Skip to main content
SpringerLink
Log in

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

  • Micropropagation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ‘Cleopatra’ mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N 6-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ‘Cleopatra’ mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ‘Cleopatra’ mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ‘Cleopatra’ mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ‘Cleopatra’ mandarin, and sour orange, by culturing nodes from mature plants, has been established.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Similar content being viewed by others

References

  • Al Bahrany AM (2002) Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing. Sci Hortic 95:285–295

    Article  CAS  Google Scholar 

  • Al Khayri JM, Al Bahrany AM (2001) In vitro micropropagation of Citrus aurantifolia (lime). Curr Sci 81:1242–1246

    CAS  Google Scholar 

  • Barlass M, Skene KGM (1982) In vitro plantlet formation from Citrus species and hybrids. Sci Hortic 17:333–341

    Article  Google Scholar 

  • Barlass M, Skene KGM (1986) Citrus (Citrus species). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry. Springer, Berlin, pp 207–219

    Google Scholar 

  • Baruah A, Nagaraju V, Parthasarathy VA (1995) Cytokinin mediated response in shoots of Citrus grandis Osbeck. Ann Plant Physiol 9:13–16

    Google Scholar 

  • Baruah A, Nagaraju V, Parthasarathy VA (1996) Micropropagation of three endangered Citrus species. 1. Shoot proliferation in vitro. Ann Plant Physiol 10:124–128

    Google Scholar 

  • Begum F, Amin MN, Islam S, Azad MAK (2004) A comparative study of axillary shoot proliferation from the nodal explants of three varieties pummelo (Citrus grandis [L.] Osb.). Biotechnology 3:46–62

    Google Scholar 

  • Beloualy N (1991) Plant regeneration from callus culture of three Citrus rootstocks. Plant Cell Tissue Organ Cult 24:29–34

    Article  CAS  Google Scholar 

  • Bonga JM (1982) Vegetative propagation in relation to juvenility; maturity and rejuvenation. In: Bonga JM, Durzan DJ (eds) Tissue culture in forestry. Marinus Nijhoff and W. Junk, The Hague, pp 387–412

  • Bordón Y, Guardiola JL, García-Luis A (2000) Genotype affects the morphogenic response in vitro of epicotyl segments of Citrus rootstocks. Ann Bot 86:159–166

    Article  Google Scholar 

  • Carimi F, De Pasquale F (2003) Micropropagation of Citrus. In: Jain SM, Ishii K (eds) Micropropagation of woody trees and fruits. Kluwer, the Netherlands, pp 589–619

    Chapter  Google Scholar 

  • Chakravarty B, Goswani BC (1999) Plantlet regeneration from long-term callus cultures of Citrus acida and the uniformity of regenerated plants. Sci Hortic 82:159–169

    Article  CAS  Google Scholar 

  • Chatuverdi HC, Singh SK, Sharma AK, Agnihotri S (2001) Citrus tissue culture employing vegetative explants. Indian J Exp Biol 39:1080–1095

    Google Scholar 

  • Chawala HS (2002) Introduction to plant biotechnology. Science, Enfield

    Google Scholar 

  • Chiariotti A, Antonelli M (1988) The effect of 6-BAP and adenine sulphate on peach shoot proliferation. Acta Hortic 227:418–420

    Google Scholar 

  • Corazza-Nunes MJ, Machado MA, Nunes WMC, Cristofani M, Targon MLPN (2002) Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima Burm. Merr.) using RAPD and SSR markers. Euphytica 126:169–176

    Article  CAS  Google Scholar 

  • Da Silva RM, Mendes BMJ, Mourao Filho FA (2008) In vitro induction and culture of adventitious buds in epicotyl segments of sour orange. Pesqui Agropecu Bras 43:1331–1337

    Article  Google Scholar 

  • Drew RA (1988) Rapid clonal propagation of papaya in vitro from mature field-grow trees. HortSci 23:609–611

    Google Scholar 

  • Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. HortSci 19:507–509

    Google Scholar 

  • El-Morsy AA, Millet B (1996) Rhythmic growth and optimization of micropropagation: the effect of excision time and position of axillary buds on in vitro culture of Citrus aurantium L. Ann Bot 78:197–202

    Article  Google Scholar 

  • Fraguas CB, Pasqual M, Pereira AM (2004) In vitro multiplication of Ficus carica L.: kinetin and gibberellic acid effects. Cienc Agrotec 28:49–55

    Article  CAS  Google Scholar 

  • George EF (ed) (1993) Plant propagation by tissue culture. Part 1: the technology. Exegetics, London, pp 231–272

    Google Scholar 

  • George EF (ed) (1996) Plant propagation by tissue culture. Part 2: in practice. Exegetics, London

