The effects of growth regulators on somaclonal variation in rye (Secale cereale L.) and selection of somaclonal variants with increased agronomic traits

The aim of this research was to characterize somaclonal variation in populations derived from embryos cultured on two types of induction medium (supplemented with either 2,4-D or dicamba), as well as to select and characterize several somaclonal lines. The sexual progenies of 40 R(0)regenerants - A somaclones (derived on the medium with 2,4-D) and B somaclones (derived on the medium with dicamba) - were analysed according to the following traits: plant height, total number of tillers, number of productive tillers, spike length, number of spikelets per spike, spike compactness, number of normally developed grains per spike, weight of grains per spike, and the weight of 1000 grains. The results for twenty-two R(1)plants surpassed the variability range for the control. The transmission of positive changes to the next generation was proved in the case of 8 originally chosen R(1) plants: 7 plants selected from the A somaclones and one plant from the B somaclones. Five out of the eight created somaclonal lines proved to be stable somaclonal variants. The absolute rate of the efficiency of positive somaclonal changes was calculated as 0.64%.     

Selection and Analysis of Polymorphisms in Somaclonal Variants of <i>Agave americana</i> Resistant to <i>Fusarium oxysporum</i> via an Ethyl Methanesulphonate Treatment

Agave americana L. callus were exposed to different concentrations of ethyl methanesulphonate (EMS) 0, 15, 30, 45 and 60 mM and to different times of exposure (2 and 4 h). The viability and capacity of shoot formation were shown to be affected when the callus were exposed to high concentrations (30–60 mM). Only the callus exposed to 15 mM EMS presented shoot formation; the exposure time of two hours produced the largest quantity of shoots regenerated per callus (21 shoots/callus). In order to generate somaclonal variants resistant to Fusarium oxysporum, a selection pressure was applied through of a culture filtrate (CF) of 100 ppm of the fungus. This was made in callus obtained in the treatment with 15 mM EMS during 2 h of exposure. The CF caused oxidation and necrosis in 71.25% of the callus; however, they were capable of generating shoots (3.5 shoots/callus). Molecular markers type RAPD, ISSR and DAMD were used to evaluate the genetic variation arising from the mutations caused by EMS on control plants and 16-month-old somaclonal variants. The polymorphic information content (PIC) for each one of the initiating groups was: 0.28 (DAMD), 0.09 (ISSR) and 0.14 (RAPD). DAMD revealed a greater percentage of polymorphism than RAPD and ISSR. Polymorphic bands were detected in the somaclonal variants. This indicated that the EMS caused genetic variation in the regenerated plants conferring resistance to them against Fusarium oxysporum.     

Seed and seedling diversity delimitation and differentiation of Indian populations of Melia dubia cav.

Melia dubia is one of the most important industrial tree species in the South East Asia. In last few decades, the populations of M. dubia has rapidly expanded in the Indian sub-continents, leading to an increase in the genetic diversity of species. However, very less information is available on intra-specific variation in Melia under the Indian subcontinent. Therefore, a present investigation was undertaken, to assess the level of diversity in seed and saplings of the Melia populations (ecotypes) collected from three agro-ecological regions of India. Results revealed that the seed and saplings of all the ecotypes are significantly different for all the traits, except for number of branches per plant, and the maximum variability was recorded in germination percentage, seed weight, internodal length, and sapling height of the species. The high heritability for seed weight (0.99), length (0.99), and width (0.97), and germination percentage (0.99) indicated that selection and genetic gain for these traits would be effective during the commencement of improvement program. Trait association analysis explained that higher seed weight significantly reduced sapling height, collar diameter, number of leaves per plant, internodal length, petiole length, and germination percentage (r = ?0.86; p < 0.001) that ultimately reduced the seedling vigor in Melia dubia. Interestingly, the number of branches per plant were not associated with any of the morphological traits. The first principal component explained 50.09% of the entire variation and all the traits contributed greatly to the variation for this principal component, except for number of branches, leaf width and seed length. The clustering approach assorted geographic variation of M. dubia populations into three main sub-clusters i.e. South, North, and North East populations each consisting of five, seven and one populations (including cultivar), respectively. Among different ecotypes, Bahumukhi, Varsha and US Nagar seed sources outperformed all others in seedling vigour (sapling height) and rest of the growth parameters. Overall, findings explained that considerable scope exists for the development of superior planting material of M. dubia through exploration of seeds and selection at the early seedling stage.     

