, Volume 31, Issue 3, pp 781–789 | Cite as

Can explant choice help resolve recalcitrance problems in in vitro propagation, a problem still acute especially for adult conifers?

  • J. M. BongaEmail author
Part of the following topical collections:
  1. Seed Biology and Micropropagation


Key message

There are tissues distributed throughout the plant that have a higher morphogenic capacity than others in the plant, and these could perhaps solve recalcitrance problems.


For many conifer species, regeneration by organogenesis or somatic embryogenesis (SE) is still difficult and is often restricted to explants taken from juvenile donors. This review is based on the premise that there are tissues in the plant that are not normally used as explant, mostly because excising them in a viable state is difficult. Nevertheless, in cases where recalcitrance is a major problem, it may be worthwhile to pay closer attention to these tissues. Recalcitrance is a general problem, and discussion of it requires examples from the general literature. However, to restrict the scope of this review, preference will be given to conifer examples whenever possible.


Asymmetric division In vitro Isolation Organogenesis Somatic embryogenesis Symplasm 



Lateral root meristem


Organizing center


Capable of forming one or more organs


Shoot apical meristem


Somatic embryogenesis


Somatic embryos


Capable of forming an embryo from a somatic cell that is similar to a zygotic embryo



I wish to thank Dr. Krystyna Klimaszewska and Dr. Patrick von Aderkas for their review of the manuscript.

Compliance with ethical standards

Conflict of interest

The author declares that he has no conflict of interest.


