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

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.

Assessment of genetic and epigenetic changes during cell culture ageing and relations with somaclonal variation in Coffea arabica

“…Polymorphisms were not found using RAPD markers in 2-year-old cultures of hop (Peredo et al 2006). Using SSR markers Smýkal et al (2007) reported no genetic variation in 24-year-old multiple shoot cultures of pea. However, in the same work, AFLP molecular markers successfully revealed 22 polymorphic and singleton fragments out of a total of 436 fragments.…”

“…In our conditions, the number of methylation changes per plant was small and the resulting total polymorphism was \1 % independently of the age of the cell culture, indicating little disruption to the epigenetic Values are means of 3-4 independent measurements ± SD status. Similarly, low levels of methylation variation were found in freesia somatic seedlings derived from short-term cultures (Gao et al 2010) and pea long-term multiple shoot cultures (Smýkal et al 2007). Many studies with methylation-sensitive molecular markers that reported a high level of epigenetic variation reported no effect on phenotype or morpho-agronomic traits (Bednarek et al 2007;Li et al 2007;Schellenbaum et al 2008;Fiuk et al 2010).…”

“…Unfortunately their involvement in SV occurrence has been reported. Studies have been undertaken either directly on cell cultures (Tanurdzic et al 2008) or on plants derived from cell cultures (Hao and Deng 2002;Smýkal et al 2007;Rodriguez-Lopez et al 2010). Early attempts to explain SV revealed the implication of chromosomal abnormalities (Karp 1991).…”

Epigenetics in plant tissue culture

“…The most common difference between field plants and in vitro plants was demethylation of cytosines in some restriction targets, representing 63% of the observed changes. Smýkal et al (2007) used various molecular methods to assess the genetic stability of long-term (24 years) propagated shoot cultures of pea. No differences were found using microsatellite, inter-simple sequence repeat (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) markers.…”

Variations in BARE-1 insertion patterns in barley callus cultures

“…This system was used to distinguish barley varieties (Kalendar et al, 1999), to map defense-related genes in barley (Manninen et al, 2000), to study grass genome evolution (Vicient et al, 2001), to detect retrotransposon integration events in allopolyploid Spartina anglica (Baumel et al, 2002), to characterize somaclonal variation in banana (Muhammad and Othman, 2005), and recently in barley (Campbell et al, 2011), to investigate genetic relationships of Diospyros kaki and related species (Guo et al, 2006), as well as to group Fusarium oxysporum f. sp. lactucae isolates (Pasquali et al, 2007), to assess stability of pea long-term cultures (Smykal et al, 2007), and to evaluate evolutionary relationships among accessions of Aegilops tauschii (Saeidi et al, 2008). In the present study, we aimed to detect any possible BARE-1 insertion variations among calli of different stages (30 days old, 45 days old and 60 days old) and mature embryo tissue.…”