Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 123, Issue 1, pp 143–154 | Cite as

Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

  • Ricardo J. Licea-Moreno
  • Angela Contreras
  • Ana V. Morales
  • Ignacio Urban
  • Marcos Daquinta
  • Luis Gomez
Original Article
First Online:
Received:
Accepted:

Abstract

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 × J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the sequential use of 0.4 and 0.2 mM phloroglucinol during shoot multiplication, but not at later stages, so as to maximize shoot growth without inhibiting root formation. Moreover, substituting FeEDTA by FeEDDHA diminished chlorotic symptoms and significantly improved the rooting ability of all genotypes, with up to 90 % microshoots developing viable roots at 6.81 mg/L Fe3+. The addition of 83.2 mM glucose during the root expression phase was particularly efficient at promoting plant survival during acclimatization, compared to equimolar amounts of the alternative sugars sucrose and fructose. At the proposed working concentrations, the aforementioned compounds did not cause any deleterious effects on the nine genotypes studied. Microscopic analysis revealed the physical continuity between adventitious roots and stem cambial tissue. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5800 ramets) have been established by applying this protocol.

Keywords

Acclimatization Adventitious rooting Clonal plantation Iron source Juglans Carbon source 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

Financial support for this work was obtained from Bosques Naturales S.A. and the National Research and Development Program (Grant AGL2007–64761/FOR). We are indebted to the technical personnel of Bosques Naturales S.A. and the Center for Plant Biotechnology and Genomics (Madrid, Spain). We also wish to thank Dr. Irene Merino (Agricultural University of Uppsala, Sweden), Dr. Julia Quintana (University of Helsinki, Finland), and Álvaro Vinuesa (Polytechnic University of Madrid) for insightful comments throughout this work. The assistance of Ms. Elke Pita with microsatellite analysis is gratefully acknowledged. We are indebted to Ms. Nikolina Valentinova-Apostolova for her help with statistical analyses.