  1. Aitken-Christie J, Singh AP, Horgan KJ, Thorpe AT (1985) Explant developmental state and shoot formation in Pinus radiata cotyledons. Bot Gaz 146:196–203CrossRefGoogle Scholar
  2. Atta R, Laurens L, Boucheron-Dubuisson E, Guivarc’h A, Carnero E, Giraudat-Pautot V, Rech P, Chriqui D (2009) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J 57:626–644. doi: 10.1111/j.1365-313X.2008.03715.x CrossRefPubMedGoogle Scholar
  3. Ball E (1946) Development in sterile culture of stem tips and subjacent regions of Tropaeolum majus L. and of Lupinus albus L. Am J Bot 33:301–318CrossRefGoogle Scholar
  4. Ballester A, Corredoira E, Vieitez AM (2016) Limitations of somatic embryogenesis in hardwood trees. In: Park YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. Korea Forest Research Institute, Seoul, pp 56–74. Google Scholar
  5. Bernard-Dagan C, Carde JP, Gleizes M (1979) Etude des composés terpéniques au cours de la croisance des aiguilles du Pin maritime: comparaison de données biochimiques et ultrastructurales. Can J Bot 57:255–263CrossRefGoogle Scholar
  6. Birnbaum KD, Alvarado AS (2008) Slicing across kingdoms: regeneration in plants and animals. Cell 132:697–710CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bonga JM (1977) Organogenesis in in vitro cultures of embryonic shoots of Abies balsamea (Balsam fir). In Vitro 13:41–53CrossRefPubMedGoogle Scholar
  8. Bonga JM (1981) Organogenesis in vitro of tissues from mature conifers. In Vitro 17:511–518CrossRefGoogle Scholar
  9. Bonga JM (1984) Adventitious shoot formation in cultures of immature female strobili of Larix decidua. Physiol Plant 62:416–421CrossRefGoogle Scholar
  10. Bonga JM (1996) Frozen storage stimulates the formation of embryo-like structures and elongating shoots in explants from mature Larix decidua and L. × eurolepis trees. Plant Cell Tissue Organ Cult 46:91–101CrossRefGoogle Scholar
  11. Bonga JM (2004) The effect of various culture media on the formation of embryo-like structures derived from explants taken from mature Larix decidua. Plant Cell Tissue Organ Cult 77:43–48. CrossRefGoogle Scholar
  12. Bonga JM (2012) Recalcitrance in the in vitro propagation of trees. In: Proceedings of the IUFRO Working Party 2.09.02 conference: Integrating vegetative propagation, biotechnologies and genetic improvement for tree production and sustainable forest management, 25–28 June 2012, Brno, pp 37–46.
  13. Bonga JM (2016) Conifer clonal propagation in tree improvement programs. In: Park YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. National Institute of Forest Science (NiFoS), Seoul, pp 3–31.
  14. Bonga JM, McInnis AH (1983) Origin and early development of roots in plantlets derived from embryo sections of Larix decidua in vitro. Can For Service Res Notes 3:12–14Google Scholar
  15. Bonga JM, von Aderkas P (1988) Attempts to micropropagate mature Larix decidua Mill. In: Ahuja MR (ed) Somatic cell genetics of woody plants. Kluwer, Dordrecht, pp 155–168CrossRefGoogle Scholar
  16. Bonnett HT Jr, Torrey JG (1966) Comparative anatomy of endogenous bud and lateral root formation in Convolvulus arvensis roots cultured in vitro. Am J Bot 53:496–507CrossRefGoogle Scholar
  17. Cardoso JC, Martinelli AP, Latado RR (2012) Somatic embryogenesis from ovaries of sweet orange cv. Tobias. Plant Cell Tissue Organ Cult 109:171–177CrossRefGoogle Scholar
  18. Colcombet J, Boisson-Dernier A, Ros-Palau R, Vera CE, Schroeder JI (2005) Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation. Plant Cell 17:3350–3361CrossRefPubMedPubMedCentralGoogle Scholar
  19. Corredoira E, San-José MC, Vieitez AM (2012) Induction of somatic embryogenesis from different explants of shoot cultures derived from young Quercus alba trees. Trees 26:881–891CrossRefGoogle Scholar
  20. Cutter EG (1972) Plant anatomy: experiment and interpretation. Part 2 Organs. William Clowes & Sons Ltd, LondonGoogle Scholar
  21. de Almeida M, de Almeida CV, Graner EM, Brondani GE, de Abreu-Tarazi MF (2012) Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study. Plant Cell Rep 31:1495–1515CrossRefPubMedGoogle Scholar
  22. De Smet I (2011) Lateral root initiation: one step at a time. New Phytol 193:867–873CrossRefGoogle Scholar
