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Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system

Abstract

A protocol for culturing chestnut axillary shoots with the basal sections continuously immersed in liquid medium was developed and the influence of various parameters was assessed: explant type and size, support material, bioreactor size and aeration regime. Shoots excised from eight selected chestnut genotypes were successfully cultured in 10 L bioreactors in liquid Murashige and Skoog medium with half strength nitrates and supplemented with 0.05 mg/L N 6-benzyladenine and 30 g/L sucrose. Forced ventilation and the use of a support material were essential for producing healthy, non-hyperhydric shoots. High proliferation rates were achieved and good quality shoots ready for multiplication, rooting and acclimatization were obtained. Larger leaves showing higher levels of photosynthetic pigments were observed in shoots cultured in bioreactors, suggesting a certain degree of photoautotrophy in shoots cultured under forced ventilation. This is the first demonstration of the production of chestnut shoots in stationary liquid medium, indicating the feasibility of the method for large-scale propagation of the species.

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References

  1. 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

  2. Arigita L, González A, Sánchez Tamés R (2002) Influence of CO2 and sucrose on photosynthesis and transpiration of Actinidia deliciosa explants cultured in vitro. Physiol Plant 115:166–173. doi:10.1034/j.1399-3054.2002.1150119.x

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

  4. 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. doi:10.1080/14620316.2003.11511671

  5. Chakrabarty D, Dewir YH, Hahn EJ, Datta SK, Paek KY (2007) The dynamics of nutrient utilization and growth of apple root stock ‘M9 EMLA’ in temporary versus continuous immersion bioreactors. Plant Growth Regul 51:11–19. doi:10.1007/s10725-006-9115-5

  6. Cournac L, Dimon B, Carrier P, Lohou A, Chagvardieff P (1991) Growth and photosynthetic characteristics of Solanum tuberosum plantlets cultivated in vitro in different conditions of aeration, sucrose supply and CO2 enrichment. Plant Physiol 97:112–117. doi:10.1104/pp.97.1.112

  7. Cui Y, Hahn E, Kozai T, Paek K (2000) Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro. Plant Cell Tiss Organ Cult 62:219–226. doi:10.1023/A:1006412321864

  8. Dimasi-Theriou K, Economou AS (1995) Ethylene enhances shoot formation in cultures of the peach rootstock GF-677 (Prunus persica × P. amygdalus). Plant Cell Rep 15:87–90. doi:10.1007/BF01690260

  9. Ermayanti TM, Imelda M, Tajuddin T, Kubota C, Kozai T (1999) Growth promotion by controlling in vitro environment in micropropagation of tropical plant species. Proc. The Tokyo Intl. Forum on Conservation and Sustainable Use of Tropical Bioresources. pp 10–25. NEDO and IBA, Tokyo

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

  11. Feito I, González A, Centeno M, 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

  12. Ferris R, Sabatti M, Miglietta F, Mills RF, Taylor G (2001) Leaf area is stimulated in Populus by free air CO2 enrichment (POPFACE), through increased cell expansion and production. Plant Cell Environ 24:305–315. doi:10.1046/j.1365-3040.2001.00684.x

  13. Fujiwara K, Kira S, Kozai T (1992) Time course of CO2 exchange of potato cultures in vitro with different sucrose concentrations in the culture medium. J Agr Met 48:49–56

  14. Goel MK, Kukreja AK, Khanuja SPS (2007) Cost-effective approaches for in vitro mass propagation of Rauwolfia serpentina Benth. Ex Kurz. Asian J Plant Sci 6:957–961. doi:10.3923/ajps.2007.957.961

  15. Goel MK, Kukreja AK, Bisht NS (2009) In vitro manipulations in St. John’s wort (Hypericum perforatum L.) for incessant and scale up micropropagation using adventitious roots in liquid medium and assessment of clonal fidelity using RAPD analysis. Plant Cell Tiss Organ Cult 96:1–9. doi:10.1007/s11240-008-9453-2

