Abstract
Ponkan mandarin (Citrus reticulata Blanco) is one of the most important commercial cultivars of mandarin orange in China. This study reports an improved and efficient protocol for in vitro plant regeneration of Ponkan mandarin. Epicotyl segments, which were cut longitudinally into two halves, were used as explants. The shoot regeneration frequency was significantly increased by longitudinal cutting. A 100% shoot regeneration frequency and 13.2 shoots per explant were obtained when cultures were maintained in darkness for 20 d before being transferred to light conditions, with bud induction by indirect organogenesis. A 72.5% shoot regeneration frequency and 7.8 shoots per explant were obtained when explants were incubated under a 16-h light photoperiod continuously with buds differentiating directly from the cutting wound surface. The optimal medium for shoot formation was Murashige and Tucker basal medium supplemented with 2 mgL−1 BA and 30 gL−1 sucrose both under light conditions. The addition of the auxin NAA reduced the frequency of regeneration. A “filter-paper bridge” technique was used for rooting in this study. The basal portion of regenerated shoots was dipped into 1,000 mgL−1 IBA solution for 15 min before placement on a filter-paper bridge that was maintained in 1/2 MS liquid medium supplemented with 10 gL−1 sucrose. Eighty percent of the shoots rooted, and an average of 2.0 roots per shoot were achieved. Survival rate through acclimatization was 100%.
References
Almeida W. A. B.; Mourao-Filho F. A. A.; Pino L. E.; Boscariol R. L.; Rodriguez A. P. M.; Mendes B. M. J. Genetic transformation and plant recovery from mature tissues of Citrus sinensis L. Osbeck. Plant Sci 164: 203–211; 2003.
Boscariol R. L. W.; Almeida A. B. The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 22: 122–128; 2003.
Cervera M.; Pina J.; Juárez J.; Navarro A.; Navarro L.; Peña L. Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18: 271–278; 1998.
de Oliveira M. L.; Febres V. J.; Costa M. G.; Moore G. A.; Otoni W. C. High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Rep 28: 387–395; 2009.
Dominguez A.; Guerri J.; Cambra M.; Navarro L.; Moreno P.; Pena L. Efficient production of transgenic citrus plants expressing of the coat protein gene of Citrus tristeza virus. Plant Cell Rep 19: 427–433; 2000.
Edriss M. H.; Burger D. W. In vitro propagation of Troyer citrange from epicotyl segments. Sci Hort 23: 159–162; 1984.
Garcia-Luis A.; Bordony Y.; Moreira-Dias J. M.; Molina R. V.; Guardiola J. L. Explant orientation and polarity determine the morphogenic response of epicotyl segments of troyer citrange. Ann Bot 84: 715–723; 1999.
Ghorbel R.; Dominguez A.; Navarro L.; Pena L. Efficiency genetic transformation of sour orange (Citrus aurantium L.) and production of transgenic trees containing the coat protein gene of Citrus tristeza virus. Tree Physiol 20: 1183–1189; 2000.
Ghorbel R.; Navarro L.; Durán-Vila N. Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C. aurantium) and alemow (C. macrophylla). J Hort Sci Biotech 73: 323–327; 1998.
Gutiérrez-E M. A.; Luth D.; Moore G. A. Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of Citrus tristeza virus. Plant Cell Rep 16: 745–753; 1997.
Huang J. Q.; Wu Zh R.; Sun Zh H. Several physiological factors influencing adventitious bud regeneration from the epicotyls of Ponkan (Citrus reticulata Blanco). Plant Physiol Communication 41(1): 37–40; 2005. In Chinese.
Kaneyoshi J.; Kobayashi S.; Nakamura Y.; Shigemoto N.; Doi Y. A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.). Plant Cell Rep 13: 541–545; 1994.