  23. Dénarié J, Debellé F, Promé J-C (1996) Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem 65:503–535CrossRefPubMedGoogle Scholar
  24. Diaz-Sala C, Hutchinson KW, Goldfarb B, Greenwood MS (1996) Maturation-related loss in rooting competence by loblolly pine stem cuttings: the role of auxin transport, metabolism and tissue sensitivity. Physiol Plant 97:481–490CrossRefGoogle Scholar
  25. Ehlers K, Kollmann R (2001) Primary and secondary plasmodesmata: structure, origin and functioning. Protoplasma 216:1–30CrossRefPubMedGoogle Scholar
  26. Ehlers K, Binding H, Kollmann R (1999) The formation of symplasmic domains by plugging of plasmodesmata: a general event in morphogenesis? Protoplasma 209:181–192CrossRefGoogle Scholar
  27. Esau K (1965) Plant anatomy, 2nd edn. Wiley, New YorkGoogle Scholar
  28. Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Organ Cult 74:201–228CrossRefGoogle Scholar
  29. Flygh G, Grönroos R, Gulin L, von Arnold S (1993) Early and late root formation in epicotyl cuttings of Pinus sylvestris after auxin treatment. Tree Physiol 12:81–92CrossRefPubMedGoogle Scholar
  30. Fowke L (2010) Creative young minds plus serendipity—a recipe for science. Botany 88:443–451CrossRefGoogle Scholar
  31. Gómez-Maldonado J, Crespillo R, Avila C, Cánovas M (2001) Efficient preparation of maritime pine (Pinus pinaster) protoplasts suitable for transgene expression analysis. Plant Mol Biol Rep 19:361–366CrossRefGoogle Scholar
  32. Guzzo F, Baldan B, Mariani P, Lo Schiavo F, Terzi M (1994) Studies on the origin of totipotent cells in explants of Daucus carota L. J Exp Bot 45:1427–1432CrossRefGoogle Scholar
  33. Hall RD, Riksen-Bruinsma T, Weyens G, Lefèbvre M, Dunwell JM, Krens FA (1996) Stomatal guard cells are totipotent. Plant Physiol 112:889–892CrossRefPubMedPubMedCentralGoogle Scholar
  34. Hall RD, Riksen-Bruinsma T, Weyens G, Lefèbvre M, Dunwell JM, van Tunen A, Krens FA (1997) Sugar beet guard cell protoplasts demonstrate a remarkable capacity for cell division enabling applications in stomatal physiology and molecular breeding. J Exp Bot 48:255–263CrossRefGoogle Scholar
  35. Halperin W (1978) Organogenesis at the shoot apex. Annu Rev Plant Physiol 29:239–262CrossRefGoogle Scholar
  36. Haywood V, Kragler F, Lacas WJ (2002) Plasmodesmata: pathways for protein and ribonucleoprotein signalling. Plant Cell 14:S303–S325PubMedPubMedCentralGoogle Scholar
  37. Hecht V, Vielle-Calzada Hartog MV, Schmidt EDL, Boutilier K, Grossniklaus U, de Vries S (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in the developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 127:803–816CrossRefPubMedPubMedCentralGoogle Scholar
  38. Irish EE, McMurray D (2006) Rejuvenation by shoot apex culture recapitulates the developmental increase of methylation at the maize gene Pl-Blotched. Plant Mol Biol 60:747–758CrossRefPubMedGoogle Scholar
  39. Kajala K, Ramakrishna P, Fisher A, Bergmann DC, De Smet I, Sozzani R, Weijers D, Brady SM (2014) Omics and modelling approaches for understanding regulation of asymmetric cell divisions in Arabidopsis and other angiosperm plants. Ann Bot 113:1083–1105CrossRefPubMedPubMedCentralGoogle Scholar
  40. Kim I, Zambryski PC (2005) Cell-to-cell communication via plasmodesmata during Arabidopsis embryogenesis. Curr Opin Plant Biol 8:593–599CrossRefPubMedGoogle Scholar
  41. Klimaszewska K, Rutledge RG (2016) Is there potential for propagation of adult spruce trees through somatic embryogenesis? In: Park YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. National Institute of Forest Science (NiFoS), Seoul, pp 195–210.
  42. Kurczyńska EU, Gaj MD, Ujczak A, Mazur E (2007) Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh. Planta 226:619–628CrossRefPubMedGoogle Scholar
  43. Larson PR (1975) Development and organization of the primary vascular system in Populus deltoides according to phyllotaxy. Am J Bot 62:1084–1099CrossRefGoogle Scholar
  44. Laux T (2003) The stem cell concept in plants: a matter of debate. Cell 113:281–283CrossRefPubMedGoogle Scholar
  45. Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, Schroeder JI (2004) Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16:596–615CrossRefPubMedPubMedCentralGoogle Scholar
