Plant Cell, Tissue and Organ Culture

, Volume 72, Issue 3, pp 253–260 | Cite as

Genetically stable regeneration of apple plants from slow growth



Shoot-tips of apple cultivar `Gala' were stored in vitrousing a low temperature slow-growth culture method. All shoot-tips survived 1-year storage, with a significant height increment over that period. Eight `Gala' single-bud sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variation in plants recovered from slow growth culture, the ploidy remained genetically stable relative to the before-storage cultures. An amplified fragment length polymorphism (AFLP) assay was performed to detect DNA sequence variation. No differences in the DNA fragment patterns were observed using 20 primer combinations between the before-storage and the stored samples. In addition, a methylation sensitive amplified polymorphism (MSAP) assay was performed to investigate the DNA methylation status in both the before-storage and stored samples. It was found that the slow-growth storage resulted in a significant DNA methylation change in the stored shoots compared with the before-storage samples.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Angel F, Barney VE, Tohme J & Roca WM (1996) Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage. Euphytica 90: 307-313Google Scholar
  2. Ashmore SE (1997) Status Report On the Development and Appli-cation of In Vitro Techniques For the Conservation and Use of Plant Genetic Resources. International Plant Genetic Resources Institute, RomeGoogle Scholar
  3. Dellaporta SL, Wood J & Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1: 19-21Google Scholar
  4. Engelmann F (1991) In vitro conservation of horticultural species.Acta Hortic. 298: 327-334Google Scholar
  5. Engelmann F (1997) In vitro conservation methods. In: Callow JA, Ford-Lloyd BV & Newbury HJ (eds) Biotechnology and PlantGenetic Resources (pp 119-161). CABI, OxonGoogle Scholar
  6. Guo W, Deng X & Shi Y (1998) Optimization of electrofusion parameters and interspecific somatic hybrid regeneration in Citrus. Acta Bot. Sin. 40: 417-424Google Scholar
  7. Harding K (1991) Molecular stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from slow growth and cryopreservation. Euphytica 55: 141-146Google Scholar
  8. Harding K (1994) The methylation status of DNA derived from potato plants recovered from slow growth. Plant Cell Tiss. Org. Cult. 37: 31-38Google Scholar
  9. Harding K (1996) Approaches to assess the genetic stability ofplants recovered from in vitro culture. In: Normah MN, Narimah MK & Clyde MM (eds) Proceedings of the International Work-shop on In Vitro Conservation of Plant Genetic Resources (pp137–170). Plant Biotechnology Laboratory, University Kebangsaan Malaysia, Kuala Lumpur, MalaysiaGoogle Scholar
  10. Harding K (1997) Stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from cryopreservation. Cryo-Letters 18: 217-230Google Scholar
  11. Harding K, Benson E & Clacher K (1997) Plant Conservation Biotechnology: An Overview. Agro-Food-Industry Hi-Tech (May/June): 24-29Google Scholar
  12. Harding K, Marzalina M, Krishnapillay B, Zaimah NAN, Normah MN & Benson EE (2000) Molecular stability assessments of trees regenerated from cryopreserved mahogany (Swietenia mac-rophylla) seed germplasm using non-radioactive techniques to examine the chromatin structure and DNA methylation status of the ribosomal RNA genes. J. Trop. For. Sci. 12: 149-163Google Scholar
  13. Lambe P, Mutambel HSN, Fouche JG, Deltour R, Foidart JM & Gaspar T (1997) DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression? In vitro Cell Dev. Biol.-Plants 33: 155-162Google Scholar
  14. McClelland M, Nelson M & Raschke E (1994) Effect of sitespecific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 22: 3640- 3659Google Scholar
  15. Morguchi T, Kozaki I, Matsuta N & Yamak S (1988) Plant regeneration from grape callus stored under a combination of low temperature and silicone treatment. Plant Cell Tiss. Org. Cult. 5: 67-71Google Scholar
  16. Moriguchi T, Kozaki I, Yamaki S & Sanada T (1990) Low temperature storage of pear shoots in vitro. Bull. Fruit Tree Res. Stn. 17: 11-18Google Scholar
  17. Murashige T & Tucker DPH (1969) Growth factor requirements of citrus tissue culture. Proc. 1st Int. Citrus Symp. 3: 1155-1161Google Scholar
  18. Oka S & Niino T (1997) Long term storage of pear (Pyrus spp.) shoot cultures in vitro by minimal growth method. JARQ 31: 1-7Google Scholar
  19. Orlikowska T (1992) Effect of in vitro storage at 4 8C on survival and proliferation of two apple rootstocks. Plant Cell Tiss. Org. Cult. 31: 1-7Google Scholar
  20. Shen D (1992) Fruit Tree Breeding. In: Fruit Tree Breeding (pp 8-48). China Agricultural Press, BeijingGoogle Scholar
  21. Van den Houwe I, De Smet K, du Monteel HT & Swennen R (1995) Variability in storage potentail of banana shoot cultures under medium term storage conditions. Plant Cell Tiss. Org. Cult. 42: 269-274Google Scholar
  22. Vos P, Hogers R, Bleeker M, Reijan M, Homes M, Frijters A, Pot J, Peleman J, Kuiper M & Zabeau M (1995) AFLP: a new tech-nique for DNA fingerprinting. Nucleic Acids Res. 23: 4407- 4414Google Scholar
  23. Withers LA (1987) Long-term preservation of plant cells, tissues and organs. Oxford Sur. Plant Mol. Cell. Biol. 4: 221-272Google Scholar
  24. Xiong LZ, Xu CG, Maroof MAS & Zhang Q (1999) Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique. Mol. Gen. Genet. 261: 439-446Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  1. 1.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina

Personalised recommendations