  16. Gonçalves JC, Diogo G, Amâncio S (1998) In vitro propagation of chestnut (Castanea sativa × 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

  17. González A, Arigita L, Majada J, Sánchez Tamés R (1997) Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L. Plant Growth Regul 22:1–6. doi:10.1023/A:1005751017498

  18. 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

  19. Hassankhah A, Vahdati K, Lotfi M, Mirmasoumi M, Preece J, Assareh M-H (2014) Effects of ventilation and sucrose concentrations on the growth and plantlet anatomy of micropropagated Persian walnut plants. Int J Hortic Sci Technol 1:111–120

  20. Kiferle C, Lucchesini M, Maggini R, Pardossi A, Mensuali-Sodi A (2014) In vitro culture of sweet basil: gas exchanges, growth and rosmarinic acid production. Biol Plant 58:601–610. doi:10.1007/s10535-014-0434-5

  21. Kirdmanee C, Kitaya Y, Kozai T (1995) Effects of CO2 enrichment and supporting material in vitro on photoautotrophic growth of Eucalyptus plantlets in vitro and ex vitro. In Vitro Cell Dev Biol Plant 31:144–149. doi:10.1007/BF02632010

  22. Kozai T (1991) Photoautotrophic micropropagation. In Vitro Cell Dev Biol Plant 27:47–51. doi:10.1007/BF02632127

  23. Kozai T, Kubota C (2001) Developing a photoautotrophic micropropagation system for woody plants. J Plant Res 114:525–537. doi:10.1007/PL00014020

  24. Kozai T, Kubota C (2005) In vitro aerial environments and their effects on growth and development of plants. In: Kozai T, Afreen F, Zobayed SMA (eds) Photoautotrophic (sugar-free medium) micropropagation as a new micropropagation and transplant production system. Springer, Dordrecht, pp 31–52

  25. Kubota C, Kakizaki N, Kozai T, Kasahara K, Nemoto J (2001) Growth and net photosynthetic rate of tomato plantlets during photoautotrophic and photomixotrophic micropropagation. Hortic Sci 36:49–52

  26. Lai CC, Lin HM, Nalawade SM, Fang W, Tsay HS (2005) Hyperhydricity in shoot cultures of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels. J Plant Physiol 162:355–361. doi:10.1016/j.jplph.2004.07.015

  27. Latawa Y, Shukla MR, Saxena PK (2016) An efficient temporary immersion system for micropropagation of hybrid hazelnut. Botany 94:1–8. doi:10.1139/cjb-2015-0111

  28. Lucchesini M, Mensuali-Sodi A, Massai R, Gucci R (2001) Development of autotrophy and tolerance to acclimatization of Myrtus communis transplants cultured in vitro under different aeration. Biol Plant 44:167–174. doi:10.1023/A:1010277403705

  29. Maner L, Merkle S (2010) Polymerized peat plugs improve American chestnut somatic embryo germination in vitro. J American Chestnut Foundation 24(3):16

  30. Mingozzi M, Montello P, Merkle S (2009) Adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides) cultured under photoautotrophic conditions. Tree Physiol 29:333–343. doi:10.1093/treephys/tpn029

  31. Miyashita Y, Kitaya Y, Kubota C, Kozai T (1996) Photoautotrophic growth of potato plantlets as affected by explant leaf area, fresh weight and stem length. Sci Hortic 65:199–202. doi:10.1016/0304-4238(96)00877-1

  32. Moncaleán P, Cañal MJ, Feito I, Rodríguez A, Fernández B (1999) Cytokinins and mineral nutrition in Actinidia deliciosa shoots cultured in vitro. J Plant Physiol 155:606–612. doi:10.1016/S0176-1617(99)80061-3

  33. Moncaleán P, Cañal MJ, Fernández H, Fernández B, Rodríguez A (2003) Nutritional and gibberellic acid requirements in kiwifruit vitroponic cultures. In Vitro Cell Dev Biol Plant 39:49–55. doi:10.1079/IVP2002371