  46. Maruyama E, Tanaka T, Hosoi Y, Ishii K, Morohoshi N (2000) Embryogenic cell culture, protoplast regeneration, cryopreservation, biolistic gene transfer and plant regeneration in Japanese cedar (Cryptomeria japonica D. Don). Plant Biotechnol 17:281–296CrossRefGoogle Scholar
  47. Merkle SA, Neu KA, Battle PJ, Baily RL (1998) Somatic embryogenesis and plantlet regeneration from immature and mature tissues of sweetgum (Liquidambar styraciflua). Plant Sci 132:169–178CrossRefGoogle Scholar
  48. Michaux-Ferrière N, Grout H, Carron MP (1992) Origin and ontogenesis of somatic embryos in Hevea brasiliensis (Euphorbiaceae). Am J Bot 79:174–180CrossRefGoogle Scholar
  49. Miyashima S, Sebastian J, Lee J-Y, Helariutta Y (2013) Stem cell function during plant vascular development. EMBO J 32:178–193. doi: 10.1038/emboj.2012.301 CrossRefPubMedGoogle Scholar
  50. Monteuuis O (1991) Rejuvenation of a 100-year-old Sequoiadendron giganteum through in vitro meristem culture. I. Organogenic and morphological arguments. Physiol Plant 81:111–115CrossRefGoogle Scholar
  51. Monteuuis O, Doulbeau S, Verdeil J-L (2008) DNA methylation in different origin clonal offspring from a mature Sequoiadendron giganteum genotype. Trees 22:779–784CrossRefGoogle Scholar
  52. Monteuuis O, Lardet L, Montoro P, Berthouly M, Verdeil J-L (2011) Somatic embryogenesis and phase change in trees. In: Proceedings of the IUFRO Working Party 2.09.02 “Somatic embryogenesis of trees” conference on “Advances in somatic embryogenesis of trees and its application for the future forests and plantations”, 19–21 August, Suwon, pp 21–28Google Scholar
  53. Nelson T, Tausta SL, Gandotra N, Liu T (2006) Laser microdissection of plant tissue: what you see is what you get. Annu Rev Plant Biol 57:181–201CrossRefPubMedGoogle Scholar
  54. Parizot B, Laplaze L, Ricaud L, Boucheron-Dubuisson E, Bayle V, Bonke M, De Smet I, Poethig SR, Helariutta Y, Haseloff J, Chirqui D, Beeckman T, Nussaume L (2008) Diarch symmetry of the vascular bundle in Arabidopsis root encompasses the pericycle and is reflected in distich lateral root initiation. Plant Physiol 146:140–148CrossRefPubMedPubMedCentralGoogle Scholar
  55. Petricka JJ, Van Norman JM (2009) Benfey PN (2009) Symmetry breaking in plants: molecular mechanisms regulating asymmetric cell division in Arabidopsis. Cold Spring Harbor Perspect Biol 1:a000497CrossRefGoogle Scholar
  56. Prehn D, Serrano C, Mercado A, Stange C, Barrales L, Arce-Johnson P (2003) Regeneration of whole plants from apical meristems of Pinus radiata. Plant Cell Tissue Organ Cult 73:91–94CrossRefGoogle Scholar
  57. Pulianmackal AJ, Kareem AVK, Durgaprasad K, Trivedi ZB, Prasad K (2014) Competence and regulatory interactions during regeneration in plants. Front Plant Sci 5:1–16. doi: 10.3389/fpls.2014.00142 CrossRefGoogle Scholar
  58. Rodrigues AP, Sérgio PM, Teixeira MR, Pais MS (2001) In vitro break of dormancy of axillary buds from woody species (Persea indica and Arbutus unedo) by sectioning with a laser beam. Plant Sci 161:173–178CrossRefGoogle Scholar
  59. Russell JA (1993) Advances in protoplast culture of woody plants. In: Ahuja MR (ed) Micropropagation of woody plants. Kluwer, Dordrecht, pp 67–91CrossRefGoogle Scholar
  60. Selby C, Harvey BMR (1985) The influence of natural and in vitro bud flushing on adventitious bud production in Sitka spruce [Picea sitchensis (Bong.) Carr.] bud and needle culture. New Phytol 100:549–562CrossRefGoogle Scholar
  61. Shabde M, Murashige T (1977) Hormonal requirements of excised Dianthus caryophyllus L. shoot apical meristem in vitro. Am J Bot 64:443–448CrossRefGoogle Scholar
  62. Smith S, De Smet I (2012) Root system architecture: insights from Arabidopsis and cereal crops. Philos Trans R Soc B 367:1441–1452. doi: 10.1098/rstb.2011.0234 CrossRefGoogle Scholar
  63. Smith RH, Murashige T (1970) In vitro development of the isolated shoot apical meristem of angiosperms. Am J Bot 57:562–568CrossRefGoogle Scholar
  64. Soyars CL, James SR, Nimchuk ZL (2016) Ready, aim, shoot: stem cell regulation of the shoot apical meristem. Curr Opin Plant Biol 29:163–168CrossRefPubMedGoogle Scholar
  65. Steeves TA, Sussex IM (1972) Patterns in plant development. Prentice Hall, New Jersey, p 302Google Scholar