Effect of in vitro culture conditions on somaclonal variation in cowpea (Vigna unguiculata Walp.) using RAPD markers

We report a high frequency regeneration protocol in cowpea (Vigna unguiculata Walp. var. C 152) via somatic embryogenesis from 10-d-old primary leaf explants. A study was conducted to examine the effect of somaclonal variations in in vitro derived cowpea plants under field conditions. The regenerated plantlets were successfully transferred to field after hardening in vitro and grown for collecting R0, R1 and R2 seeds. The seeds of R1 and R2 generations were subsequently, grown under field conditions and their various biometrical traits were compared and evaluated with non-tissue cultured cowpea plants as check. There was no detectable somaclonal variation induced in R0-R2 in any of the biometrical traits. The results indicate that the inclusion of different plant growth promoters at specified concentrations and duration in our earlier tissue culture work did not induce any detectable mutation. The RAPD analysis also shows that there is no genetic variation among R2 cowpea plants. The somatic embryogenesis protocol we report could thus be safely applied for high frequency true-to-type regeneration and transformations protocols without any somaclonal variation.     

EMS对三个玉米自交系的诱变效应分析

EMS诱变玉米花粉是玉米化学诱变的主要技术。该研究以生产上3个常用的玉米自交系K305、21-ES、R08为材料,对其花粉用不同浓度的EMS诱变处理,探讨其EMS诱变的最佳浓度范围,明确其诱变效应。结果表明：3个自交系经过不同浓度的EMS诱变后,其结实率随着浓度的增大表现出减小的趋势,从其半致死剂量来看,EMS诱变花粉的适宜浓度范围自交系K305和R08均为0.67~1.0 mL?L-1,21-ES在1.67 mL?L-1附近。 M1代不同性状其变异幅度和变异系数与对照相比主要表现出增大的趋势,其不同性状的生物学效应在材料间表现不一致,表明性状在不同材料间对EMS的敏感性不一样,生育期表现为21-ES>K305>R08;主要株型性状表现为R08>21-ES>K305;主要雄穗性状K305和21-ES比R08敏感;主要果穗性状表现为21-ES>K305>R08。 M2代整体表现为变异谱扩大,其株高、穗位高和叶面积以及主要果穗性状的变异表现复杂,主要雄穗性状中除K305的M2株系雄穗分枝数呈双向变异外,其余M2株系整体偏向于雄穗变短,雄穗分枝数减小。该研究结果为后续研究和应用打下了基础。     

Bacterial contamination of in vitro plant cultures: confounding effects on somaclonal variation and detection of contamination in plant tissues

Bacterial contamination represents a serious problem for plant tissue culture research and applications. Bacterial interference with normal plant physiology and morphology can generate misleading conclusions if the presence of bacteria is ignored. Bacterial contaminants in in vitro plant culture are typically detected by direct observation; thus, it is assumed that cultures without visible symptoms are bacteria free. Here, we demonstrate that contaminating Bacillus DNA in plant DNA solutions from asymptomatic plants can interfere with the analysis of somaclonal variation in chrysanthemum. We studied somaclonal variation in chrysanthemum using short semi-specific PCR primers based on conserved motifs in NBS–LRR disease resistance genes and in mobile elements. Instead of true somaclonal variation we found three polymorphic bands derived from contaminant bacterial DNA in plant extracts. Although the detection of asymptomatic bacteria in in vitro plant cultures is a major issue, we found that it has not been adequately addressed to date, particularly for studies on somaclonal variation. We reviewed the most commonly cited contaminant bacteria in in vitro plant culture and designed specific 16S rRNA gene-based PCR primers for the main genera causing contamination (Bacillus, Pseudomonas, Staphylococcus, Lactobacillus, Erwinia/Enterobacter and Xanthomonas). Using a panel of pure bacterial DNAs, artificial mixes of bacterial/plant DNAs, and in vitro plant cultures with and without visible contamination we demonstrated that our primers are in most instances both reliable and sensitive, and appropriate for the identification and tracking of the most frequent bacterial contaminants in plant in vitro cultures. Implications of bacterial identification to molecular analysis of somaclonal variation and plant culture decontamination are discussed.     

