Skip to main content
SpringerLink
Log in

A temporary immersion system for micropropagation of axillary shoots of hybrid chestnut

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

A protocol for culturing chestnut axillary shoots by temporary immersion in liquid medium was developed. The influence of type of explant, support material, bioreactor, and immersion was investigated for five artificial hybrids and five natural hybrids of Asian and European chestnut selected for resistance to ink disease. The type of explant influenced shoot quality and proliferation rates, and basal explants with callus produced more and longer shoots than apical and nodal segments. Use of rockwool cubes as support material prevented hyperhydricity and allowed proliferation of explants in Murashige and Skoog medium with half-strength nitrates supplemented with 0.22 µM BA and 3 % sucrose, cultured both in plantform™ and RITA® vessels with three or six immersions per day and additional aeration of 1 min per hour in the case of plantform™ bioreactors. Basal explants cultured in plantform™ for 5 weeks produced long shoots suitable for rooting, whereas apical and nodal explants cultured in plantform™ or RITA® produced shorter shoots that were suitable for maintenance of stock. For most of the clones, similar or higher proliferation rates were observed when cultured in liquid medium than in semisolid medium, with the additional benefit of cost-reduction of the former system. Shoots developed in liquid medium were submitted to ex vitro root induction by dipping in indole-3-butyric acid, and acclimatized under greenhouse conditions. This is the first demonstration of the production of chestnut plantlets from shoots cultured in liquid medium, and the protocol presented here shows good potential for application in large-scale propagation.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Afreen F, Zobayed SMA (2005) Photoautotrophic plant conversion in the process of somatic embryogenesis. In: Kozai T et al (eds) Photoautotrophic (sugar-free medium) micropropagation as a new propagation and transplant production system. Springer, The Netherlands, pp 91–122

    Chapter  Google Scholar 

  • Akdemir H, Süzerer V, Onay A, Tilkat E, Ersali Y, Çiftçi YO (2014) Micropropagation of the pistachio and its rootstocks by temporary immersion system. Plant Cell Tissue Organ Cult 117:65–76. doi:10.1007/s11240-013-0421-0

    Article  CAS  Google Scholar 

  • Aragón CE, Escalona M, Capote I, Pina D, Cejas I, Rodríguez R, Cañal MJ, Sandoval J, Roels S, Debergh P, González-Olmedo JL (2005) Photosynthesis and carbon metabolism in plantain (Musa AAB) growing in temporary immersion bioreactor (TIB) and ex vitro acclimatization. In Vitro Cell Dev Biol Plant 41:550–554. doi:10.1079/IVP2005640

    Article  Google Scholar 

  • Ballester A, San-José MC, Vidal N, Fernández-Lorenzo JL, Vieitez AM (1999) Anatomical and biochemical events during in vitro rooting of microcuttings from juvenile and mature phases of chestnut. Ann Bot 83:619–629. doi:10.1006/anbo.1999.0865

    Article  CAS  Google Scholar 

  • Carvalho L, Amâncio S (2002) Effect of ex vitro conditions on growth and acquisition of autotrophic behaviour during acclimatisation of chestnut regenerated in vitro. Sci Hortic 95:151–164. doi:10.1016/S0304-4238(02)00037-7

    Article  Google Scholar 

  • Chakrabarty D, Hahn EJ, Yoon YS, Paek KY (2003) Micropropagation of apple root stock ‘M9 EMLA’ using bioreactor. J Hortic Sci Biotechnol 78:605–609

    CAS  Google Scholar 

  • Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2006) Hyperhydricity in apple: physiological and ultrastructural aspects. Tree Physiol 26:377–388. doi:10.1093/treephys/26.3.377

    Article  CAS  PubMed  Google Scholar 

  • Debnath SC (2007) A two-step procedure for in vitro multiplication of cloudberry (Rubus chamaemorus L.) shoots using bioreactor. Plant Cell Tissue Organ Cult 88:185–191. doi:10.1007/s11240-006-9188-x

    Article  CAS  Google Scholar 

  • Debnath SC (2009) A scale-up system for lowbush blueberry micropropagation using a bioreactor. HortScience 44:1962–1966

    Google Scholar 

  • Escalona M, Lorenzo JC, González B, Daquinta M, Borroto CG, González JL, Desjardins Y (1999) Pineapple micropropagation in temporary immersion systems. Plant Cell Rep 18:743–748. doi:10.1007/s002990050653

