Global DNA methylation profiles of somatic embryos of peach palm (Bactris gasipaes Kunth) are influenced by cryoprotectants and droplet-vitrification cryopreservation

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.     

马铃薯（Solanum tuberosum L.）茎尖的超低温保存及其遗传变异的初步观察

研究马铃薯茎尖超低温保存技术的结果表明,4℃低温下锻炼6d,在添加二甲基亚砜（DMSO）和乙酰胺的培养基中预培养5d,60％PVS2于室温下装载30min,0℃下PVS2脱水40min时,茎尖成活率最高（71．6％）,再生植株生长分化正常。进一步对再生植株进行AFLP分析,6对引物组合共扩增出385条带,超低温保存前后的材料之间未见到明显差的异带,但用MSAP技术分析超低温保存前后植株甲基化的结果显示：超低温保存后的材料均有不同程度的甲基化。在扩增的624条带中,处理与否之间完全一致的带型为584条;有变化的带型为40条,处理2（茎尖经过完整的超低温保存过程,区别于处理1,增加了冷冻、解冻和洗涤后恢复培养）有13个位点的甲基化增加,21个位点去甲基化。     

Field performance evaluation and genetic integrity assessment of cryopreserved papaya clones

This paper is the first report of field performance and evaluation of morphological traits following cryopreservation in four genotypes of Carica papaya (Z6, 97, TS2 and 35). It also describes the successful establishment of in vitro plantlets following vitrification-based cryopreservation of shoot tips and their acclimatisation through to field establishment. Cloned plants resulting from untreated controls, as well as controls taken at three other stages of the cryopreservation process (dissection, pre-treatment, plant vitrification solution 2 (PVS2) treatment) and cryopreserved plants were established to ensure a rigorous appraisal of any variation. Results indicate no differences between any of the control plants or cryopreserved plants for either growth performance or morphology. In addition, both randomly amplified DNA fingerprinting and amplified DNA methylation polymorphism markers were used to assess any genomic or methylation changes in genotype 97 at four different developmental stages post cryopreservation (in vitro, acclimatisation and field). Only small genomic DNA modifications (0–8.3%) were detected in field stage plants and methylation modifications (0–4.3%) were detected at both the in vitro and field stages for samples treated with PVS2 or cryopreservation.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

Epigenetics in plant tissue culture

Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at the molecular level, viz., changes in DNA methylation and alterations of histone methylation or acetylation. Various studies have been carried out with animals, and with plant cells or tissues that have grown in tissue culture but only little work has been done with shoots generated by axillary branching. We present various molecular methods that are being used to measure epigenetic variation. In micropropagated plants mostly differences in DNA methylation have been examined. Epigenetic changes are thought to underlie various well-known tissue-culture phenomena including rejuvenation, habituation, and morphological changes such as flower abnormalities, bushiness, and tumorous outgrowths in, among others, oil palm, gerbera, Zantedeschia and rhododendron.     

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.     

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.     

A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control

Earlier experiments demonstrated that DNA from young plants of 5-azacytidine-induced flax (Linum usitatissimum) lines that flower earlier-than-normal is hypomethylated relative to DNA from their control lines and detected differences in methylation level between plants sampled at different ages, which suggested that the methylation level in flax changes during development. To investigate this possibility, and its potential impact on the difference in methylation level between early-flowering and control lines, developmental profiles were established for the cytosine methylation levels in DNA from post-germination seedlings and from the shoot tips of main stems and the cotyledons sampled throughout vegetative phase. The methylation profiles for two early-flowering lines and their control lines were compared. The methylation profiles were then compared to profiles for DNA content, tissue weight and chlorophyll content (green tissues); these additional parameters provided information on tissue status in terms of cell division, tissue expansion and/or photosynthetic maturity. With one exception, methylation levels were either static or increased with plant age and/or tissue maturity; the highest methylation levels were seen in senescent cotyledons. Although DNA from immature plants or tissues of the early-flowering lines was usually hypomethylated, the hypomethylation was not always apparent in tissues from older plants.     