Kayim M.; Ceccardi T. L.; Berretta M. J. G.; Barthe G. A.; Derrick K. S. Introduction of a citrus blight-associated gene into Carrizo citrange [C. sinensis (L.) Osbc. X Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation. Plant Cell Rep 23: 377–385; 2004.
Khawale R. N.; Singh S. K.; Garg G.; Baranwal V. K.; Alizadeh S. A. Agrobacterium-mediated genetic transformation of Nagpur mandarin (Citrus reticulata Blanco). Curr Sci 91(12): 1700–1705; 2006.
Li D. D.; Shi W.; Deng X. X. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Rep 21: 153–156; 2002.
Maggon R.; Singh B. D. Promotion of adventitious bud regeneration by ABA in combination with BAP in epicotyl and hypocotyl explants of sweet orange (Citrus sinensis L. Osbeck). Sci Hort 63: 123–128; 1995.
Moore G. A.; Jacono C. C.; Neidigh J. L.; Lawrence S. D.; Cline K. Agrobacterium-mediated transformation of Citrus stem explants and regeneration of transgenic plants. Plant Cell Rep 11: 238–242; 1992.
Moreira-Dias J. M.; Molina R. V.; Bordõn Y. J. L.; Guardiola J. L.; Garcîa-Luis A. 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: 103–110; 2000.
Murashige T.; Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–479; 1962.
Murashige T.; Tucker D. P. H. Growth factor requirements of citrus tissue culture. Proc First Int Citrus Symp 3: 1155–1161; 1969.
Peña L.; Cervera M.; Juárez J.; Navarro A.; Pina J. A.; Durán-Vila N.; Navarro L. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Rep 14: 616–619; 1995.
Peña L.; Martín-Trillo M.; Juárez J.; Pina J. A.; Navarro L.; Martinez-Zapater J. M. Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nature Biotech 19(3): 263–267; 2001.
Peña L. A.; Peãrez R.; Cervera M.; Jose Â. A.; Juaã R. Navarro L. Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann Bot 94: 67–74; 2004.
Pérez-Molphe-Balch E.; Ochoa-Alejo N. In vitro plant regeneration of Mexican lime and mandarin by direct organogenesis. Hortsci 32(5): 931–934; 1997.
Sim G. E.; Goh C. J.; Loh C. S. Micropropagation of Citrus mitis Blanco. Multiple bud formation from shoot and root explants in the presence of 6-benzylaminopurine. Plant Sci 59: 203–210; 1989.
Shen Z. M. Present conditions and development of Ponkan in China. Friend of Fruit Planter 7: 8–9; 2006. In Chinese.
Vardi A.; Bleichman S.; Aviv D. Genetic transformation of citrus protoplasts and regeneration of transgenic plants. Plant Sci 69: 199–206; 1990.
Yang Z. N.; Ingelbrecht I. L.; Louzada E.; Skaria M.; Mirkov T. E. Agrobacterium-mediated transformation of the commercially important grapefruit cultivar ‘Rio Red’ (Citrus paradisi Macf). Plant Cell Rep 19: 1203–1211; 2000.
Yu Ch H.; Huang Sh.; Chen Ch X.; Deng Zh A.; Ling P.; Gmitter F. J. J. Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange. Plant Cell Tiss Org Cult 71: 147–155; 2002.
Zeng L. H.; Xu H. F.; Wang H. Q.; Wu Sh H.; Zhu Y. X. Construction of plant expression vectors with PMI gene as selection marker and utilization in transformation of Citrus sinensis L. Osbeck. J Agri Biotech 16(5): 858–864; 2008. In Chinese.
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Zeng, L., Xu, H., Zeng, Y. et al. High efficiency in vitro plant regeneration from epicotyl explants of Ponkan Mandarin (Citrus reticulata Blanco). In Vitro Cell.Dev.Biol.-Plant 45, 559–564 (2009). https://doi.org/10.1007/s11627-009-9248-0
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DOI: https://doi.org/10.1007/s11627-009-9248-0