Ex vitro rooting using a mini growth chamber increases root induction and accelerates acclimatization of Kopyor coconut (Cocos nucifera L.) embryo culture-derived seedlings

  • Sisunandar
  • Alkhikmah
  • Arief Husin
  • Teguh Julianto
  • Alice Yuniaty
  • Alain Rival
  • Steve W. Adkins
Embryo Culture


Ex vitro rooting using a mini growth chamber to maintain relative humidity has been used to mass produce true-to-type Kopyor coconut (Cocos nucifera L.) seedlings through embryo culture. This new process was found to (1) improve the proportion of seedlings successfully transferred to soil (from 40 to 90% for seedlings with roots); (2) achieve this step in the shortest time possible (a reduction from 10 to 4 mo in in vitro culture); (3) improve root formation using indolebutyric acid (IBA; an improvement in the number of primary roots from 2 to 5); and (4) improve the vigor of seedlings ex vitro (improvements of fresh weight, shoot length, number of opened leaves, leaf thickness, amount of epicuticular wax, and stomatal density). The best ex vitro rooting and rapid acclimatization protocol was obtained when 4-mo-old seedlings with two opened leaves were kept in the mini growth chamber for 3 mo before being transferred into soil, and when the mini growth chamber was flooded with a quarter strength hybrid embryo culture (HEC) medium with 1 μM IBA but depleted of vitamins and sugar. This protocol was efficient in delivering high-value Kopyor seedlings to the field (90% success rate), with a decreased risk of contamination and lower labor cost. The improved process was found applicable to both tall and dwarf Kopyor and other coconut types.


Embryo culture Coconut mutant Morphological analysis Indolebutyric acid 

Supplementary material

11627_2018_9897_MOESM1_ESM.docx (13 kb)
Table S1 (DOCX 13 kb)