    Article  CAS  Google Scholar 

  • Escalona M, Samson G, Borroto C, Desjardins Y (2003) Physiology of effects of temporary immersion bioreactors on micropropagated pineapple plantlets. In Vitro Cell Dev Biol Plant 39:651–656. doi:10.1079/IVP2003473

    Article  CAS  Google Scholar 

  • Etienne H, Berthouly M (2002) Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult 69:215–231. doi:10.1023/A:1015668610465

    Article  Google Scholar 

  • Etienne H, Lartaud M, Michaux-Ferrière N, Carron MP, Berthouly M, Teisson C (1997) Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique. In Vitro Cell Dev Biol Plant 33:81–87. doi:10.1007/s11627-997-0001-2

    Article  Google Scholar 

  • Etienne-Barry D, Bertrand B, Vasquez N, Etienne H (1999) Direct sowing of Coffea arabica somatic embryos mass-produced in a bioreactor and regeneration of plants. Plant Cell Rep 19:111–117. doi:10.1007/s002990050720

    Article  CAS  Google Scholar 

  • Feito I, González A, Centeno ML, Fernández B, Rodríguez A (2001) Transport and distribution of benzyladenine in Actinidia deliciosa explants cultured in liquid and solid media. Plant Physiol Biochem 39:909–916. doi:10.1016/S0981-9428(01)01309-2

    Article  CAS  Google Scholar 

  • Gatica-Arias A, Weber G (2013) Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system. In Vitro Cell Dev Biol Plant 49:656–664. doi:10.1007/s11627-013-9574-0

    Article  CAS  Google Scholar 

  • Gonçalves JC, Diogo G, Amâncio S (1998) In vitro propagation of chestnut (Castanea sativa x C. crenata): effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation. Sci Hortic 72:265–275. doi:10.1016/S0304-4238(97)00136-2

    Article  Google Scholar 

  • González MV, Cuenca B, López M, Prado MJ, Rey M (2011) Molecular characterization of chestnut plants selected for putative resistance to Phytophthora cinnamomi using SSR markers. Sci Hortic 130:459–467. doi:10.1016/j.scienta.2011.07.020

    Article  Google Scholar 

  • Gresshoff PM, Doy CH (1972) Development and differentiation of haploid Lycopersicon esculentum. Planta 107:161–170. doi:10.1007/BF00387721

    Article  CAS  PubMed  Google Scholar 

  • Hahn EJ, Paek KY (2005) Multiplication of Chrysanthemum shoots in bioreactors as affected by culture method and inoculation density of single node stems. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 143–153

    Chapter  Google Scholar 

  • Jackson MB (2005) Aeration stress in plant tissue cultures. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 459–473

    Chapter  Google Scholar 

  • Kong LS, Holtz CT, Nairn CJ, Houke H, Powell WA, Baier K, Merkle SA (2014) Application of airlift bioreactors to accelerate genetic transformation in American chestnut. Plant Cell Tissue Organ Cult 117:39–50. doi:10.1007/s11240-013-0418-8

    Article  CAS  Google Scholar 

  • Kozai T, Kubota C (2005) In vitro aerial environments and their effects on growth and development of plants. In: Kozai T et al (eds) Photoautotrophic (sugar-free medium) micropropagation as a new propagation and transplant production system. Springer, The Netherlands, pp 31–52

    Chapter  Google Scholar 

  • Lorenzo JC, Gonzalez BL, Escalona M, Teisson C, Espinosa P, Borroto C (1998) Sugarcane shoot formation in an improved temporary immersion system. Plant Cell Tissue Organ Cult 54:197–200. doi:10.1023/A:1006168700556

    Article  CAS  Google Scholar 

  • Lowe K, Anthony P, Power B, Davey MR (2003) Novel approaches for regulating gas supply to plant systems in vitro: application and benefits of artificial gas carriers. In Vitro Cell Dev Biol Plant 39:557–566. doi:10.1079/IVP2003469

    Article  CAS  Google Scholar 

  • Luna C, Collavino M, Mroginski L, Sansberro P (2008) Identification and control of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion bioreactor system using 16S rDNA analysis. Plant Cell Tissue Organ Cult 95:13–19. doi:10.1007/s11240-008-9408-7

    Article  CAS  Google Scholar 

  • Luna C, Acevedo R, Collavino M, González A, Mroginski L, Sansberro P (2013) Endophytic bacteria from Ilex paraguariensis shoot cultures: localization, characterization, and response to isothiazolone biocides. In Vitro Cell Dev Biol Plant 49:326–332. doi:10.1007/s11627-013-9500-5

