Abstract Different genotypes and source tissues in sugarcane were used for the initiation of somatic tissue cultures

Different genotypes and source tissues in sugarcane were used for the initiation of somatic tissue cultures. Alterations in the level of 5-methyl-cytosine in DNA between somatic tissues throughout tissue culture were determined by HPLC analysis. Several factors influenced the 5-methyl-cytosine content: the tissue used as source for the explant, the 2,4-D concentration in the medium and the genotype. Sugarcane meristematic tissue showed significantly higher levels of methylation than roots or leaves. The level of 5-methyl-cytosine in genomic and mitochondrial DNA, was positively correlated with the concentration of 2,4-D in the culture medium, but showed an inverse relationship with the ability of somatic cells to regenerate. The genotypes B80-84 and Co 997 produced the largest amount of callus, mostly embryogenic in appearance, while C437-68 callus production was much lower and mainly non-embryogenic. The remaining genotypes were classified as intermediate and produced both embryogenic and non-embryogenic callus types, but predominantly embryogenic. The highest levels of cytosine methylation were observed in the genotypes with the lowest embryogenic and morphogenic capacity. DNA methylation was significantly lower in the cultivars B80-84, Co997, C87-51 and Co421. The genotypes C120-78 and C266-70 had high levels of methylation, even at low 2,4-D concentration in the medium and were classified as morphogenic but their calli differed in color and consistency. There was an apparent correlation between genotype and callus morphology in terms of DNA methylation levels: calli with similar characteristics share similar 5-methyl cytosine content. Differences in DNA methylation were also detected between morphologically distinguished calli from various sugarcane genotypes.

Keywords: DNA methylation; sugarcane; somatic embryogenesis; 2,4-D; HPLC

Abbreviations: 2,4-D: 2,4-dichlorophenoxiacetic acid; HPLC: high performance liquid chromatography; 5-mC: 5-methyl-cytosine; MS: Murashige & Skoog; Mt: mitochondrial; E: embryogenic; SE: somatic embryogenesis