Authors contribution statement

R.L., A.C. and A.V.M. conducted all in vitro and acclimatization experiments. I.U. contributed to genotype selection and data collection. M.D. participated in data analysis. R.L. and L.G. designed the study and wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Aletá N, Ninot A, Voltas J (2004) Retrospective evaluation of parental selection in nursery tests of Juglans regia L. using a mixed model analysis. Silvae Genet 53:26–32Google Scholar
  2. Al-Khateeb AA (2008) Regulation of in vitro bud formation of date palm (Phoenix dactylifera L.) cv. Khanezi by different carbon sources. Bioresour Technol 99:6550–6555CrossRefPubMedGoogle Scholar
  3. Ashrafi EN, Vahdati K, Ebrahimzadeh H, Mirmasoumi M (2010) Analysis of in vitro explants mineral contents to modify medium mineral composition for enhancing growth of Persian walnut (Juglans regia L.). J Food Agric Environ 8:325–329Google Scholar
  4. Becquey J (1997) Les noyers à bois. Institut pour le Développement Forestier, ParisGoogle Scholar
  5. Bosela MJ, Michler CH (2008) Media effects on black walnut (Juglans nigra) shoot growth in vitro: evaluation of multiple nutrient formulations and cytokinin types. In vitro Cell Dev Biol Plant 44:316–329CrossRefGoogle Scholar
  6. Carra A, Sajeva M, Abbate L, Siragusa M, Sottile F, Carimi F (2012) In vitro plant regeneration of caper (Capparis spinosa L.) from floral explants and genetic stability of regenerants. Plant Cell Tissue Organ Cult 109:373–381CrossRefGoogle Scholar
  7. Clark J, Hemery G (2010) Walnut hybrids in the UK: fast growing quality hardwoods. Q J For 104:43–46Google Scholar
  8. Cornu D, Jay-Allemand C (1989) Micropropagation of hybrid walnut trees (Juglans nigra × Juglans regia) through culture and multiplication of embryos. Ann Sci For 46S:113–116CrossRefGoogle Scholar
  9. Custodio L, Martins-Loução MA, Romano A (2004) Influence of sugars on in vitro rooting and acclimatization of carob tree. Biol Plant 48:469–472CrossRefGoogle Scholar
  10. da Rocha Corrêa L, da Rocha Corrêa L, Paim DC, Schwambach J, Fett-Neto AG (2005) Carbohydrates as regulatory factors on the rooting of Eucalyptus saligna Smith and Eucalyptus globulus Labill. Plant Growth Regul 45:63–73CrossRefGoogle Scholar
  11. De Klerk GJ, Guan H, Huisman P, Marinova S (2011) Effects of phenolic compounds on adventitious root formation and oxidative decarboxilation of applied indolacetic acid in Malus Jork 9. Plant Growth Regul 63:175–185CrossRefGoogle Scholar
  12. Dolcet-Sanjuan R, Claveria E, Gruselle R, Meier-Dinkel A, Jay-Allemand C, Gaspar T (2004) Practical factors controlling in vitro adventitious root formation from walnut shoots microcuttings. J Am Soc Hortic Sci 129:198–203Google Scholar
  13. Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. Hortic Sci 19:507–509Google Scholar
  14. Find J, Grace L, Krogstrup P (2002) Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana). Physiol Plant 116:231–237. doi: 10.1034/j.1399-3054.2002.1160213.x CrossRefPubMedGoogle Scholar
  15. Gibson SI (2005) Control of plant development and gene expression by sugar signaling. Curr Opin Plant Biol 8:93–102CrossRefPubMedGoogle Scholar
  16. Jay-Allemand C, Capelli P, Cornu D (1992) Root development of in vitro hybrid walnut microcuttings in a vermiculite-containing gelrite medium. Sci Hortic 51:335–342CrossRefGoogle Scholar
  17. Kumar V, Gururaj HB, Narasimha Prasad BC, Giridhar P, Ravishankar GA (2005) Direct shoot organogenesis on shoot apex from seedling explants of Capsicum annuum L. Sci Hortic 106:237–246CrossRefGoogle Scholar
  18. Leal DR, Sánchez-Olate M, Avilés F, Materan ME, Uribe M, Hasbún R, Rodríguez R (2007) Micropropagation of Juglans regia L. In: Jain SM, Häggman H (eds) Protocols for micropropagation of woody trees and fruits. Springer, Heidelberg, pp 381–390CrossRefGoogle Scholar
  19. Leslie CA, Hackett WP, Bujazha D, Hirbod S, McGranahan GH (2005) Adventitious rooting and clonal plant production of hybrid walnut (Juglans) rootstock selections. Acta Hortic 705:325–328Google Scholar
  20. Leslie CA, Hackett WP, McGranahan GH (2009) Improved rooting methods for walnut (Juglans) microshoots. Acta Hortic 861:365–372Google Scholar
  21. McGranahan G, Leslie CA (1988) In vitro propagation of mature Persian walnut cultivars. Hortic Sci 23:220Google Scholar
  22. McGranahan GH, Driver JA, Tulecke W (1987) Tissue culture of Juglans. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry, vol 3. Martinus Nijhoff, Boston, pp 261–271CrossRefGoogle Scholar
  23. Moreno RJL, Morales AV, Daquinta M, Gomez L (2012) Towards scaling-up the micropropagation of Juglans major (Torrey) Heller var. 209 × J. regia L., a hybrid walnut of commercial interest. In: Park YS, Bonga JM (eds) Integrating vegetative propagation, biotechnologies and genetic improvement for tree production and sustainable forest management. IUFRO, Czech Republic, pp 80–91Google Scholar
  24. Nas MN, Read PE (2001) Micropropagation of hybrid hazelnut: medium composition, physical state and iron source affect shoot morphogenesis, multiplication and explant vitality. Acta Hortic 556:251–258Google Scholar
  25. Phan CT, Hegedus P (1985) Possible metabolic basis for the developmental anomaly observed in in vitro culture, called ‘vitreous plants’. Plant Cell Tissue Org Cult 6:83–94CrossRefGoogle Scholar
  26. Philips RL, Kaeppler SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal control. Proc Natl Sci USA 91:5222–5226CrossRefGoogle Scholar
  27. Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Ann Rev Plant Biol 57:675–709CrossRefGoogle Scholar
  28. Sarkar D, Naik PS (2000) Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro. Plant Cell Tissue Organ Cult 60:139–149CrossRefGoogle Scholar
  29. Tallón CI, Porras I, Pérez-Tornero O (2012) Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators. In vitro Cell Dev Biol Plant 48:488–499CrossRefGoogle Scholar
  30. Teixeira da Silva JA, Dobránzki J, Ross S (2013) Phloroglucinol in plant tissue culture. In vitro Cell Dev Biol Plant 49:1–16CrossRefGoogle Scholar
  31. Toosi S, Dilmagani K (2010) Proliferation of Juglans regia L. by in vitro embryo culture. J Cell Biol Genet 1:12–19Google Scholar
  32. 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. Hortic Sci 39:324–327Google Scholar
  33. Vahdati K, Razaee R, Mirmasoomi M (2009) Micropropagation of some dwarf and early mature walnut genotypes. Biotechnology 8:171–175CrossRefGoogle Scholar
  34. Van der Salm TPM, Van der Toorn CJG, Hänish Ten Cate CH, Dubois LAM, De Vries DP, Dons HJM (1994) Importance of the iron chelate formula for micropropagation of Rosa hybrida L. “Moneyway”. Plant Cell Tissue Organ Cult 37:73–77CrossRefGoogle Scholar
  35. Victory ER, Glaubitz JC, Rhodes OE, Woeste KE (2006) Genetic homogeneity in Juglans nigra (Juglandaceae) at nuclear microsatellites. Am J Bot 93:118–126CrossRefGoogle Scholar
  36. Woeste K, Michler C (2011) Juglans. In: Chittaranjan K (ed) Forest trees Wild crop relatives: genomic and breeding resources. Springer, Berlin, pp 77–88CrossRefGoogle Scholar
  37. Zawadzka M, Orlikowska T (2009) Influence of FeEDDHA on in vitro rooting and acclimatization of red raspberry (Rubus idaeus L.) in peat and vermiculite. J Hortic Sci Biotechnol 84:599–603Google Scholar
  38. 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

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Ricardo J. Licea-Moreno
    • 1
    • 2
  • Angela Contreras
    • 1
    • 3
  • Ana V. Morales
    • 2
  • Ignacio Urban
    • 4
  • Marcos Daquinta
    • 5
  • Luis Gomez
    • 1
    • 3
  1. 1.Center for Plant Biotechnology and Genomics (CBGP)Universidad Politécnica de MadridPozuelo de AlarconSpain
  2. 2.Department of Biotechnology, Micropropagation UnitBosques Naturales S.A.AlcobendasSpain
  3. 3.Department of Natural Resources and Systems, Forestry SchoolUniversidad Politécnica de MadridMadridSpain
  4. 4.Department of ForestryBosques Naturales S.A.AlcobendasSpain
  5. 5.Bioplant CenterUniversidad Ciego de ÁvilaCiego de ÁvilaCuba

Personalised recommendations