    Article  CAS  Google Scholar 

  • Mallón R, Covelo P, Vieitez AM (2012) Improving secondary embryogenesis in Quercus robur: application of temporary immersion for mass propagation. Trees 26:731–741. doi:10.1007/s00468-011-0639-6

    Article  Google Scholar 

  • Mallón R, Vieitez AM, Vidal N (2013) High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR. Plant Cell Tissue Organ Cult 114:171–185. doi:10.1007/s11240-013-0313-3

    Article  Google Scholar 

  • McAlister B, Finnie J, Watt MP, Blakeway F (2005) Use of temporary immersion system (RITA®) for production of commercial Eucalyptus clones in Mondi forests (SA). Plant Cell Tissue Organ Cult 81:347–358. doi:10.1007/s11240-004-6658-x

    Article  Google Scholar 

  • McCown BH (1988) Adventitious rooting of tissue cultured plants. In: Davis TD, Haissig BE, Sankhla N (eds) Adventitious root formation in cuttings. Dioscorides Press, Portland, pp 289–302

    Google Scholar 

  • Moreno RJ, Morales AV, Daquinta M, Gómez L (2012) Towards scaling-up the micropropagation of Juglans major (Torrey) Heller var. 209 x J. regia L., a hybrid walnut of commercial interest. 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” June 25–28, 2012. Brno, Czech Republic. pp. 80–91

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  • Murch SJ, Chunzhao L, Romero RM, Saxena PK (2004) In vitro culture and temporary immersion bioreactor production of Crescentia cujete. Plant Cell Tiss Org Cult 78:36–68. doi:10.1023/B:TICU.0000020397.01895.3e

    Article  Google Scholar 

  • Nagae S, Takamura T, Watanabe T, Murakami A, Murakami K, Tanaka M (1996) In vitro shoot development of Eucalyptus citriodora on rockwool in the film culture vessel under CO2 enrichment. J Forest Res 1:227–230. doi:10.1007/BF02348330

    Article  Google Scholar 

  • Nehra NS, Becwar MR, Rottmann WH, Pearson L, Chowdhury K, Chang S, Wilde HD, Kodrzycki RJ, Zhang C, Gause KC, Parks DW, Hinchee MA (2005) Forest biotechnology: innovative methods, emerging opportunities. In Vitro Cell Dev Biol Plant 41:701–717. doi:10.1079/IVP2005691

    Article  CAS  Google Scholar 

  • Niemenak N, Saare-Surminski K, Rohsius C, Omokolo Ndoumou D, Lieberei R (2008) Regeneration of somatic embryos in Theobroma cacao L. in temporary immersion bioreactor and analyses of free amino acids in different tissues. Plant Cell Rep 27:667–676. doi:10.1007/s00299-007-0497-2

    Article  CAS  PubMed  Google Scholar 

  • Norikane A, Takamura T, Morokuma M, Tanaka M (2010) In vitro growth and single-leaf photosynthetic response of Cymbidium plantlets to super-elevated CO2 under cold cathode fluorescent lamps. Plant Cell Rep 29:273–383. doi:10.1007/s00299-010-0820-1

    Article  CAS  PubMed  Google Scholar 

  • Norikane A, da Silva JAT, Tanaka M (2013) Growth of in vitro Oncidesa plantlets cultured under cold cathode fluorescent lamps (CCFLs) with super-elevated CO2 enrichment. AoB Plants 5:plt044. doi:10.1093/aobpla/plt044

    Article  PubMed Central  Google Scholar 

  • Pérez M, Bueno MA, Escalona M, Toorop P, Rodríguez R, Cañal MJ (2013) Temporary immersion systems (RITA©) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production. Trees 27:1277–1284. doi:10.1007/s00468-013-0876-y

    Article  Google Scholar 

  • Pijut PM, Lawson SS, Michler CH (2011) Biotechnological efforts for preserving and enhancing temperate hardwood tree biodiversity, health, and productivity. In Vitro Cell Dev Biol Plant 47:123–147. doi:10.1007/s11627-010-9332-5

    Article  Google Scholar 

  • Quiala E, Cañal MJ, Meijón M, Rodriguez R, Chavéz M, Valledon L, Feria M, Barbón R (2012) Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments. Plant Cell Tissue Organ Cult 109:223–234. doi:10.1007/s11240-011-0088-3