NATURAL VARIATION IN EPIGENETIC GENE REGULATION AND ITS EFFECTS ON PLANT DEVELOPMENTAL TRAITS

In plants, epigenetic variation contributes to phenotypic differences in developmental traits. At the mechanistic level, this variation is conferred by DNA methylation and histone modifications. We describe several examples in which changes in gene expression caused by variation in DNA methylation lead to alterations in plant development. In these examples, the presence of repeated sequences or transposons within the promoters of the affected genes are associated with DNA methylation and gene inactivation. Small interfering RNAs expressed from these sequences recruit DNA methylation to the gene. Some of these methylated alleles are unstable giving rise to revertant sectors during mitosis and to progeny in which the methylated state is lost. However, others are stable for many generations and persist through speciation. These examples indicate that although DNA methylation influences gene expression, this is frequently dependent on classical changes to DNA sequence such as transposon insertions. By contrast, forms of histone methylation cause repression of gene expression that is stably inherited through mitosis but that can also be erased over time or during meiosis. A striking example involves the induction of flowering by exposure to low winter temperatures in Arabidopsis thaliana and its relatives. Histone methylation participates in repression of expression of an inhibitor of flowering during cold. In annual, semelparous species such as A. thaliana, this histone methylation is stably inherited through mitosis after return from cold to warm temperatures allowing the plant to flower continuously during spring and summer until it senesces. However, in perennial, iteroparous relatives the histone modification rapidly disappears when temperatures rise, allowing expression of the floral inhibitor to increase and limiting flowering to a short interval. In this case, epigenetic histone modifications control a key adaptive trait, and their pattern changes rapidly during evolution associated with life‐history strategy. We discuss these examples of epigenetic developmental traits with emphasis on the underlying mechanisms, their stability, and adaptive value.     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥(Arabidopsis thaliana)幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明, 在扩增的662条带中, 对照和2个处理及其第2代间完全一致的带型有598条; 发生变化的带型有64条, 其中能遗传给第2代的有48条, 占变异条带的75%。与对照相比, 经超低温保存的样品新产生的甲基化位点有14个, 而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条, 差异条带有38条, 占5.7%, 而对照与未冷冻处理组的差异率是7.45%, 对照与冷冻处理组之间的差异率是6.63%。可见, 拟南芥在超低温保存中, 无论是经液氮冷冻还是未经冷冻处理, 对材料的甲基化状态均有影响, 而这种甲基化变化大部分是可以遗传的。     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥（Arabidopsis thaliana）幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明,在扩增的662条带中,对照和2个处理及其第2代间完全一致的带型有598条：发生变化的带型有64条,其中能遗传给第2代的有48条,占变异条带的75％。与对照相比,经超低温保存的样品新产生的甲基化位点有14个,而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条,差异条带有38条,占5.7％,而对照与未冷冻处理组的差异率是7．45％,对照与冷冻处理组之间的差异率是6。63％。可见,拟南芥在超低温保存中,无论是经液氮冷冻还是未经冷冻处理,对材料的甲基化状态均有影响,而这种甲基化变化大部分是可以遗传的。     

Analysis of target sequences of DDM1s in Brassica rapa by MSAP

DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.     

Changes in DNA-methylation during zygotic embryogenesis in interspecific hybrids of beans (<Emphasis Type="Italic">Phaseolus</Emphasis> ssp.)

Hybrid embryos resulting from crosses between Phaseolus species often fail to reach maturity and some combinations frequently abort at early developmental stages. The genetic or molecular basis for these consistent developmental defects is at present not clear. However, an extremely complex genetic system, thought to be caused by major epigenetic changes associated with gene expression changes, has been shown to be active in plant species. We have investigated DNA methylation in two interspecific hybrids, Phaseolus vulgaris × Phaseolus coccineus and its reciprocal crosses, using methylation sensitive amplification polymorphism (MSAP). The potential use of MSAP for detecting methylation variation during embryogenesis in interspecific hybrids is discussed. Significant differences in the DNA methylation patterns were observed in abortive (interspecific hybrids) and non abortive (parental) genotypes. Taken together, our results strongly suggest that generalized alterations in DNA methylation profiles could play a causative role in early interspecific embryo abortion in vivo. A considerable change in the methylation pattern during embryogenesis could be involved in the disruption of the regulation or maintenance of the embryogenesis process of Phaseolus interspecific hybrids. The results also support the earlier hypothesis that DNA methylation is critical for the regulation of plant embryogenesis and gene expression.     

Variable DNA methylation changes during differentiation of human melanoma cells

The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.