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Plant Cell Reports

, Volume 26, Issue 11, pp 1985–1998 | Cite as

Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea (Pisum sativum L.)

  • P. Smýkal
  • L. Valledor
  • R. Rodríguez
  • M. Griga
Genetics and Genomics

Abstract

In vitro clonal propagation of plants should generate identical copies of the selected genotype. However, associated stress might result in a breakdown of control mechanisms and consequent instability of the genome. We have used several molecular methods to assess the genetic stability of long-term propagated (24 years) multiple shoot in vitro culture of pea (Pisum sativum L.). We focused on assessing the stability of repetitive sequences, such as simple sequence repeats (SSR) and retrotransposons, both comprising a large part of genome. No differences were found when seedlings (Co-2004) or original seed (Co-1982) controls and long-term or newly established in vitro (one subculture cycle) samples were investigated by the SSR, inter-repeats (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) method. However, the more global amplified fragment length polymorphism (AFLP) and particularly the methylation sensitive MSAP methods detected 11 and 18% polymorphism among samples, respectively. Interestingly, investigation of the global cytosine methylation status by HPCE measurement revealed no statistically significant differences. Some evidence of retrotransposon re-arrangement was observed by sequence-specific amplification polymorphism. This occurred mostly in the abundant Ty3-gypsy type Cyclop element and to a smaller extent in the Ogre element. Alternatively, no polymorphism was detected among the PDR-1 element of the Ty1-copia type retrotransposon. Based on these results, multiple shoot culture of pea maintained over a long period may be considered as a true to type multiplication method of the original genotype.

Keywords

Clonal propagation Genetic stability DNA methylation Pisum sativum Retrotransposons Somaclonal variation 

Abbreviations

5mdC

5-Methyldeoxycytidine

HPCE

High performance capillary electrophoresis

HPLC

High performance liquid chromatography

ISSR

Inter-simple sequence repeats

IRAP

Inter retrotransposon amplification polymorphism

MSAP

Methylation sensitive amplified polymorphism

MSB

Murashige and Skoog (1962) basal medium with vitamins after Gamborg et al. (1968)

PAGE

Polyacrylamide gel electrophoresis

SD

Standard deviation

SSR

Simple sequence repeats

AFLP

Amplified fragment length polymorphism

SSAP

Sequence specific amplified polymorphism

Notes

Acknowledgments

This work was supported by the Ministry of Education of Czech Republic, projects OC 843.70, MSM 267842460 project and AGL-2004-00810 from Ministry of Education of Spain. Part of this work was performed during STSM-843-00977 granted to P.S. within the frame of COST-843 Action of European Union. Excellent technical support of Ms. L. Vítámvásová, E. Fialová and J. Vysloužilová is greatly acknowledged.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • P. Smýkal
    • 1
  • L. Valledor
    • 2
  • R. Rodríguez
    • 2
  • M. Griga
    • 1
  1. 1.Plant Biotechnology DepartmentAGRITEC Plant Research LtdŠumperkCzech Republic
  2. 2.Department of Biology of Organisms and Systems, Plant Physiology, Epiphysage Research GroupUniversity of OviedoOviedoSpain

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