    Article  CAS  Google Scholar 

  • Sacco E, Mascarello C, Pamato M, Musso V, Ruffoni B (2015) Evaluation of temporary immersion system for in vitro propagation of Stevia rebaudiana Bertoni. Acta Hortic 1083:327–333

    Article  Google Scholar 

  • Sáez PL, Bravo LA, Latsague MI, Toneatti MJ, Coopman RE, Álvarez CE, Sánchez-Olate M, Ríos DG (2015) Influence of in vitro growth conditions on the photosynthesis and survival of Castanea sativa plantlets during ex vitro transfer. Plant Growth Regul 75:625–639. doi:10.1007/s10725-014-9965-1

    Article  Google Scholar 

  • Sánchez MC, San-José MC, Ferro E, Ballester A, Vieitez AM (1997) Improving micropropagation conditions for adult-phase shoots of chestnut. J Hortic Sci 72:433–443

    Google Scholar 

  • Takayama S, Akita M (1998) Bioreactor techniques for large-scale culture of plant propagules. Adv Hortic Sci 12:93–100

    Google Scholar 

  • Tanaka M, Nagae S, Fukai S, Goi M (1992) Growth of tissue cultured Spathiphyllum on rockwool in a novel film culture vessel under high CO2. Acta Hortic 314:139–146

    Article  Google Scholar 

  • Troch V, Sapeta H, Werbrouck S, Geelen D, Van Labeke MC (2010) In vitro culture of chestnut (Castanea sativa Mill.) using temporary immersion bioreactors. Acta Hortic 885:383–390

    Article  Google Scholar 

  • Vidal N, Vieitez AM, Fernández MR, Cuenca B, Ballester A (2010) Establishment of cryopreserved gene banks of European chestnut and cork oak. Eur J Forest Res 129:635–643. doi:10.1007/s10342-010-0364-5

    Article  CAS  Google Scholar 

  • Vidal N, Correa B, Rial E, Regueira M, Sánchez C, Cuenca B (2015) Comparison of temporary and continuous immersion systems for micropropagation of axillary shoots of chestnut and willow. Acta Hortic 1083:227–233

    Article  Google Scholar 

  • Vieitez AM, Ballester A, San Jose MC, Vieitez E (1985) Anatomical and chemical studies on vitrified shoots of chestnut regenerated in vitro. Physiol Plant 65:177–184. doi:10.1111/j.1399-3054.1985.tb02379.x

    Article  CAS  Google Scholar 

  • Vieitez AM, Sánchez MC, García-Nimo ML, Ballester A (2007) Protocol for micropropagation of Castanea sativa Mill. In: Jain SM, Häggman H (eds) Protocols for micropropagation of woody trees and fruits. Springer, Heidelberg, pp 299–312

    Chapter  Google Scholar 

  • Watt MP (2012) The status of temporary immersion system (TIS) technology for plant micropropagation. Afr J Biotechn 11:14025–14035. doi:10.5897/AJB12.1693

    CAS  Google Scholar 

  • Welander M, Persson J, Asp H, Zhu LH (2014) Evaluation of a new vessel system based on temporary immersion system for micropropagation. Sci Hortic 179:227–232. doi:10.1016/j.scienta.2014.09.035

    Article  CAS  Google Scholar 

  • Xiao Y, Niu G, Kozai T (2011) Development and application of photoautotrophic micropropagation plant system. Plant Cell Tissue Organ Cult 105:149–158. doi:10.1007/s11240-010-9863-9

    Article  CAS  Google Scholar 

  • Zhu LH, Li XY, Welander M (2005) Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle. Plant Cell Tissue Organ Cult 81:313–318. doi:10.1007/s11240-004-6659-9

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank Maite García, Begoña Pato, Begoña Correa and Estiben Becerra for technical assistance. This research was partially funded by the Xunta de Galicia (Spain) through project 09MRU016E.

Author information

Authors and Affiliations

  1. Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apdo 122, 15780, Santiago de Compostela, Spain

    N. Vidal & B. Blanco

  2. TRAGSA, Vivero de Ourense, Ctra. Maceda-Valdrey km 2, 32700, Maceda, Ourense, Spain

    B. Cuenca

Authors
  1. N. Vidal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. Blanco
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Cuenca
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N. Vidal.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vidal, N., Blanco, B. & Cuenca, B. A temporary immersion system for micropropagation of axillary shoots of hybrid chestnut. Plant Cell Tiss Organ Cult 123, 229–243 (2015). https://doi.org/10.1007/s11240-015-0827-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0827-y

Keywords

Search

Navigation