Somaclonal variability as a source for creation of new varieties of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

Data on selection and characteristics of somaclonal variants of finger millet (Eleusine coracana (L.) Gaertn.), obtained as a result of regeneration of plants from callus tissue, are presented. Among the genetically stable lines tested, the somaclonal variant SE-7 was the most interesting owing to its acquired important agricultural traits, such as a higher seed and biomass yield, rapid seed germination at lower temperatures, and shortening of the main plant development stages. Data analysis showed that somaclonal variability can serve as a source of initial material for further selection of new finger millet varieties.     

Differential salt-stress response during germination and vegetative growth in in vitro selected somaclonal mutants of Cenchrus ciliaris L.

Four somaclonal mutants (S1, S4, S6 and M10) and their parental Cenchrus ciliaris L. cultivar Biloela were characterized under salinity conditions at germination and vegetative growth stages. Seeds of all somaclonal mutants had higher germination percentages than cv. Biloela seeds in the control and salt treatments. At 150 mM, germination was significantly higher in M10, S6 and S4 (72.3%, 66.3% and 61.8%, respectively) than in cv. Biloela (35.5%). Mutants grown under salinity along with cv. Biloela for 35 days had a different relative growth rate. S6 had the highest growth rate, indicating its potential tolerance to salt stress, whereas M10 was the most sensitive, with Bi, S4 and S1 being intermediate tolerant genotypes. Catalase enzyme activity (CAT) in M10 decreased in response to salt stress and was significantly associated with malondialdehide content, suggesting salt injury, whereas higher levels of CAT activity in S6 during salt stress were associated with increased salinity tolerance. The present results indicate that somaclonal variation and in vitro mutagenesis offer an effective tool for improvement of C. ciliaris because the somaclonal mutants showed differential tolerance to salt stress with respect to their parental and could be a better choice for use in a breeding program.     

Prickly Poppies Can Get Pricklier: Ontogenetic Patterns in the Induction of Physical Defense Traits

Plant ontogeny is a common source of variation in defense and herbivory. Yet, few studies have investigated how the induction of physical defense traits changes across plant ontogeny. Physical defense traits are costly to produce, and thus, it was predicted that induction as a cost-saving strategy would be particularly favorable for seedlings, leading to ontogenetic declines in the inducibility of these traits. We tested for induction of three different physical defense traits (prickles, latex and leaf toughness) in response to mechanical defoliation and jasmonic acid application using prickly poppies (Argemone glauca and A. mexicana, Papaveraceae) as a model system. Genetic variation in the induction of physical defenses was tested using maternal sib-ships sampled from multiple populations. Both species induced higher densities of laminar prickles, although the magnitude of induction was much higher in the endemic Hawaiian prickly poppy, A. glauca, than in the cosmopolitan A. mexicana. The magnitude of prickle induction was also higher in young compared to older juvenile plant stages in A. glauca, demonstrating a strong role of ontogeny. Neither latex exudation nor leaf toughness was induced in either species. Although significant genetic variation was detected within and among populations for constitutive expression of physical defense traits in Argemone, there was no evidence for genetic variation in the induction of these traits. This study provides the first evidence for the induction of physical defenses in prickly poppies, emphasizing how an ontogenetically explicit framework can reveal new insights into plant defense. Moreover, this study illustrates how sister species comparisons between island vs. continental plants can provide new insights into plant functional and evolutionary ecology, highlighting a fruitful area for future research on more species pairs.     

