Micropropagation of Citrus spp. by Organogenesis and Somatic Embryogenesis

Part of the Methods in Molecular Biology book series (MIMB, volume 994)


Citrus spp., the largest fruit crops produced worldwide, are usually asexually propagated by cuttings or grafting onto seedling rootstocks. Most of Citrus genotypes are characterized by polyembryony due to the occurrence of adventive nucellar embryos, which lead to the production of true-to-type plants by seed germination. Tissue culture and micropropagation, in particular, are valuable alternatives to traditional propagation to obtain a high number of uniform and healthy plants in a short time and in a small space. Moreover, in vitro propagation provides a rapid system to multiply the progeny obtained by breeding programs, allows the use of monoembryonic and seedless genotypes as rootstocks, and it is very useful also for breeding and germplasm preservation.

In this chapter, two protocols regarding organogenesis of a rootstock and somatic embryogenesis of a cultivar have been described.

Key words

Anther culture Citrus Epicotyl cuttings Organogenesis Somatic embryogenesis 


  1. 1.
  2. 2.
    Barlass M, Skene KGM (1986) Citrus (Citrus species). In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, Trees I, vol 1. Springer, BerlinGoogle Scholar
  3. 3.
    Cervera M, Navarro A, Navarro L, Peña L (2008) Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol 28:55–66PubMedCrossRefGoogle Scholar
  4. 4.
    Bordòn Y, Guardiola JL, Garcia-Luis A (2000) Genotype affects the morphogenic response in vitro of epicotyl segments of Citrus rootstocks. Ann Bot 86:159–166CrossRefGoogle Scholar
  5. 5.
    Edris MH, Burger DW (1984) In vitro propagation of Troyer citrange from epicotyl segments. Sci Hortic 23:159–162CrossRefGoogle Scholar
  6. 6.
    Garcia-Luis A, Molina RV, Varona V, Castello S, Guardiola JL (2006) The influence of explant orientation and contact with the medium on the pathway of shoot regeneration in vitro in epicotyl cuttings of Troyer citrange. Plant Cell Tiss Org 85:137–144CrossRefGoogle Scholar
  7. 7.
    Ghorbel R, Navarro L, Duran-Vila N (1998) Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C. aurantium) and alemow (C. macrophylla). J Hortic Sci Biotech 73:323–327Google Scholar
  8. 8.
    Goh CJ, Sim GE, Morales CL, Loh CS (1995) Plantlet regeneration through different morphogenic pathways in pommelo tissue culture. Plant Cell Tiss Org 43:301–303Google Scholar
  9. 9.
    Grinblat U (1972) Differentiation of Citrus stem in vitro. J Am Soc Hort Sci 97:599–603Google Scholar
  10. 10.
    Maggon R, Singh BD (1995) Promotion of adventitious bud regeneration by ABA in combination with BAP in epicotyl and hypocotyl explants of sweet orange (Citrus sinensis L. Osbeck). Scientia Hortic 63:123–128CrossRefGoogle Scholar
  11. 11.
    Moreira-Dias JM, Molina RV, Bordon N, Guardiola JL, Garcia-Luis A (2000) Direct and indirect shoot organogenic pathways in epicotyl cuttings of Troyer citrange differ in hormone requirements and in their response to light. Ann Bot 85:103–110CrossRefGoogle Scholar
  12. 12.
    Perez-Molphe-Balch E, Ochoa-Alejo N (1997) In vitro plant regeneration of Mexican lime and mandarin by direct organogenesis. Hortscience 32:931–934Google Scholar
  13. 13.
    Sim GE, Goh CJ, Loh CS (1989) Micropropagation of Citrus mitis Blanc. Multiple bud formation from shoot and root explants in the presence of 6-benzylaminopurine. Plant Sci 59:203–210CrossRefGoogle Scholar
  14. 14.
    Van Le B, Thanh Ha N, Anh Hong LT, Van Tran Thanh K (1999) High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell later method. CR Acad Sci Paris Life Sci 322:1105–1111Google Scholar
  15. 15.
    Bhat SR, Chitralekha P, Chandel KPS (1992) Regeneration of plants from long term root culture of lime, Citrus aurantifolia (Christm) Swing. Plant Cell Tiss Org 29:19–25CrossRefGoogle Scholar
  16. 16.
    Gill M, Singh Z, Dhillon BS, Gosal SS (1994) Somatic embryogenesis and plantlet regeneration on calluses derived from seedling explants of Kinnow mandarin (Citrus nobilis Lour. x Citrus deliciosa Ten.). J Hort Sci 69(2):231–236Google Scholar
  17. 17.
    Carimi F, De Pasquale F (2003) Micropropagation of Citrus. In: Mohan Jain S, Katsuaki I (eds) Micropropagation of woody trees and fruits. Kluwer Academic, Dordrecht, pp 589–619Google Scholar
  18. 18.
    Germanà MA (2003) Somatic embryogenesis and plant regeneration from anther culture of Citrus aurantium and Citrus reticulata. Biologia Bratislava 58(4):843–850Google Scholar
  19. 19.
    Germanà MA (2005) Protocol of somatic embryogenesis from Citrus spp. anther culture. In: Jain SM, Gupta PK (eds) Protocols of somatic embryogenesis-woody plants. ISBN: 1-4020-2984-5. Dordrecht: Springer, Netherlands, pp. 191–207Google Scholar