  34. Mosaleeyanon K, Cha-um S, Kirdmanee C (2004) Enhanced growth and photosynthesis of rain tree (Samanea saman Merr.) plantlets in vitro under a CO2-enriched condition with decreased sucrose concentrations in the medium. Sci Hortic 103:51–63. doi:10.1016/j.scienta.2004.02.010

  35. 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

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

  37. 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 For Res 1:227–230. doi:10.1007/BF02348330

  38. Nguyen QT, Kozai T (2001) Photoautotrophic micropropagation of tropical and subtropical woody plants. In: Morohoshi N, Komamine A (eds) Molecular breeding of woody plants. Elsevier, Amsterdam, pp 335–344

  39. Nguyen QT, Kozai T (2005) Photoautotrophic micropropagation of woody species. In: Kozai T, Afreen F, Zobayed SMA (eds) Photoautotrophic (sugar-free medium) micropropagation as a new mipropagation and transplant production system. Springer, Dordrecht, pp 123–146

  40. Nguyen QT, Kozai T, Heo J, Thai DX (2001) Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition. Plant Cell Tiss Org Cult 66:217–225. doi:10.1023/A:1010662413486

  41. Nguyen QT, Xiao Y, Kozai T (2016) Photoautotrophic micropropagation. In: Kozai T, Niu G, Takagaki M (eds) Plant factory—an indoor vertical farming system for efficient quality food production, 1 edn. Chapter 20, Elsevier, Amsterdam, pp. 271–283. doi:10.1016/B978-0-12-801775-3.00020-2

  42. 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

  43. Norikane A, Teixeira da Silva JA, 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

  44. Nour KA, Thorpe TA (1994) The effect of the gaseous state on bud induction and shoot multiplication in vitro in eastern white cedar. Physiol Plant 90:163–172. doi:10.1111/j.1399-3054.1994.tb02207.x

  45. Petrova M, Zayova E, Todorova M, Stanilova M (2014) Enhancement of Arnica montana in vitro shoot multiplication and sesquiterpene lactones production using temporary immersion system. IJPSR 5:5170–5176. doi:10.13040/IJPSR.0975-8232.5(12).5170-76

  46. Pierik RLM (1987) The influence of plant material on growth and development. In: In Vitro culture of higher plants. Martinus Nijhoff Publishers, Dordrecht, pp 107–114

  47. Preece JE (2010) Micropropagation in stationary liquid media. Propag Ornam Plants 10:183–187

  48. Rathore JS, Rai MK, Phulwaria M, Shekhawat NS (2014) A liquid culture system for improved micropropagation of mature Acacia nilotica (L.) Del. ssp. indica and ex vitro rooting. Proc Natl Acad Sci India Sect B 84:193–200. doi:10.1007/s40011-013-0204-8

  49. Reed S, Schnell R, Moore JM, Dunn C (2012) Chlorophyll a + b content and chlorophyll fluorescence in avocado. J Agric Sci 4:29–36. doi:10.5539/jas.v4n4p29

  50. Rojas-Martínez L, Visser RGF, de Klerk G-J (2010) The hyperhydricity syndrome: waterlogging of plant tissues as a major cause. Propag Ornam Plants 10: 169–175

  51. Sáez P, Bravo L, Latsague M, Sanchez-Olate M, Ríos D (2012) Increased light intensity during in vitro culture improves water loss control and photosynthetic performance of Castanea sativa grown in ventilated vessels. Sci Hortic 130:7–16. doi:10.1016/j.scienta.2012.02.005

  52. 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

  53. Saldanha CW, Otoni CG, Azevedo JLF, Dias LLC, Rêgo MM, Otoni WC (2012) A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tiss Org Cult 110:413–422. doi:10.1007/s11240-012-0162-5