Assessment of genetic instabilities induced by tissue culture in alkaligrass

Alkaligrass (Puccinellia chinampoensis Ohwi), one of the important forage grasses in saline-alkalieroded grasslands, has been proved to be invaluable for improving saline-alkali soils. However, little is known of its genetic instabilities during in vitro culture for its artificial breeding. In this paper, a simple and efficient regeneration system of mature seed-induced calli in alkaligrass was established, and the somaclonal variation in the regenerated plants was assessed by inter-simple sequence repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. 18 randomly chosen regenerants were subjected to ISSR and REMAP analysis with the shoot from the same grain of seed as the control. ISSR analysis showed that of the 145 scored bands, 13 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 8.97%. REMAP analysis revealed that 4 out of 127 scored bands were polymorphic, a genetic variation frequency of 3.15% occurred. Cluster analysis indicated that the genetic similarity index calculated on the basis of ISSR data or REMAP data among the 18 regenerated plants and the donor plant was 0.974 and 0.996 respectively. All the results confirmed that somaclonal variation was induced by tissue culture in alkaligrass at a higher frequency, and indicated that the regeneration system could be a viable option for genetic improvement of alkaligrass by biotechniques.     

Somaclonal variation in plants: causes and detection methods

Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.     

Comparative study of morphological parameters of Grand Nain banana (<Emphasis Type="Italic">Musa</Emphasis> AAA) after <Emphasis Type="Italic">in vitro</Emphasis> multiplication with growth retardants

Variation in morphological traits and increased disease susceptibility were observed in micropropagated plants of rhubarb (Rheum rhaponticum L.), PC49. Our investigations have demonstrated that micropropagated plants can vary substantially in morphological traits and the variation of quantitative traits was substantially greater than conventionally propagated plants. Micropropagated plants produced significantly more leaves than conventional plants under the same growth period, with a more bushy growth habit and/or recumbence. A higher incidence of disease (petiole spotting) was also observed in micropropagated plants. It is concluded that micropropagated PC49 had substantially higher incidence of somaclonal variation and regenerants were not suitable to establish an economic crop comparable to the conventional plants.     

A 12-deoxywithastramonolide-rich somaclonal variant in Withania somnifera (L.) Dunal—molecular cytogenetic analysis and significance as a chemotypic resource

Withania somnifera, commonly known as ashwagandha or Indian ginseng, is a valuable medicinal plant, synthesizing a wide array of pharmacologically active secondary metabolites known as withanolides. In this study, we investigated variation among 54 regenerated plants attained through indirect organogenesis from leaf explants. Organogenic calli were induced on Murashige and Skoog medium containing 2?mg?l^?1 kinetin and 1?mg?l^?1 indole-3-butyric acid. High-performance liquid chromatography was used for quantitative determination of the major withanolides in the somaclones. One somaclone (WS-R-1) showed significantly higher accumulation of 12-deoxywithastramonolide (WS-12D; 0.516%) compared to the explant donor mother plant (0.002%). The incidence of somaclonal variation at the cytological level was investigated by studying mitosis and meiosis in relation to chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic chromosome count, or meiotic behavior. Fidelity at genomic level was evaluated by random amplification of polymorphic DNA (RAPD) analyses, which revealed multiple genetic polymorphisms between the WS-12D over-producing somaclone and the explant donor mother plant. This study demonstrates the capability of inducing chemotypic variability for the development of high-yielding clones due to molecular instability in W. somnifera using an in vitro approach.     

