Liliana Marum

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.

Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l−1 polyethylene glycol (PEG) and 10 g l−1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.

Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber

The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

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.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

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< or =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.

Physiological characterization and true-to-typeness evaluation of in vitro and ex vitro seedlings of Pinus elliottii: A contribution to breeding programs

Pinus elliottii var. elliottii is a pine species with enormous economic value particularly for timber and resin industries, and is subject of high pressure for genetic improvement and cloning elite genotypes. We have recently developed a robust micropropagation protocol for this species. Plantlets performance needs to be evaluated to validate this protocol for further mass propagation. Micropropagated plantlets and seed-derived plants with similar age and shoot length were compared regarding photosynthesis, carbohydrates and pigments content, water status, DNA content and cell cycle dynamics. Micropropagated plantlets had an overall physiological performance similar to seed-derived plants. In particular, except for the transpiration rate (E), CO2 assimilation rate (A) and total soluble sugars (TSS) content, no major differences between plantlets and seedlings in terms of relative water content (RWC), chlorophyll a fluorescence and pigments content were found. Genetic fidelity analyses support that the micropropagation protocol neither induce DNA content changes nor alterations in cell cycle dynamics.

Identification of GPAT acyltransferases in cork oak

Background Acyltransferases are enzymes with an important role in the synthesis of both cutin and suberin which are part of the lipophilic barriers, such as epidermis and periderm that protect terrestrial plants against water loss and other external aggressions. During secondary growth in woody plants such as cork oak (Quercus suber L.), the epidermis is replaced by a suberized periderm that includes the phellem (cork), phellogen (cork cambium) and phelloderm tissues. In Q. suber the successive formation of phellem following removal at periodic intervals (every 9 years) allows for exploitation of cork oak on a sustainable basis. The main component of cork (45-50%) is suberin, a complex polymer comprising both aliphatic and aromatic domains and associated waxes [1,2]. Despite the physiological importance of suberin, its biosynthetic pathway as well as its deposition remains largely unknown. Since cork oak is a unique species among terrestrial plants due to its remarkable capacity for cork production, it is expected that suberin biosynthesis and deposition are tightly controlled mechanisms. As a first step to start unraveling these control mechanisms we intend to identify and characterize genes coding for the acyltransferases of the GPAT (glycerol-3-phosphate acyltransferase) family, involved in suberin and cutin synthesis in cork oak. Two ESTs highly similar to GPAT5 (EE 743864 and EE 743865) and one EST (EE743668) highly similar to GPAT4 shown to be strongly up-regulated in the suberin-rich phellem of cork oak tree (Q. suber) were first identified by Soler et al. [3]. Material and methods In this work, phellem tissues from small branches with increasing age (1 to 7 years old) were harvested from cork oak and holm oak (a related but cork non-producing species)at the Instituto Superior de Agronomia (Portugal). Tissues collected during different growth periods were also used for analysis: samples collected during a period of high phellogen activity (April – June, 2009 and 2010) and samples collected during the inactive growth period (January, 2010). Total RNA was successfully extracted from these tissues using a protocol described by Reid et al. [4], with minor modifications. cDNA was synthesized using standard procedures and 5’and 3’-RACE are being performed in order to determine the full-length of putative GPAT coding sequences from Q. suber transcriptome. The expression level of GPAT4 and GPAT5 genes was assessed by quantitative RT_PCR in two different seasonal stages (April and June) in periderm cells from 3 year old branches of Q. suber. The Cp values were converted into relative quantities, using the formula, Q=E, where E (the efficiency of the gene amplification for each primer pair) was calculated using the Real-time PCR Miner algorithm.

Cryopreservation of Hybrid Pinus elliottii × P. caribaea

The cryopreservation of embryogenic cultures within operational forestry proves to be a crucial tool, to store clones without loss of juvenility, while field tests, to identify genotypes with significant genetic gain, are being conducted. Besides several conifers species have been cryopreserved till now, only few studies have been reported for hybrid species. The interspecific pine hybrid, P. elliottii var. elliottii x P. caribaea var. hondurensis has a major economic importance mainly in South America, West Africa and Australia. The hybrid superiority appears to be derived from a complementary recombination of traits from the two parental species–growth rate and high yield of resin production from P. caribaea var. hondurensis, combined with wind-firmness, adaptability to wet sites, high wood-density and stem straightness of P. elliottii var. elliottii. This chapter describes a complete cryopreservation procedure for embryonal mass of the hybrid P.elliottii var. elliottii x P. caribaea var. hondurensis, and a methodology to analyse the ploidy stability by flow cytometry.