  54. Saldanha CW, Otoni CG, Notini MM, Kuki KN, Cruz ACF, Rubio Neto A, Dias LLC, Otoni WC (2013) A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. In Vitro Cell Dev Biol Plant 49:433–444. doi:10.1007/s11627-013-9529-5

  55. 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. doi:10.1080/14620316.1997.11515531

  56. Savio LEB, Astarita LV, Santarém ER (2012) Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium. Plant Cell Tiss Organ Cult 108:465–472. doi:10.1007/s11240-011-0058-9

  57. 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. doi:10.17660/ActaHortic.1992.314.16

  58. Tanaka M, Giang DTT, Murakami A (2005) Application of a novel disposable film culture system to photoautotrophic micropropagation of Eucalyptus uro-grandis (Urophylia × grandis). In Vitro Cell Dev Biol Plant 41:173–180. doi:10.1079/IVP2004622

  59. Thorpe T, Stasolla C, Yeung EC, De Klerk GJ, Roberts A, George EF (2008) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture, volume 1. The background, 3 edn. Springer, Dordrecht, pp. 115–173

  60. 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. doi:10.17660/ActaHortic.2010.885.54

  61. Vibha JB, Shekhawat NS, Mehandru P, Dinesh R (2014) Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting. Physiol Mol Biol Plant 20:81–87. doi:10.1007/s12298-013-0213-3

  62. 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

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

  64. Vidal N, Correa B, Rial E, Regueira M, Sánchez C, Cuenca B (2015b) Comparison of temporary and continuous immersion systems for micropropagation of axillary shoots of chestnut and willow. Acta Hortic 1083:227–233. doi:10.17660/ActaHortic.2015.1083.27

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

  66. 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

  67. Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313. doi:10.1016/S0176-1617(11)81192-2

  68. Xiao Y, Zhao J, Kozai T (2000) Practical sugar-free micropropagation system using large vessels with forced ventilation. In: Kubota C, Chun C. (eds) Transplant production in the 21st century. Kluwer Academic Publishers, Dordrecht, pp. 266–273

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

  70. Zobayed SMA, Armstrong J, Armstrong W (1999) Cauliflower shoot-culture: effects of different types of ventilation on growth and physiology. Plant Sci 141:209–217. doi:10.1016/S0168-9452(98)00232-5

  71. Zobayed SMA, Afreen-Zobayed F, Kubota C, Kozai T (2000) Mass propagation of Eucalyptus camaldulensis in a scaled-up vessel under in vitro photoautotrophic condition. Ann Bot 85:587–592. doi:10.1006/anbo.1999.1106

  72. Zobayed SMA (2005) Ventilation in micropropagation. In: Kozai T, Afreen F, Zobayed SMA (eds) Photoautotrophic (sugar-free medium) micropropagation as a new micropropagation and transplant production system. Springer, Dordrecht, pp 147–186

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Acknowledgements

We acknowledge the contribution of our dear colleague and friend, Brais Bogo Graña, who died before completion of the manuscript. We thank Alejandro Díaz, Rafael Sánchez and Patricia Val for technical assistance. This research was partly funded through the FEDER INNTERCONECTA 2013/2014 programme (Project INTEGRACASTANEA EXP00064828/ITC-20133040).

Author information

BC, CS and NV conceived and designed the experiment. NV, AA, BB, BB and BC performed the experiments and analysed and interpreted the data. NV and CS wrote the manuscript. All authors read and approved the final manuscript.

Correspondence to N. Vidal.

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The authors declare that they have no conflict of interest.

Additional information

Communicated by Francisco de Assis Alves Mourão Filho.

Deceased: B. Bogo.

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Cuenca, B., Sánchez, C., Aldrey, A. et al. Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system. Plant Cell Tiss Organ Cult 131, 307–320 (2017). https://doi.org/10.1007/s11240-017-1285-5

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Keywords

  • Bioreactors
  • Castanea
  • Forced ventilation
  • Large-scale propagation