Field level evaluation of rice introgression lines for heat tolerance and validation of markers linked to spikelet fertility

Rice lines derived from wild species and mutants can serve as a good resource for favorable alleles for heat tolerance. In all, 48 stable lines including 17 KMR3/O. rufipogon introgression lines (KMR3 ILs), 15 Swarna/O. nivara ILs (Swarna ILs) along with their parents, Nagina 22 (N22) and its 4 EMS induced mutants and 7 varieties were evaluated for heat tolerance under irrigated conditions under field in two seasons, wet season 2012 using poly cover house method and dry season 2013 using late sown method. Spikelet fertility (SF), yield per plant (YP) and heat susceptibility index (HSI) for these two traits were considered as criteria to assess heat tolerance compared to control. Four KMR3 ILs and eight Swarna ILs were identified as heat tolerant based on SF and YP and their HSIs in both wet and dry seasons. S-65 and S-70 showed low SF and high YP consistently in response to heat in both seasons. We provide evidence that SF alone may not be the best criterion to assess heat tolerance and including YP is important as lines with low SF but high YP and vice versa were identified under heat stress. Out of 49 SSR markers linked to spikelet fertility, 18 were validated for five traits. RM229 in wet season and RM430 and RM210 in dry season were significantly associated with both SF and its HSI under heat stress. RM430 was also significantly associated with both YP and its HSI in dry season. Thirty two candidate genes were identified close to nine markers associated with traits under heat stress.     

高皂苷轮叶党参EMS诱变突变体研究

以轮叶党参为材料,采用甲基磺酸乙酯(EMS)处理离体叶片和愈伤组织对轮叶党参进行诱变,选择最佳诱变组合,并对诱变再生群体进行遗传分析。结果表明:(1)轮叶党参叶片和愈伤组织经EMS处理的存活率和分化率均低于对照,并且随EMS浓度的升高和处理时间的延长而下降。(2)叶片和愈伤组织的致死处理组合分别是0.4%EMS处理4h和0.3%EMS处理4h,半致死组合分别为0.3%EMS处理2h和0.2%EMS处理2h,愈伤组织是EMS诱变轮叶党参的的最佳材料。(3)筛选出的6号变异株皂苷含量为5.061mg/g,较对照平均值提高了5.48%。(4)对诱变再生苗进行遗传分析,8个特异引物对10个供试材料共扩增出59条带,具有多态性的谱带数为44条,占74.6%。材料间的相似系数变化范围0.453～0.912,其中3号、7号株与其他8株达到了品种间遗传差异。研究认为,EMS处理可应用于轮叶党参无性变异系的诱变,3号、6号、7号植株为诱变产生的具有较高皂苷含量的初选植株。     

Epigenetic variation in tissue cultured Gaura lindheimeri

High levels of variability were observed in tissue cultured Gaura lindheimeri genotypes when flowered in situ. Tissue culture treatments for chromosome doubling (colchicine: 0, 0.25, 1.25 mM; trifluralin: 0, 15, 30 μM) were all highly variable for morphological traits. Experiment No. 1 tested the tissue culture protocols used. In the first control (C1), plantlets were subcultured continuously. Nodes were excised and placed on solid medium for the second control (C2). In the third control (C3), nodes were excised and put in liquid medium for 24 h at room temperature. The fourth control (C4) was the same as the third control except liquid cultures were moved to 4°C for 48 h after treatment at room temperature. Experiment No. 2 examined the stability of the variation. Representative plants with different traits were selected for clonal propagation and grown in a replicated trial in the greenhouse. Several morphological traits (flower size, leaf length:width ratios, petal length:width ratios, and flower color) were measured. All of the controls had as much variability as the treated plants. Flower size of the first flower for plant number 01G-02 was significantly different in C1 compared with C3, but not with C2 and C4. Plant number 443-1 (white) was more stable in the replicated trial for the flower size than plant number 01G-02 (pink). All traits measured for plant number 01G-02 were unstable; most flower colors and patterns reverted back to the original color of the non-tissue cultured plants. The somaclonal variation observed was epigenetic in nature, indicating changes in DNA methylation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.     

