ГЕНЕТИКА, 2014, том 50, № 11, с. 1338-1344
ГЕНЕТИКА РАСТЕНИЙ
УДК 575.17:582.683.2
ASSESSMENT OF DNA METHYLATION CHANGES IN TISSUE CULTURE OF Brassica napus
© 2014 Y. Gao1, L. Ran1, Y. Kong1, J. Jiang1, V. Sokolov2,3, and Y. Wang1
1Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou, 225009 China
e-mail: wangyp@yzu.edu.cn
2Institute of Molecular and Cell Biology, Siberian Branch Russian Academy of Sciences,
Novosibirsk, 630090 Russia
3Vavilov Institute of Plant Industry, St. Petersburg, 190000 Russia
Received April 30, 2014
Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA me-thylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rape-seed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.
DOI: 10.7868/S001667581410004X
Epigenetic plays important role in gene regulation of plants, through modulation of DNA activity without altering the basic nucleotide structure (including DNA methylation and histone modification) [1]. And it is proved that epigenetic information is heritable between generations and provides memory of environmental stresses [2, 3]. Recent studies revealed that epigenetic variation exceeds genetic diversity, and this can lead directly to a variant phenotype [4]. Epigenetic changes are of utmost importance during natural development of plant populations, as well as plant response to environmental stimulations [5, 6]. Recently, epigenetic modification mechanisms have been implicated regulating transcription, replication, gene transposition and DNA repair. And small RNA-directed modification in transcriptional and post-transcrip-tional control of gene expression has been proved important to chromatin modifications [7]. These modifications include DNA methylation, chromatin reshaping, histone modification and RNA interference [8]. Methylation, especially cytosine methylation at CpG, CpNpG and asymmetric CpHpH sites, can be induced by cis- or trans-acting DNA polymorphisms or by epigenetic phenomena [9, 10]. Several proteins, including Domains rearranged methylase 1 (DRM1) and DRM2, DNA methyltransferase 1 (MET1), DNA methyltransferase chromomethylase 3 (CMT3), DNA
methylation 1 (DDM1) are associated with plant de novo methylation [11—13]. Cytosine methylation level differs among species. For example, mammalian methylation is generally restricted to CpG sites, whereas plant has extensive asymmetric and CpNpG methyla-tion. Plant methylated cytosines in genome may up to 30%, much higher than methylation in animals. Furthermore, cytosine methylation also differs among tissues and developmental stages of each plant species [14], indicating DNA methylation is species and tissue specific, which varies during cellular differentiation and tissue development [15—17].
Phenotypic and genetic variations have been greatly observed during plant tissue culture, which is more frequent than natural variation [18]. Methylation induced variation during tissue culture has been considered as part of the molecular mechanism for soma-clonal variation [10, 14]. Steward et al. [19] reported the heritable variation induced by DNA methylation during regeneration of maize tissue culture. Bardini et al. [20] discovered extensive methylation changes in calluses of Arabidopsis thaliana exposed to kanamycin. Xu et al. [14] observed methylation alterations during somatic embryogenesis in rose, and found the highest methylation level during differentiation of callus. Tre-jgell et al. [21] reported different methylation in 18S
Callus induction rate of B. napus hypocotyl under different composition of hormones
Medium composition No. of total explants No. of calli Induction rate, %
0.05 mg/L 2,4-D 138 113
93 77 82 ± 2a*
161 138
MS +1.0 mg/L 6-BA 0.1 mg/L 2,4-D 142 75 162 131 67 147 91 ± 2b
0.2 mg/L 2,4-D 133 117
104 95 89 ± 2ab
154 138
0.5 mg/L 6-BA 139 110
79 59 79 ± 4a
156 129
MS + 0.1 1.0 mg/L 6-BA 142 131
mg/L 2,4-D 75 162 67 147 91 ± 2b
2.0 mg/L 6-BA 135 96 116 69 81 ± 8ab
* a, b stands for significant difference at the 0.05 level.
rRNA and 25S RNA, when studying in vitro regeneration of Carlina acaulis. Recently, Rival et al. [22] found the positive correlation between methylation level and suspension cultivation time of Elaeis guineensis. Moreover, researches on DNA methylation and somaclonal variation have also been documented on crops including banana, grape and rice [23—25].
