An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond

Epigenetic mechanisms are highly dynamic events that modulate gene expression. As more accurate and powerful tools for epigenetic analysis become available for application in a broader range of plant species, analysis of the epigenetic landscape of plant cell cultures may turn out to be crucial for understanding variant phenotypes. In vitro plant cell and tissue culture methodologies are important for many ongoing plant propagation and breeding programmes as well as for cutting-edge research in several plant model species. Although it has long been known that in vitro conditions induce variation at several levels, most studies using such conditions rely on the assumption that in vitro cultured plant cells/tissues mostly conform genotypically and phenotypically. However, when large-scale clonal propagation is the aim, there has been a concern in confirming true-to-typeness using molecular markers for evaluating stability. While in most reports genetic variation has been found to occur at relatively modest frequencies, variation in DNA methylation patterns seems to be much more frequent and in some cases it has been directly implicated in phenotypic variation. Recent advances in the field of epigenetics have uncovered highly dynamic mechanisms of chromatin remodelling occurring during cell dedifferentiation and differentiation processes on which in vitro adventitious plant regeneration systems are based. Here, an overview of recent findings related to developmental switches occurring during in vitro culture is presented. Additionally, an update on the detection of epigenetic variation in plant cell cultures will be provided and discussed in the light of recent progress in the plant epigenetics field.     

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Determining DNA strand breakage from embryogenic cell cultures of a conifer species using the single-cell gel electrophoresis assay

Although a routine procedure to detect mutagenesis by DNA strand breakage in animal cells, the single-cell gel electrophoresis (“comet”) assay is difficult to apply in plant material due to constraints in obtaining suitable nucleoids (formed by DNA trapped in the agarose matrix after the cell lysis process) in either quality or quantity. A suitable protocol is described for the first time to perform the comet assay in conifer somatic embryogenic cultures by determining total DNA strand breakage in protoplasts, after having failed to acquire nuclei by standard mechanical techniques. The results show that protoplasts obtained from embryogenic cultures of the Norway spruce (Picea abies) are suitable to be lysed and surveyed for DNA damage through the standard alkaline version of the comet assay. Several common comet metrics were compared and all were found suitable for analysis, with the percentage of DNA in the comets' tail (constituted by DNA fragments that migrated during electrophoresis), given by the proportion between tail fluorescence intensity and total nucleoid intensity, being simplest and the most sensitive to compare between control and hydrogen peroxide-treated cells. The established procedures may be useful, for instance, for a comparative evaluation of somatic embryogenesis protocols and selection of less damaging treatments for clonal propagation or for mutagenesis-related studies with conifer cell cultures.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Efficient protocol for <Emphasis Type="Italic">in vitro</Emphasis> mass micropropagation of slash pine

An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~?7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~?43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~?89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.