Somatic embryogenesis is the process by which somatic cells develop into plants through characteristic morphological stages that resemble zygotic embryo development. Under in vitro conditions somatic embryogenesis either proceeds from explant cells that are already pre-embryogenically determined (Thorpe, 2012), or the process begins with an initiation phase in which a dedifferentiation of cells occurs followed by these same cells becoming competent for embryogenesis (Lyndon, 2013). Somatic embryogenesis can also be triggered by non-hormonal factors for example, various stress conditions (Leljak-Levanic et al., 2004). Treatment with phytohormones, especially the synthetic auxin 2,4-D, during the induction of embryogenesis has shown to increase the frequency of these modifications.
In sugarcane (Saccharum spp. hybrid), embryogenic callus culture and regenerable cell suspension cultures can be obtained from somatic embryos cultured on MS medium (Murashige and Skoog, 1962) supplemented with organic compounds and 2,4-D (MSC+2,4-D), as described in the literature (Saad and Elshahed, 2012). However, establishment of embryogenic cell suspensions is very difficult and problems with plant regeneration are frequently observed.
Jaligot et al. (2000) demonstrated that a correlation exists between DNA hypomethylation and hormone effects. These authors demonstrated a direct cause-effect relationship between DNA methylation and the stress induced by tissue culture. Peng and Zhang (2009) determined that abiotic stresses and successive rounds of subculture generally decrease the levels of DNA methylation. Lo Schiavo et al. (1989) found a positive correlation between exogenously added auxin and cytosine methylation in carrot. A high level of cytosine methylation occurred during the induction of embryogenesis by exogenous auxin. Following induction and removal of the auxin the level of cytosine methylation rapidly decreased. Subsequent development of the embryos could be correlated to a modest increase in cytosine methylation.
DNA methylation is one of the modes of DNA modification which is involved in important biological functions. This is an essential epigenetic mechanism involved in gene regulation and disease, but little is known about the mechanisms underlying inter-individual variation in methylation profiles (Bell et al., 2011). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication
(Vanyushin, 2006)
Several mechanisms have been proposed to elucidate the role of methylation in gene expression. Methylation of cytosines in plant DNA plays a key role in the regulation of gene expression (Zhang et al., 2006, Berdasco et al., 2008) and non-coding factors (Tsukahara et al., 2009). Methylation is a wide-spread and significant form of regulatory factor, with genome-wide studies in plants reporting between 5-25% (Pradhan et al., 2008, Zemach et al., 2010) of cytosine as methylated.
HPLC is an efficient and versatile method for detecting DNA methylation, in spite that is not capable of detecting DNA-methylation at single-nucleotide resolution. HPLC can be a useful alternative, because it provides excellent output for determining % DNA methylation.
Interestingly, significant changes in the level of methylation of genomic DNA have been observed during somatic tissue culture of carrot (Arnholdt-Schmitt, 1993). The changes of the methylation pattern have been shown to strongly affect the embryogenesis process in plants (LoSchiavo et al., 1989). Molecular analysis of sugarcane embryogenic callus pointed to a close relationship between 2,4-D concentration, DNA methylation and plant regeneration in previous experiments.
A possible hypothesis of this work is related with the role of 2,4-D concentration on embryogenic capacity and occurrence of DNA Methylation. In that sense, the purpose of the investigation reported here was to examine and compare changes in cytosine methylation in genomic and mitochondrial DNAs of seven sugarcane cultivars with different embryogenic capacity, hypothesizing the effect caused by the auxin 2,4-D. The effect of different source of explants and different 2,4-D concentrations is also discussed. Using this system we were able to determine whether the high level of cytosine methylation observed in early embryogenesis was a consequence of exogenous auxin addition or whether methylation has a primary role in the control of gene expression during the early stages of embryo development.
Materials and Methods
Plant Material
The experiments were designed on the basis of the following factors: three tissue sources, three different concentrations of the hormone 2,4-D in the medium of callus induction and subculture, and seven sugarcane genotypes.
Tissue source
Samples of roots, leaves and meristematic tissues (spindles) by triplicates were collected from three different plants of each sugarcane cultivar. This procedure for obtaining tissue source was performed for each genotype as biological replicates.
Somatic tissue culture
Spindles from a group of sugarcane cultivars with different response were used in the somatic tissue culture experiment: C437-68, C266-70, C120-78, Co 421, C87-51, Co 997 and B80-84. Callus morphology of the different cultivars was recorded according to callogenesis, color and main appearance of the callus culture. Callus culture were initiated and sub-cultured in the presence of 0.5, 3 and 5 mg.L-1 2,4-D in the culture medium, as described by Díaz et al. (1991). Briefly, young rolled leaves were surface-sterilized and cultured on Murashige & Skoog (MS) medium, supplemented with 10 mg.L-1 kinetin, 2.5 mg.L-1 indole acetic acid (IAA), 1 mg.L-1 2,4-D and solidified with 7 g.L-1 Oxoid agar. Calli were harvested after four weeks and sub-cultured in the same medium but using 0.5, 3 and 5 mg.L-1 2,4-D. At the end of the second subculture, samples were collected for DNA analysis. Additionally, ten pieces of calli were randomly selected and placed onto H- (2,4-D-free MS) and SH (hormone-free MS) medium. Plant regeneration was scored after six weeks and expressed as number of regenerating plants per total number of calli.
Preparation for Scanning Electron Microscopy
Callus tissue was fixed in 3.2% glutaraldehyde and post-fixed in 1% osmium tetroxide buffered in sodium cacodylate solution 0.1 M, pH 7.4. Fixed tissues were dehydrated in ethanol, critical point dried and sputter coated with gold. Samples were analyzed using a JEOL JEM 100 CXII-ASID 4D Scanning Electron Microscope.
Genomic DNA isolation
High-molecular weight genomic DNA was extracted from sugarcane tissues (roots, leaves and spindles) of different cultivars, as described by Doyle and Doyle (1987). This was followed by RNase treatment overnight at 4oC and extraction with phenol/chloroform-isoamyl-alcohol. The quantitative estimation of DNA concentration was performed by measuring UV lectures at 260 and 280 nm using a UV/vis spectrophotometer Shimadzu model.
Mitochondrial DNA isolation
Mitochondrial (Mt) DNA was isolated according to Erickson et al. (1985), from calli of the cultivar C 87-51 grown under three different 2,4-D concentrations.
Determination of 5-methyl-cytosine content in DNA.
Determination of 5-methyl-cytosine content in DNA was performed using ten microgram purified DNA and hydrolyzed in sealed glass vials with formic acid for 30 min at 175oC. The samples were lyophilized and stored at -20oC. Prior to use, they were dissolved in 200 µL H2O and subjected to analysis of base composition by reversed phase-high performance liquid chromatography (RP-HPLC) as described by Ngernprasirtsiré et al. (1988). The equipment consisted of a Reversed phase column C-18, Eurospher 100 (250 mm, Knauer) without pre-column, LKB 2150 pump, UV-vis Spectrophotometer Shimadzu and Register (Phillips PM 8251A). Elution was carried out with 6.5 mM NH4H2PO4 pH 4/ 8% (vol/vol) methanol at a flow rate of 1.2 mL.min-1 at 25oC. Eluates were monitored at 254 nm. Methylation percent was calculated according to Palmgren et al. (1990). Standard bases from Sigma (highest available purity) were kindly supplied by Dr. D.C.W. Brown.
Data analysis
Analysis of variance was conducted for each of the three experimental factors. Means of treatments were compared according to Newman-Keuls multiple range test (P