Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

Vishal Gupta A. J. Bijo Manoj Kumar C. R. K. Reddy Bhavanath Jha Regeneration of protoplasts into de novo plants was reported for a large number of seaweed species. The regeneration of protoplasts into different morphotypes as a result of epigenetic variations was discussed for the first time in this study. The loci assessed for methylation modifications in normal filamentous thalli showed a frequency of 32.43% as unmethylated DNA, 24.32% as a hemimethylated, and 20.27% as a methylation of internal cytosine at both the strands. The corresponding methylation values for disk-type thalli were 27.02%, 32.43%, and 14.86%, respectively. The hypermethylation condition was apparent in the disk-type thalli with methylation ratio of 72.97% compared to that of normal filamentous thalli with 67.56%. The frequency of methylation polymorphic sites among the two morphotypes was 53%. The present study reveals the distinct expression of cytosine methylation and is thus correlated to differential morphogenesis of plants regenerated from cultured cells. The number of protoplasts regenerating into filamentous thalli declined with increasing temperature from 15C, 20C, 25C, and 30C. The disk-type variant had higher thermal stability at 30C over normal filamentous thalli. Further, this variant could maintain itself for more than a year in the laboratory indicating its suitability for in vitro germplasm maintenance and propagation. - Plant protoplasts have been employed for investigating the various aspects of developmental biology and in vitro genetic manipulation techniques aimed at development of genetically improved strains of agronomic crops. There have been numerous studies on the isolation and regeneration of protoplasts from a wide variety of seaweeds ranging from morphologically simple leafy thallus to anatomically complex thallus (see review Reddy et al. 2008). Unlike higher plants, seaweed protoplasts regenerate and differentiate into a full thallus without any amendments of phytohormones to culture medium. Nevertheless, the protoplasts from green seaweeds followed different types of regeneration patterns and gave rise to several phenotypically variable morphotypes such as sporangia, microthalli, saccate (or spherical), tubular (or spindle), irregular, and frondose with various life spans (Reddy et al. 1989; Huang et al. 1996; Chen 1998; Krishnakumar et al. 1999; Chen and Shih 2000; Rusig and Cosson 2001). Also, in the red alga Porphyra, three types of protoplast regeneration patterns, i.e., callus, filamentous, and conchocelis have been described (PolneFuller and Gibor 1984; Fujita and Migita 1985; Waaland et al. 1990; Dipakkore et al. 2005). The reasons offered for differentiation of cells into such variable morphotypes in vitro were mostly speculative and primarily attributed to either axenic culture conditions employed (Singh et al. 2011), age of source material used, or physical culture conditions such as temperature and irradiance (Chen 1998). Although in vitro conditions do induce phenotypic variations, it is not well understood how various morphotypes develop from a single genotype when cultured under the same conditions (Vogel 2005). Studies on higher plants have shown that the epigenetic mechanisms such as DNA methylation, histone modifications, chromatin remodeling, and RNA interference regulate the differentiation and development of cells or tissues cultured in vitro. These epigenetic mechanisms influence the expression of genes that in turn triggers the signals involved in development program eventually leading to formation of different phenotypes. It has also been suggested that in vitro conditions induce genotypic variations at modest frequency while variation in degree of DNA methylation seems to be frequent and occasionally directly linked with phenotypic variations (Miguel and Marum 2011). Studies on terrestrial plants have revealed that epigenetic regulation controlled by frequency and distribution of DNA methylation causing pleiotropic effect on morphology and development (Zhang et al. 2006). The DNA methylation mostly occurs at cytosine particularly at CG dinucleotides, although significant levels of methylation at CNG and CNN has also been reported (Cao and Jacobsen 2002; Hsieh and Fischer 2005). Differential DNA methylation regulates the specific gene expression by getting associated with the 5 upstream promoter sequence either in one or both alleles of tissue-specific genes (Fraga et al. 2002). Significant differences in cytosine methylation have been observed in different parts of a species such as tomato (Messeguer et al. 1991), rice (Xiong et al. 1999), Silene latifolia (Zluvova et al. 2001), and even in the different developmental phases of Pinus (Fraga et al. 2002) and Prunus (Bitonti et al. 2002). The earlier study with Arabidopsis has reported that young seedlings have lower DNA methylation levels than mature leaves (Finnegan et al. 1998). It has also been well evidenced that the change in DNA methylation do occur among the plants derived from in vitro tissue culture (Chen et al. 2009; Park et al. 2009) and somatic embryogenesis (Chakrabarty et al. 2003). The molecular genetics for green seaweeds is still in their inception, and these organisms are useful source for investigating the molecular mechanisms underpinning the developmental processes. In the present study, genome-wide distribution and pattern of DNA methylation sites was studied for the first time in seaweed to investigate the epigenetic variations arising from protoplast-derived morphotypes. Further, the regenerated thallus types were shown to have interesting growth properties making their suitability for germplasm storage and propagation applications. Materials and Methods Collection of Seaweed Sample and Protoplasts Isolation Thalli of Ulva reticulata Forsskl C were collected from Okha (2227.04 N; 6903.58 E), Gujarat, along the west coast of India and brought to the laboratory under cool conditions. The seawater temperature at collection site was 21C. Selected thalli were thoroughly rinsed with autoclaved seawater to remove dirt and epiphytes. The unialgal culture of this alga was established by growing it in sterile enriched seawater media (Provasoli 1968) with GeO2 (10 mg L1) for a week under white fluorescent lamps of irradiance intensity about 15 mol photon m2 s1 with a 12:12 h light/dark photoperiod. During this period, the culture media were changed every 2 days. Thereafter, the algal thalli were made axenic and protoplasts isolation was carried out following the protocol described by Reddy et al. (2006) for green seaweeds. For protoplasts culture, a density of 1.0 105 cells were dispensed into 10 ml of enriched seawater medium and incubated at a temperature gradient of 20 1C, 25 1C, 30 1C, and 35 1C under white fluorescent lamps of irradiance intensity about 15 mol photon m2 s1 with a 12:12-h light/dark photoperiod in a plant growth chamber (Eyela, Japan). Cell Wall R (...truncated)