Eight apple ( <i> Malus x domestica </i> ) and two pear ( <i> Pyrus communis </i> ) traditional cultivars of commercial interest were selected from the germplasm bank CIAM (Centro de Investigaciones Agrarias de Mabegondo, NW of Spain) one of the most important collections of The Iberian Peninsula. In order to define the establishment protocol, apical buds 1.0 - 1.5 millimetre length of the selected cultivars were established in solid mineral medium Murashige and Skoog (MS) with modified vitamins (thiamine 10X) and supplemented with 1 mg · L -1 6-benzyladenine (BA), 0.2 mg · L - 1 gibberellic acid (GA 3 ) and 0.3 mg · L - 1 of indole-3-butyric acid (IBA). The highest shoot initiation rates were achieved with apple cultivar “ Principe Grande ” (55%) and “ Barburiña ” pear cultivar (50%); the lowest rate was obtained with apple cultivar “ Camoesa ” (11%). Initiation rates for shoots culture varied between 22% and 37% for the other cultivars. The cytokinins BA, zeatin, 2-isopentyladenine (2iP) and thidiazuron (TDZ) were tested at concentrations of 0, 0.25, 0.5 and 1 mg · L - 1 in the absence or presence of IBA (0.1 mg · L - 1 ) for the multiplication step. For all cultivars, the highest multiplication rates were obtained with medium supplemented with TDZ and also in media containing BA. The <i> in vitro </i> rooting success of shoots in medium with 0.1 mg · L - 1 IBA without cytokinins, and the survival rate of plantlets acclimatized under greenhouse conditions depended on the genotype of the different cultivars. <i> In vitro </i> propagation of these ten traditional apple and pear cultivars in NW Spain has not previously been reported.
KeywordsApple ShootsPear ShootsAxillary BudMultiplicationCytokininGene Bank
FAO (2013) Fuente. http://faostat3.fao.org
Flachowsky, H. and Höfer, M. (2010) The German Fruit Genebank, a Decentral Network for Sustainable Preservation of Fruit Genetic Resources. Journal für Kulturpflanzen, 62, 9-16.
Pereira-Lorenzo, S., Ramos-Cabrer, A.M., Ascasíbar-Errasti, J. and Piñeiro-Andión, J. (2003) Analysis of Apple Germplasm in Northwestern Spain. Journal of the American Society for Horticultural Science, 128, 67-84.
Pereira-Lorenzo, S., Ramos-Cabrer, A.M. and Díaz-Hernández, M.B. (2007) Evaluation of Genetic Identity and Variation of Local Apple Cultivars (Malus x domestica Borkh.) from Spain Using Microsatellite Markers. Genetic Resources Crop Evolution, 54, 405-420. https://doi.org/10.1007/s10722-006-0003-7
European Plant Protection Organization (1999) EPPO Standars PM 4/27(1) Certification Schemes Malus, Pyrusand cydonia. European Plant Organization, Paris.
Panattoni, A., Luvisi, A. and Triolo, E. (2013) Review. Elimination of Viruses in Plants: Twenty Years of Progress. Spanish Journal of Agriculture Research, 11, 173-188. https://doi.org/10.5424/sjar/2013111-3201
Manganaris, G.A., Economou, A.S., Boubourakas, I.N. and Katis, N.I. (2003) Elimination of PPV and PNRSV through Thermotherapy and Meristem-Tip Culture in Nectarine. Plant Cell Reports, 22, 195-200. https://doi.org/10.1007/s00299-003-0681-y
Paprstein, F., Sèdlak, J., Polak, J., Svobodova, L., Hassan, M. and Bryxiova, M. (2008) Results of in vitro Thermotherapy of Apple Cultivars. Plant Cell Tissue and Organ Culture, 94, 347-352. https://doi.org/10.1007/s11240-008-9342-8
Deberegh, P.C. and Zimmerman, R.H. (2002) Micropropagation Technology and Application. Kluwer Academic Publishers, Boston.
Sèdlak, J. and Paprstein, F. (2016) In Vitro Establishment and Proliferation of Apple Cultivars. Acta Horticulturae (ISHS), 1113, 107-112. https://doi.org/10.17660/ActaHortic.2016.1113.15
Xu, J., Wang, Y., Zhang, Y. and Chai, T.Y. (2008) Rapid in Vitro Multiplication and ex Vitro Rooting of Malus zumi (Matsumura) Rehd. Acta Physiologiae Plantarum, 30, 129-132. https://doi.org/10.1007/s11738-007-0075-9
Muniz, A.W., Luiz de Sá, E., Dalagnol, G.L. and Filho, J.A. (2013) Rooting and Acclimatization of Micropropagated Marubakai Do Apple Rootstock Using Adesmialatifolia rhizobia. SpringerPlus, 2, 437. https://doi.org/10.1186/2193-1801-2-437
Dobránszki, J. and Teixeira da Silva, J.A. (2010) Micropropagation of Apple—A Review. Biotechnology Advances, 28, 462-488. https://doi.org/10.1016/j.biotechadv.2010.02.008
Hirabayashi, T., Moriguchi, T., Kozaki, I., Yamamoto, Y. and Matsuzaki, S. (1987) In Vitro Propagation of Pyrus Shoots Tips. International Society for Horticultural Science VII International Symposium on Pear, 14, 9-16.
