Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation

Abstract

This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

“…Harding [ 77 ] and Peredo et al [ 80 , 81 ] attributed the DNA methylation changes detected in slow growth to the in vitro culture procedures. Studies of the effect of tissue culture on the DNA methylation stability have detected significant changes, as for example the work of Gimenez et al [ 89 ], on in vitro propagated garlic, a species usually conserved in germplasm banks through slow growth storage. These authors, using MSAP, detected changes, mainly demethylations, in plants under prolonged in vitro culture.…”

Genetic and Global Epigenetic Modification, Which Determines the Phenotype of Transgenic Rice?

“…For instance, Rhee et al [7] demonstrated the silencing of epialleles by epigenetic modifications and showed that the pericarp color1 (p1) epialleles were capable of functioning in the presence of the correct trans-acting factors in maize. Furthermore, there are many reports on epigenetic changes caused by plant regeneration in rice [9], garlic [10], triticale [11], pineapple [12], torenia, and rye [13,14].…”

Dynamics of the Methylome and Transcriptome during the Regeneration of Rice

“…In addition to genotype, the culture medium also induces variations in gene regulation, chromatin structure and the epigenome [19][20][21]. Epigenetic changes are reported during the regeneration of rye [22], triticale [23], garlic and torenia [24,25]. Furthermore, the use of demethylating agents, such as 5-azacytidine (5-azaC), has led to differential transcriptional activities in regenerants, implying that changes in DNA methylation induced through in vitro culture may regulate gene expression, resulting in phenotypic variations [26][27][28][29][30].…”