  20. 20.
    Germanà MA, Wang YY, Barbagallo MG, Iannolino G, Crescimanno FG (1994) Recovery of haploid and diploid plantlets from anther culture of Citrus clementina Hort. ex Tan. and Citrus reticulata Blanco. J Hort Sci 69(3):473–480Google Scholar
  21. 21.
    Germanà MA, Macaluso L, Patricolo G, Chiancone B (2008) Morphogenic response in vitro of epicotyl segments of Citrus macrophylla. Plant Biosyst 142(3):661–664CrossRefGoogle Scholar
  22. 22.
    Germanà MA, Micheli M, Chiancone B, Macaluso L, Standardi A (2011) Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf). Plant Cell Tiss Org Cult 106:299–307CrossRefGoogle Scholar
  23. 23.
    Bar-Joseph M, Marcus R, Lee RF (1989) The continuous challenge of citrus tristeza virus control. Annu Rev Phytopathol 27:291–316CrossRefGoogle Scholar
  24. 24.
    Bajaj YPS (ed) (1995) Somatic embryogenesis and its applications for crop improvement. In: Biotechnology in agriculture and forestry. Somatic embryogenesis and synthetic seed I, vol 30. Springer, Berlin, pp 105–125Google Scholar
  25. 25.
    Henry RJ (1998) Molecular and biochemical characterization of somaclonal variation. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Kluwer Academic Publishers, Dordrecht, pp 485–499Google Scholar
  26. 26.
    Germanà MA, Piccioni E, Standardi A (1999) Effects of encapsulation on Citrus reticulata Blanco somatic embryos conversion. Plant Cell Tiss Org 55:235–237Google Scholar
  27. 27.
    Germanà MA, Hafiz IA, Micheli M, Standardi A (2007) In vitro and ex vitro conversion of encapsulated somatic embryos of Citrus reticulata Blanco, cv. Mandarino Tardivo di Ciaculli Plant Cell Tiss Org 88:117–120CrossRefGoogle Scholar
  28. 28.
    Singh B, Sharma S, Rani G (2007) In vitro response of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora). Plant Biotechnol Rep 1:101–107CrossRefGoogle Scholar
  29. 29.
    Germanà MA, Lambardi M, Ozudogru EA (2011) Preliminary results on desiccation and PVS2-vitrification of carrizo citrange synthetic seeds as pre-treatments to cryopreservation. Acta Hortic 892:311–318Google Scholar
  30. 30.
    Malik SK, Chaudhury R (2006) The cryopreservation of embryonic axes of two wild and endangered Citrus species. Plant genetic resources: characterization and utilization 4:204–209CrossRefGoogle Scholar
  31. 31.
    Germanà MA (1997) Haploidy in Citrus. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 5. Academic, Dordrecht, 5:195–217Google Scholar
  32. 32.
    Germanà MA (2006) Doubled Haploid production in fruit crops. Plant Cell Tiss Org 86:131–146CrossRefGoogle Scholar
  33. 33.
    Germanà MA (2009) Haploid and doubled haploids in fruit trees. In: Touraev A, Forster B, Jain M (eds) Advances in haploid production in higher plants. Springer, Dordrecht, pp. 241–263Google Scholar
  34. 34.
    Hidaka T, Yamada Y, Shichijo T (1981) Plantlet formation from anthers of Citrus aurantium L. Proc Int Soc Citriculture 1:153–155Google Scholar
  35. 35.
    Chaturvedi HC, Sharma AK (1985) Androgenesis in Citrus aurantifolia (Christm.) Swingle. Planta 165:142–144CrossRefGoogle Scholar
  36. 36.
    Ling J, Iwamasa M, Nito N (1988) Plantlet regeneration by anther culture of Calamondin (C. madurensis Lour.). Proc Sixth Intl Soc Citricult 1:251–256Google Scholar
  37. 37.
    Deng XX, Deng ZA, Xiao SY, Zhang WC (1992) Pollen derived plantlets from anther culture of Ichang papeda hybrids No.14 and Trifoliate orange. Proc Intl Soc Citricult 1:190–192Google Scholar
  38. 38.
    Germanà MA, Chiancone B, Lain O, Testolin R (2005) Anther culture in Citrus c1ementina: a way to regenerate tri-haploids. Austr J Agric Res 56:839–845CrossRefGoogle Scholar
  39. 39.
    Lillie RD (1951) Simplification of the manufacture of Schiff reagent for use in histochemical procedures. Stain Tech 26:163–165Google Scholar
  40. 40.
    Torres AM, Soost RK, Diedenhofen U (1978) Leaf isozymes as genetic markers in Citrus. American J Bot 65:869–881CrossRefGoogle Scholar
  41. 41.
    Kijas JMH, Thomas MR, Fowler JCS, Roose ML (1997) Integration of trinucleotide microsatellites into a linkage map of Citrus. Theor Appl Genet 94:701–706CrossRefGoogle Scholar
  42. 42.
    Murashige T, Skoog FA (1962) Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  43. 43.
    Al-Bahrany AM (2002) Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing. Sci Hortic 95:285–295CrossRefGoogle Scholar
  44. 44.
    Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Benedetta Chiancone
    • 1
  • Maria Antonietta Germanà
    • 1
  1. 1.Dipartimento DEMETRA, Facoltà di AgrariaUniversità degli Studi di PalermoPalermoItaly

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