Brassica napus, as one of the important oil crops, is of great economic value to human life. Genetic transformation and tissue culture are essential techniques for improvement of B. napus [26]. Hitherto, DNA methylation during tissue culture of B. napus exposed to different hormones has never been reported. Here we investigated tissue culture-induced epigenetic alterations, especially DNA methylation, in a set of dissected tissues of B. napus, including hypocotyl, callus and regenerated plantlets. Since antibodies are necessary components of tissue culture medium, which may cause hypomethylation in plants [27], assessments of methylation status under different composition of hormones may provide a useful criterion for tissue culture ofB. napus. By applying HPLC-assisted measurement of cytosine methylation in different tissues of B. napus exposed to different concentration of 2,4-D and 6-BA, we successfully elaborated the relationship between DNA methylation and callus differentiation during tissue culture, as well as the function of hormones to tissue differentiation. This study may support the improvement of tissue culture in B. napus, and contribute to explication of epigenetic divergence of regenerants of tissue culture.
MATERIALS AND METHODS
Preparation of experimental materials. Brassica napus L. (cv. Yangyou 9) was kindly provided by Jiangsu Institute of Agricultural Science in the Lixiahe District. Seeds of B. napus were sterilized in 70% alcohol for 1 min and 2% NaClO for 20 min, then washed in sterilized ddH2O for five times. Sterilized rapeseeds were germinated on MS medium and incubated at 24°C under a 16 h photoperiod (50 ^mol m-2 s-1, cool-white fluorescent tubes). After 5 to 7 d, hypocot-yls of shoots were cut into 1 cm as explants, which were then incubated on mediums containing different composition hormones (table). Explants were sub-cultured every 2 weeks, and plant materials for DNA me-thylation analysis were collected every 3 days after first subculture. Generally, 9 time points including 3 developmental stages, hypocotyls, callus and regenerated plantlets were collected.
DNA extraction and hydrolysis. The protocol for DNA extraction was a modification of CTAB method by Doyle and Doyle [28]. Chloroform and CTAB were used to eliminate proteins and carbohydrates, while CTAB-DNA was precipitated and collected. Extracted DNA was dissolved by ddH2O containing RNase, concentration and purity of DNA was determined by electrophoresis and spectrophotometry. According to Demeulemeester et al. [29], approximately 50 ^g DNA was hydrolyzed by 200 ^L 70% perchloric acid for 1 h at 100°C, and the pH was adjusted to 3~5 with 1 mol/L KOH. Finally, the incoming KClO4 precipi-
Fig. 1. Callus differentiation of B. napus hypocotyl in medium with different hormones. a, 1.0 mg/L 6-BA + 0.05 mg/L 2,4-D, b, 1.0 mg/L 6-BA + 0.1 mg/L 2,4-D, c, 1.0 mg/L 6-BA + 0.2 mg/L 2,4-D, d, 0.1 mg/L 2,4-D + 0.5 mg/L 6-BA, e, 0.1 mg/L 2,4-D + 2.0 mg/L 6-BA.
tate was centrifuged at 12000 rpm for 5 min, and the hydrolysate was collected for HPLC analysis.
HPLC analysis of DNA methylation. HPLC analysis of DNA hydrolysate was automatically injected on an HPLC system (Agilent 1200, USA), coupling with an Alltima C18 column (with a granulation and size of 5 ^m and 250 mm x 4.6 mm). Chromatographic separation was performed with a flow rate of 0.8 mL min-1 and oven temperature of 40°C using a mixture of two solvents: 10% methanol, 0.1 mol/L sodium pentane-sulfonate in 0.2% triethylamine. And the UV-spectra were recorded at 273 nm. Relative quantification was performed by comparing peak areas of similar retention times, using the calibration curves of available cy-tosine and 5-methyl cytosine (5-MeC) as standard. The percentage of 5-MeC in each sample was calculated as: concentration of 5-MeC/(concentration of 5-MeC + cytosine). All analysis were repeated 3 times and the mean ± S.E. was calculated.
RESULTS AND DISCUSSION
Degree of DNA methylation in callus cultured in mediums with different composition of hormones
In previous researches of B. napus tissue culture, we used to select MS + 0.1 mg/L 2,4-D + 1.0 mg/L 6-BA for callus induction [26]. As listed in table, here we try to analyze the induction rate of callus from B. napus
hypocotyl exposed to different concentration of 2,4-D (0.05 mg/L, 0.1 mg/L, 0.2 mg/L) and 6-BA (0.5 mg/L, 1.0 mg/L, 2.0 mg/L). When setting 1 mg/L 6-BA as fixed
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.