedtkich R, Baumann G (1989) Elimination of apple mosaic virus and raspberry bushy dwarf virus from infected red raspberry (Rubus idaeus L.) by tissue culture. J Phytopathol 127:193–199CrossRefGoogle Scholar
  40. Walkey DGA, Webb MJW (1968) Virus in plant apical meristems. J Gen Virol 3:311–313CrossRefGoogle Scholar
  41. Zapata EV, Morales GS, Lauzardo ANH, Bonfil BM, Tapia GT, Sánchez ADJ, Valle MVD, Aparicio AJ (2003) In-vitro regeneration and acclimation of plants of Turmeric (Curcuma longa L.) in a hydroponic system. Biotechnol Appl 20:25–31Google Scholar
  42. Zimmerman RH (1984) Rooting apple cultivars in vitro: interactions among light, temperature, phloroglucinol and auxin. Plant Cell Tissue Organ Cult 3:301–311CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Animal, Horticultural and Food SciencesChungbuk National UniversityCheongjuKorea
  2. 2.Brain Korea 21 Center for Bio-Resource DevelopmentChungbuk National UniversityCheongjuKorea

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