    Google Scholar 

  • George EF, Puttock DJ, George HJ (1987) Plant culture media. Exegetics, Edington

    Google Scholar 

  • Ghorbel R, Navarro L, Durán-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 73:323–327

    Google Scholar 

  • Goh CJ, Sim GE, Morales CL, Loh CS (1995) Plantlet regeneration through different morphogenic pathways in pummelo tissue culture. Plant Cell Tissue Organ Cult 43:301–303

    Google Scholar 

  • Grosser JW, Chandler JL (1986) In vitro multiplication of Swingle citrumelo rootstock with coumarin. HortSci 21:518–520

    Google Scholar 

  • Grosser JW, Medina-Urrutia V, Ananthakrishnan G, Serrano P (2004) Building a replacement sour orange rootstock: Somatic hybridization of selected mandarin + pummelo combinations. J Am Soc Hort Sci 129:530–534

    Google Scholar 

  • Harada H, Murai Y (1996) Clonal propagation of Poncirus trifoliata through culture of shoot primordia. J Hortic Sci 71:887–892

    Google Scholar 

  • Hartman HH, Kester DE, Davies FTD, Geneve RL (eds) (2004) Plant propagation. Prentice Hall, New Delhi

    Google Scholar 

  • Hassanein AM, Azooz MM (2003) Propagation of Citrus reticulata via in vitro seed germination and shoot cuttings. Biol Plant 47:173–177

    Article  Google Scholar 

  • Hirai M, Kozaki I, Kajiura I (1986) The rate of spontaneous inbreeding of trifoliate orange and some characteristics of the inbred seedling. Japan J Breed 36:138–146

    Google Scholar 

  • Khan IA, Roose ML (1988) Frequency and characteristics of nucellar and zigotic seedlings in three cultivars of trifoliate orange. J Am Soc Hort Sci 113:105–110

    Google Scholar 

  • Kitto SL, Young MJ (1981) In vitro propagation of Carrizo citrange. HortSci 16:305–306

    CAS  Google Scholar 

  • Kotsias D, Roussos PA (2001) An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings. Sci Hortic 89:115–128

    Article  CAS  Google Scholar 

  • Lane WD (1978) Regeneration of apple plants from shoot meristem tips. Plant Sci Lett 13:281–285

    Article  CAS  Google Scholar 

  • Lloyd G, McCown B (1981) Commercially feasible micropropagation of mountain laurel; Kolmia latifolia; by use of shoot tip culture. Comb Proc Intl Plant Prop Soc 30:421–427

    Google Scholar 

  • Lucchesini M, Bertoli A, Mensuali-Sodi A, Cappelli E, Noccioli C, Luciardi L, Pistelli L (2010) Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production. Cent Eur J Biol 5:111–120

    Article  CAS  Google Scholar 

  • 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). Sci Hortic 63:123–128

    Article  CAS  Google Scholar 

  • MAPA (1999) Spanish technical rules of nursery plant fruit tree control and certification (Real Decreto 1678/1999). In: de la Presidencia M (ed) Boletín Oficial del Estado, vol 276. Gobierno de España, Madrid, pp 40077–40079

    Google Scholar 

  • Marín ML, Durán-Vila N (1991) Conservation of Citrus germplasm in vitro. J Am Soc Hort Sci 116:740–746

    Google Scholar 

  • Marques NT, Nolasco GB, Leitao JP (2011) Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian. Sci Hortic 129:176–182

    Article  CAS  Google Scholar 

  • Mendes AF, Cidade LC, Otoni WC, Soares-Filho WS, Costa MG (2011) Role of auxins, polyamines and ethylene in root formation and growth in sweet orange. Biol Plant 55:375–378

    Article  CAS  Google Scholar 

  • Montoliu A, Gómez-Cadenas A, Pérez-Clemente R (2010) In vitro adventitious rooting of Carrizo citrange microshoots. HortSci 45:988–990

    Google Scholar 

  • Moore GA (1986) In vitro propagation of citrus rootstocks. HortSci 21:300–301

    CAS  Google Scholar 

  • Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant growth regulators III: gibberellins; ethylene; abscisic acid; their analogues and inhibitors; miscellaneous compounds. In: George EF, De Klerk GJ, Hall MA (eds) Plant propagation by tissue culture. Springer, the Netherlands, pp 227–282

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murashige T, Tucker DPH (1969) Growth factor requirements of citrus tissue culture. Proc First Intl Citrus Symp 3:1155–1161

    CAS  Google Scholar 

  • Murkute AA, Sharma S, Singh SK (2008) Rapid clonal in vitro multiplication of Citrus jambhiri and Citrus karna. Indian J Hortic 65:127–133