Banno, K., Yoshida, K., Hayashi, S. and Tanabe, K. (1989) In Vitro Propagation of Japanese Pear Cultivars. Journal Japanese Society Horticultural Science, 58, 37-42. https://doi.org/10.2503/jjshs.58.37
Erig, A.C. and Fortes, G.R. (2002) In Vitro Establishment of Pear (Pyrus spp.) Starting from Meristems and Buds. Ciencia Rural, 32, 577-582. https://doi.org/10.1590/S0103-84782002000400005
Thakur, A. and Kanwar, J.S. (2008) Mircropropagation of “Wild Pear” Pyrus pyrifolia (Burm F) Nakai I Explant Establishment and Shoot Multiplication. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 36, 103-108.
Lane, W.D., Iketani, H. and Hayashi, T. (1998) Shoot Regeneration from Cultured Leaves of Japanese Pear (Pyrus pyrifolia). Plant Cell Tissue and Organ Culture, 54, 9-14. https://doi.org/10.1023/A:1006032707849
Caboni, E., D’Angeli, S., Chiappetta, A., Innocenti, A.M., Van Onckelen, H. and Damiano, C. (2002) Adventitious Shoot Regeneration from Vegetative Shoot Apices in Pear and Putative Role of Cytokinin Accumulation in the Morphogenetic Process. Plant Cell Tissue and Organ Culture, 70, 199-206. https://doi.org/10.1023/A:1016304106529
Poudyal, B.K., Zhang, Y. and Du, G. (2008) Adventitious Shoot Regeneration from the Leaves of Some Pear Varieties (Pyrus spp.) Grown in Vitro. Frontiers of Agriculture in China, 2, 82-92. https://doi.org/10.1007/s11703-008-0016-4
Yepes, L.M. and Aldwinckle, S.H. (1994) Micropropagation of Thirteen Malus Cultivars and Rootstocks, and Effect of Antibiotics on Proliferation. Plant Grow Regulation, 15, 55-67. https://doi.org/10.1007/BF00024677
Tang, H., Luo, Y. and Liu, C. (2008) Plant Regeneration from in Vitro Leaves of Four Commercial Pyrus Species. Plant, Soil and Environment, 54, 140-148.
George, E.F. and Davies, W. (2008) Effects of the Physical Environment. In: George, E.F., Hall, M.A. and De Klerk, G.J., Eds., Plant Propagation by Tissue Culture, 3rd Edition, Netherlands Springer, Dordrecht, 423-464.
Berardi, G., Infante, R. and Neri, D. (1993) Micropropagation of Pyrus calleryana DCN from Seedlings. Scientia Horticulturae, 53, 157-165. https://doi.org/10.1016/0304-4238(93)90146-H
Sarwar, M. and Skirvin, R.M. (1997) Effect of Thidiazuron and 6-Benzylaminopurine on Adventitious Shoot Regeneration from Leaves of Three Strains of ‘Mcintosh’ Apple (Malus x domestica Borkh.) in Vitro. Scientia Horticulturae, 68, 95-10. https://doi.org/10.1016/S0304-4238(96)00971-5
Shibli, R.A., Ajlouni, M.M., Jaradat, A., Aljanabi, S. and Shatnawi, M. (1997) Micropropagation in Wild Pear (Pyrus syrica). Scientia Horticulturae, 68, 237-242. https://doi.org/10.1016/S0304-4238(96)00972-7
Dobránszki, J., Hudák, I., Magyar-Tábori, K., Jámbor-Benczúr, E., Galli, Z.S. and Kiss, E. (2004) Effects of Different Cytokinins on the Shoot Regeneration from Apple Leaves of ‘Royal Gala’ and ‘M.26’. Horticultural Science, 10, 69-75.