环境及遗传背景对延河流域植物叶片和细根功能性状变异的影响

研究环境筛选作用和植物系统发育背景对植物群落构建产生的影响,有助于理解植物在生长过程中对资源的分配利用和对环境的适应规律。以延河流域3个植被带(森林带、森林草原带及典型草原带)稳定的自然植物群落为研究对象,调查了31个样地107种植物,隶属于35个科78个属,测量了6种叶片和3种细根性状。分别对3个植被带和不同植物科植物的叶片和细根性状做单因素方差分析,结果表明:叶片氮含量和细根氮含量在3个植被带间无显著差异,叶厚度、比叶面积、叶组织密度、叶片磷含量、叶片氮磷比、比根长、根组织密度在3个植被带间差异极显著。由南向北随着气候干旱的加剧,植物通过调节叶片和细根性状,表现出了不同的适应策略:森林带植物叶片相对生长速率高,根系防御力强;森林草原带植物叶片防御力强,根系相对生长速率快。不同科的植物在相同的环境条件下,对于资源的竞争力和胁迫的忍耐力也有所不同,比如豆科植物具有远远高于其他科的叶片和细根氮含量,但是对养分的利用效率并不高。GLM分析结果说明,所涉及的植物功能性状的空间变异主要来自于年均降雨量的变化及植物科的差异,如16.26%的比叶面积的变异可由年均降雨量变化解释,4.02%可由植物科的差异解释。物种水平上,叶厚度、比叶面积、叶组织密度、比根长、根组织密度、叶片磷含量是对气候干燥度变化响应敏感的植物功能性状,其空间变异主要由环境差异所致。延河流域的植物群落在形成过程中,存在明显的环境筛选效应。这表明,环境异质性在植被恢复实践中必须予以考虑。     

Somaclonal variation in tomato: effect of explant source and a comparison with chemical mutagenesis

Summary Plants were regenerated from leaf, cotyledon, and hypocotyl explants of tomato cv Moneymaker. Various phenotypic alterations were observed among regenerated plants (R₁), but were not transmitted to the progenies, except for ploidy variation. Variation in ploidy level, mainly tetraploidy, occurred in R₁ plants and their R₂ progenies, and the frequency of polyploid plants depended on the explant source. More than 50% of the regenerants derived from hypocotyl explants were found to be polyploid. A correlation was observed between the percentage of polyploid cells present in the explant material in vivo and the frequency of polyploid plants. Several monogenic mutations were recovered in the R₂, four of which were shown to be allelic to known, recessive, single-gene mutants. No significant effect of explant source or duration of tissue culture period on mutant frequency or spectrum was found. For several mutant types that could be scored unambiguously, somaclonal variation was compared to variation induced by treatment of seeds with ethyl methane sulphonate (EMS). The results showed that the mutant frequencies were higher after EMS treatment than those generated through tissue culture. With respect to the mutant spectrum, no clear differences were observed between the spectra obtained after EMS treatment and those after tissue culture. However, tissue culture gave rise to polyploid plants, whereas no ploidy variants occurred after EMS treatment.     

Analysis of genomic DNA methylation patterns in regenerated and control plants of rye (Secale cereale L.)

Rye (Secale cereale L.) is a species that has shown high rates of somaclonal variation when plants obtained by in vitro culture were analysed using different techniques. In this study, using methylation-sensitive amplified polymorphism (MSAP) markers, we analysed the cytosine methylation status at genomic level of regenerated plants of rye that were obtained by somatic embryogenesis. Such plants were originated from three different cell lines and the results were compared with the data obtained from the control plants grown from seeds of the same cultivar and lot. A similar total number of MSAP markers was observed in the regenerated (937) and control plants (1,022), while the mean number detected per plant was significantly higher in regenerated (554.43) than in control plants (356.00). The analysis indicated conservation of the number of partially-methylated CCGG/GGCC sites for all type of plants. However the mean number of non-methylated sites was near twofold in the regenerated plants (442.48) than in control plants (248.19). Methylation changes have been detected in all the regenerated plants when compared within cell lines, with an average frequency of 9.01 % of the detected markers. We also observed that regenerated plants from one or several cell lines shared methylation changes at the same locus pointing to a non-random behaviour of the changes in genomic methylation.