    Google Scholar 

  • Nitsch JP, Nitsch C, Rossini LME, Bui Dang Ha D (1967) The role of adenine in bud differentiation. Phytomorphology 17:446–453

    CAS  Google Scholar 

  • Normah MN, Hamidah S, Ghani FD (1997) Micropropagation of Citrus halimii—an endangered species of South-east Asia. Plant Cell Tissue Organ Cult 50:225–227

    Article  Google Scholar 

  • Nwankwo BA, Krikorian AD (1983) Morphogenetic potential of embryo- and seedling-derived callus of Elaeis guineensis Jacq. var. pisifera Becc. Ann Bot 51:65–76

    Google Scholar 

  • Omura M, Hidaka T (1992) Shoot tip culture of Citrus. I. Longevity of cultured shoots. Bull Fruit Tree Res Stn 22:37–47

    Google Scholar 

  • Pattnaik SK, Chand PK (1997) Rapid clonal propagation of three mulberries; Morus cathayana (Hemsl), M. ihou (Koiz) and M. serrata (Roxb) through in vitro culture of apical shoot buds and nodal explants from mature trees. Plant Cell Rep 16:503–508

    CAS  Google Scholar 

  • Paudyal KP, Haq N (2000) In vitro propagation of pummelo (Citrus grandis L. Osbeck). In Vitro Cell Dev Biol Plant 36:511–516

    Article  CAS  Google Scholar 

  • Pérez-Molphe-Balch E, Ochoa-Alejo N (1997) In vitro plant regeneration of Mexican lime and mandarin by direct organogenesis. HortSci 32:931–934

    Google Scholar 

  • Pérez-Tornero O, Tallón CI, Porras I (2010) An efficient protocol for micropropagation of lemon from mature nodal segments. Plant Cell Tissue Organ Cult 100:263–271

    Article  Google Scholar 

  • Rathore JS, Rathore MS, Singh M, Singh RP, Shekhawat NS (2007) Micropropagation of mature tree of Citrus limon. Indian J Biotechnol 6:239–244

    CAS  Google Scholar 

  • Ruzic D, Saric M, Cerovic R, Culafic LJ (2004) Contents of macronutrients and growth of sweet cherry rootstock in vitro. Biol Plant 47:463–465

    Article  Google Scholar 

  • Sharma S, Prakash A, Tele A (2009) In vitro propagation of Citrus rootstocks. Not Bot Horti Agrobot Cluj-Na 37:84–88

    CAS  Google Scholar 

  • Shawkat A, Bushra M (2006) Micropropagation of rough lemon (Citrus jambhiri Lush.): effect of explant type and hormone concentration. Acta Bot Croat 65:137–146

    Google Scholar 

  • Singh IP (2001) Citrus improvement. In: Singh S, Naqvi S (eds) Citrus. International book distributing Co, Lucknow, pp 83–98

    Google Scholar 

  • Singh S, Ray BK, Bhattacharya S, Deka PC (1994) In vitro propagation of Citrus reticulata Blanco and Citrus limon Burm F. HortSci 29:214–216

    Google Scholar 

  • Starrantino A, Caruso A (1988) In vitro culture for Citrus micropropagation. Acta Hortic 227:444–446

    Google Scholar 

  • Start NG, Cumming BG (1976) In vitro propagation of Saintpaulia ionantha Wendl. HortSci 11:204–206

    CAS  Google Scholar 

  • Usman M, Sana M, Fatima B (2005) In vitro multiple shoot induction from nodal explants of Citrus cultivars. J Cent Eur Agr 6:435–442

    Google Scholar 

  • Wochok RJ, Sluis CJ (1980) In vitro propagation and establishment of wax current (Ribes inebrians). J Hortic Sci 55:355–357

    Google Scholar 

Download references

Acknowledgments

The authors thank Mr. Fernando Córdoba and Mrs. Silvia Andújar for technical assistance in the laboratory. This work was supported by the Ministerio de Ciencia e Innovación, through the project AGL2007-65437-C04-04/AGR.

Author information

Authors and Affiliations

  1. Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor, s/n., 30150, Murcia, Spain

    Carlos I. Tallón, Ignacio Porras & Olaya Pérez-Tornero

Authors
  1. Carlos I. Tallón
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ignacio Porras
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olaya Pérez-Tornero
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Olaya Pérez-Tornero.

Additional information

Editor: J. Forster

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tallón, C.I., Porras, I. & Pérez-Tornero, O. Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators. In Vitro Cell.Dev.Biol.-Plant 48, 488–499 (2012). https://doi.org/10.1007/s11627-012-9457-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-012-9457-9

Keywords

Search

Navigation