  66. Steinmacher DA, Clement CR, Guerra MP (2007a) Somatic embryogenesis from immature peach palm inflorescence explants: towards development of an efficient protocol. Plant Cell Tissue Organ Cult 89:15–22CrossRefGoogle Scholar
  67. Steinmacher DA, Krohn NG, Dantas ACM, Stefenon VM, Clement CR, Guerra MP (2007b) Somatic embryogenesis in peach palm using thin cell layer technique: induction, morpho-histological aspects and AFPL analysis of somaclonal variation. Ann Bot 100:699–709CrossRefPubMedPubMedCentralGoogle Scholar
  68. Steward FC (1961) Vistas in plant physiology: problems of organization, growth, and morphogenesis. Can J Bot 39:441–460CrossRefGoogle Scholar
  69. Steward FC (1968) Growth and organization in plants. Addison-Wesley Publishing Company, Reading, p 564Google Scholar
  70. Steward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Am J Bot 45:653–704CrossRefGoogle Scholar
  71. Steward FC, Mapes MO, Kent AE, Holsten RD (1964) Growth and development of cultured plant cells. Science 143:20–27CrossRefPubMedGoogle Scholar
  72. Stich M, Thalhammer S, Burgemeister R, Friedemann G, Ehnle S, Lüthy C, Schütze K (2003) Live cell catapulting and recultivation. Pathol Res Pract 199:405–409CrossRefPubMedGoogle Scholar
  73. Stone GN, Schönrogge K (2003) The adaptive significance of insect gall morphology. Trends Ecol Evol 18:512–522CrossRefGoogle Scholar
  74. Sugimoto K, Jiao Y, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471. doi: 10.1016/j.devcel.2010.02.004 CrossRefPubMedGoogle Scholar
  75. Teixeira da Silva J, Dobránszki J (2013) Plant thin cell layers: a 40-year celebration. J Plant Growth Regul 32:922–943CrossRefGoogle Scholar
  76. Tomaz ML, Mendes BMJ, De Assis F, Filho M, Demétrio CGB, Jansakul N, Rodriguez APM (2001) Somatic embryogenesis in Citrus spp.: carbohydrate stimulation and histodifferentiation. In Vitro Cell Dev Biol Plant 37:446–452CrossRefGoogle Scholar
  77. Tran Than Van K, Bui VL (2000) Current status of thin cell layer method for the induction of organogenesis or somatic embryogenesis. In: Mohan Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 6. Kluwer, Dordrecht, pp 51–92CrossRefGoogle Scholar
  78. Tran Thanh Van K, Yilmaz-Lentz D, Trinh TH (1987) In vitro control of morphogenesis in conifers. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry, vol 2., Specific principles and methods: growth and developmentsMartinus Nijhoff Publishers, Dordrecht, pp 168–182CrossRefGoogle Scholar
  79. Trontin J-F, Aronen T, Hargreaves C, Montalbán IA, Moncaleán P, Reeves C, Quoniou S, Lelu-Walter M-A, Klimaszewska K (2016) International effort to induce somatic embryogenesis in adult pines. In: Park YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. National Institute of Forest Science (NIFoS), Seoul, pp 211–260.
  80. Tucker MR, Okada T, Hu Y, Scholefield A, Taylor JM, Koltunow AMG (2012) Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis. Development 139:1399–1404CrossRefPubMedGoogle Scholar
  81. Uchida N, Tasaka M (2013) Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases. J Exp Bot 64:5335–5343. doi: 10.1093/jxb/ert196 CrossRefPubMedGoogle Scholar
  82. von Arnold S, Hawes C (1989) Differentiation of bud meristems and cataphylls during adventitious bud formation on embryos of Picea abies. Can J Bot 67:422–428CrossRefGoogle Scholar
  83. von Arnold S, Alsterborg E, Walles B (1988) Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin. Physiol Plant 72:248–256CrossRefGoogle Scholar
  84. Wang KX, Karnosky DF, Timmis R (1991) Adventitious bud production from mature Picea abies: rejuvenation associated with female strobili formation. In: Ahuja MR (ed) Woody plant biotechnology. Plenum Press, New York, pp 83–90CrossRefGoogle Scholar
  85. Wu H, Hu Z-h (1997) Comparative anatomy of resin ducts of the Pinaceae. Trees 11:135–143CrossRefGoogle Scholar
  86. Yang JL, Seong ES, Kim MJ, Ghimire BK, Kang WH, Yu CY, Li CH (2010) Direct somatic embryogenesis from pericycle cells of broccoli (Brassica oleracea L. var. italica) root explants. Plant Cell Tiss Org Cult 100:49–58CrossRefGoogle Scholar

Copyright information

© Her Majesty the Queen in Right of Canada 2016

Authors and Affiliations

  1. 1.Natural Resources Canada, Canadian Forest Service, Atlantic Forestry CentreFrederictonCanada

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