  1. Benmahioul B, Dorion N, Kaid-Harche M, Daguin F (2012) Micropropagation and ex vitro rooting of pistachio (Pistacia vera L.) Plant Cell Tissue Organ Cult 108:353–358CrossRefGoogle Scholar
  2. Cati M, Gennari F, Marino G (2014) Effect of culture jar seal on in vitro rooting and subsequent acclimatization of three Italian apricot varieties. Sci Hortic 168:120–123CrossRefGoogle Scholar
  3. Clapa D, Fira A, Joshee N (2013) An efficient ex vitro rooting and acclimatization method for horticultural plants using float hydroculture. Hortscience 48(9):1159–1167Google Scholar
  4. Cuenca B, Sanchez C, Aldrey A, Bogo B, Blanco B, Correa B, Vidal N (2017) Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system. Plant Cell Tissue Organ Cult 131:307–320CrossRefGoogle Scholar
  5. Engelmann F, Malaurie B, N’Nan O (2011) In vitro culture of coconut (C. nucifera L.) zygotic embryos. In: Thorpe TA, Yeung EC (eds) Plant embryo culture: methods and protocols, Methods in molecular biology. Springer, Berlin, pp 63–72CrossRefGoogle Scholar
  6. Hazarika BN (2003) Acclimatization of tissue cultured plants. Curr Sci 85(12):1704–1712Google Scholar
  7. Ilczuk A, Jacygrad E (2016) In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers. In vitro Cell Dev Biol Plant 52:379–390CrossRefPubMedPubMedCentralGoogle Scholar
  8. Johansen DA (1940) Plant microtechnique. McGraw-Hill, New YorkGoogle Scholar
  9. Karunaratne S, Kurukulaarachchi C, Gamage C (1985) A report on the culture of embryos of dwarf coconut, C. nucifera L. var nana in vitro. Cocos 3:1–8CrossRefGoogle Scholar
  10. Larraburu EE, Apostolo NM, Llorente BE (2010) Anatomy and morphology of photinia (Photinia X fraseri Dress) in vitro plants inoculated with rhizobacteria. Trees 24:635–642CrossRefGoogle Scholar
  11. Magdalita PM, Damasco OP, Adkins SW (2010) Effect of medium replenishment and acclimatization technique on growth and survival of embryo cultured coconut seedlings. Philipp Sci Lett 3:1–9Google Scholar
  12. Mukhopadhyay M, Mondal TK, Chand PK (2016) Biotechnological advances in tea (Camellia sinensis [L.] O. Kuntze): a review. Plant Cell Rep 35:255–287CrossRefPubMedGoogle Scholar
  13. Nguyen QT, Bandupriya HDD, Foale M, Adkins SW (2016) Biology, propagation and utilization of elite coconut varieties (makapuno and aromatics). Plant Physiol Biochem 109:579–589CrossRefPubMedGoogle Scholar
  14. Orense OD, Rillo EP, Imperial LAP, Cueto CA, Lobos AA, Areza-Ubaldo MB (2011) Rapid and cost-effective embryo culture technique for commercial production of Makapuno seedlings. Cord 27:20–41Google Scholar
  15. Patel AK, Phulwaria M, Rai MK, Gupta AK, Shekhawat S, Shekawat NS (2014) In vitro propagation and ex vitro rooting of Caralluma edulis (Edgew.) Benth. & Hook. f. : An endemic and endagered edible plant species of Thar Desert. Sci Hortic 165:175–180CrossRefGoogle Scholar
  16. Perveen S, Anis M, Aref IM (2013) Lipid peroxidation, H2O2 content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions. Biol Plant 57:417–424CrossRefGoogle Scholar
  17. Pospisilova J, Wilhelmova N, Synkova H, Catsky J, Krebs D, Ticha I, Hanackova B, Snopek J (1998) Acclimation of tobacco plantlets to ex vitro conditions as affected by application of abscisic acid. J Exp Bot 49(322):863–869CrossRefGoogle Scholar
  18. Pospisilova J, Synkova H, Haisel D, Semoradova S (2007) Acclimation of plantlets to ex vitro conditions : effect of air humidity, irradiance, CO2 concentration and absisic acid (a review). Acta Hortic 748:29–38CrossRefGoogle Scholar
  19. Preece JE, West TP (2006) Greenhouse growth and acclimatization of encapsulated Hibiscus moscheutos nodal segments. Plant Cell Tissue Organ Cult 87:127–138CrossRefGoogle Scholar
  20. Ranaweera KK, Gunasekara MTK, Eeswara JP (2013) Ex vitro rooting: a low cost micropropagation technique for Tea (Camellia sinensis (L.) O. Kuntz) hybrids. Sci Hortic 2013:8–14CrossRefGoogle Scholar
  21. Rillo EP (2004) Importing and growing embryos for the coconut genebank. In: Ikin R, Batugal P (eds) Germplasm health management for COGENT's multi-site international coconut genebank. IPGRI-APO, Serdang, Selangor DE, Malaysia, pp 62–68Google Scholar
  22. Samosir Y, Adkins SW (2014) Improving acclimatization through the photoautotropic culture of coconut (C. nucifera) seedlings: an in vitro system for the efficient exchange of germplasm. In: In Vitro Cell Dev Biol Plant, vol 31, pp 144–149Google Scholar
  23. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImangeJ : 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedPubMedCentralGoogle Scholar
  24. Sisunandar RA, Turquay P, Samosir Y, Adkins SW (2010) Cryopreservation of coconut (C. nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings. Planta 232:435–447CrossRefPubMedGoogle Scholar
  25. Steinmacher DA, Guerra MP, Saare-Surminski K, Lieberei R (2011) A temporary immersion system improves in vitro regeneration of peach palm trough secondary somatic embryogenesis. Ann Bot 108:1463–1475CrossRefPubMedPubMedCentralGoogle Scholar
  26. Talavera C, Contreras F, Espadas F, Fuentes G, Santamaria JM (2005) Cultivating in vitro coconut palms (C. nucifera) under glasshouse conditions with natural light, improves in vitro photosynthesis nursery survival and growth. Plant Cell Tissue Organ Cult 83:287–292CrossRefGoogle Scholar
  27. Wang SM, Piao XC, Park SY, Lian ML (2013) Improved micropropagation of Gypsophila paniculata with bioreactor and factors affecting ex vitro rooting in microponic system. In vitro Cell Dev Biol Plant 49:70–78CrossRefGoogle Scholar
  28. Yokota S, Karim MZ, Azad MAK, Rahman MM, Eizawa J, Saito Y, Ishiguri F, Lizuka K, S Y YN (2007) Histological observation of changes in leaf structure during successive micropropagation stages in Aralia elata and Phellodendron amurense. Plant Biotechnol 24:221–226CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.Coconut Research Center, Biology Education DepartmentThe University of Muhammadiyah PurwokertoPurwokertoIndonesia
  2. 2.Faculty of BiologyThe University of Jenderal SoedirmanPurwokertoIndonesia
  3. 3.Cirad – DGDRSJakartaIndonesia
  4. 4.School of Agriculture and Food Sciences, Center for Plant Science, Queensland Alliance for Agriculture and Food InnovationThe University of QueenslandSt LuciaAustralia

Personalised recommendations