Magyar-Tábori, K., Dobránszki, J., Teixeira da Silva, A., Bulley, S.M. and Hudák, I. (2010) The Role of Cytokinins in Shoot Organogenesis in Apple. Plant Cell Tissue and Organ Culture, 101, 251-267. https://doi.org/10.1007/s11240-010-9696-6
Kalinina, A. and Brown, D.C.W. (2007) Micropropagation of Ornamental Prunus spp. and GF305 Peach, a Prunus Viral Indicator. Plant Cell Reports, 26, 927-935. https://doi.org/10.1007/s00299-007-0315-x
Murashige, T. and Skoog, F. (1962) A Revised Medium for Rapid Growth and Bioassay with Tobacco Tissues Cultures. Physiologiae Plantarum, 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Li, X., Xu, M. and Korban, S.S. (2002) DNA Methylation Profiles Differ between Field and in Vitro-Grown Leaves of Apple. Journal of Plant Physiology, 159, 1229-1234. https://doi.org/10.1078/0176-1617-00899
International Business Machines Corporation (2011) IBM SPSS Statistics for Windows, Version 20.0. IBM Corp., Armonk.
Chakrabarty, D., Hahn, E.J., Yoon, Y.J. and Paek, K.Y. (2003) Micropropagation of Apple Rootstock M.9 EMLA Using Bioreactor. Journal of Horticultural Science and Biotechnology, 78, 605-609. https://doi.org/10.1080/14620316.2003.11511671
Rodriguez, R., Diaz-Sala, C., Cuozzo, L. and Ancora, G. (1991) Pear in Vitro Propagation Using a Double-Phase Culture System. HortScience, 26, 62-64.
Wang, L.P., Wang, G.P., Hong, N., Tang, R.R. and Deng, X.Y. (2006) Effect of Thermotherapy on Elimination of Apple Stem Grooving Virus and Apple Chlorotic Leaf Spot Virus for in Vitro Cultured Pear Shoot Tips. HortScience, 41, 729-732.
Dalal, M.A., Das, B., Sharma, A.K., Mir, M.A. and Sounduri, A.S. (2006) In Vitro Cloning of Apple (Malus domestica Borkh) Employing Forced Shoot Tip Cultures of M9 Rootstock. Indian Journal of Biotechnology, 5, 543-550.
Moretti, C., Scozzoli, A., Pasini, D. and Paganelli, F. (1992) In Vitro Propagation of Pear Cultivars. Acta Horticulturae, 300, 115-118. https://doi.org/10.17660/ActaHortic.1992.300.12
Kadota, M. and Niimi, Y. (2003) Effects of Cytokinin Types and Their Concentrations on Shoot Proliferation and Hyperhydricity in in Vitro Pear Cultivar Shoots. Plant Cell Tissue and Organ Culture, 72, 261-265. https://doi.org/10.1023/A:1022378511659
Wang, M.R., Li, B.Q., Feng, C.H. and Wang, Q.C. (2016) Culture of Shoot Tips from Adventitious Shoots Can Eradicate Apple Stem Pitting Virus but Fails in Apple Stem Grooving Virus. Plant Cell Tissue and Organ Culture, 125, 283-291. https://doi.org/10.1007/s11240-016-0948-y
Tan, R.R., Wang, L.P., Hong, N. and Wang, G.P. (2010) Enhanced Efficiency of Virus Eradication Following Thermotherapy of Shoot-Tip Cultures of Pear. Plant Cell Tissue and Organ Culture, 101, 229-235. https://doi.org/10.1007/s11240-010-9681-0
Fasolo, F., Zimmerman, R.H. and Fordham, I. (1989) Adventitious Shoots Formation on Excised Leaves of in Vitro Grown Shoots of Apple Cultivars. Plant Cell Tissue and Organ Culture, 16, 75-87. https://doi.org/10.1007/BF00036516
Pawlicki, N. and Welander, M. (1994) Adventitious Shoot Regeneration from Leaf Segments of in Vitro Cultured Shoots of the Apple Rootstock Jork 9. Horticultural Science, 69, 687-696. https://doi.org/10.1080/14620316.1994.11516501
Hanke, V., Rohde, A. and Grafe, C. (1991) Studies on Regeneration from Somatic Tissue in Vitro. Adventitious Shoot Formation on Leaf Explants in Apple (Malus x domestica Borkh). Dresden-Pillnitz, 56, 214-220.
Huetteman, C.A. and Preece, J.E. (1993) Thidiazuron: A Potent Cytokinin for Woody Plant Tissue Culture. Plant Cell Tissue and Organ Culture, 33, 105-119. https://doi.org/10.1007/BF01983223
Xiao, Z., Ji, N., Zhang, X., Zhang, Y., Wang, Y., Wu, T., Xu, X. and Han, Z. (2014) The Loss of Juvenility Elicits Adventitious Rooting Recalcitrance in Apple Rootstocks. Plant Cell Tissue and Organ Culture, 119, 51-63. https://doi.org/10.1007/s11240-014-0513-5
Damiano, C., Frattarelli, A. and Giorgioni, M. (2000) Micropropagation of Temperate Fruit Trees through Temporary Immersion Technique: The Case of Pear. Bulletin of the Fruit Tree Research Station. Series A, 120-121.