Difference in Cd2+ flux around the root tips of different soybean (Glycine max L.) cultivars and physiological response under mild cadmium stress.

The purpose of the study was to compare differences in Cd2+ flux in the vicinity of root tips of 20 soybean cultivars under mild Cd stress conditions using non-invasive micro-test technology (NMT). The results indicated that Cd2+ influx to the root tips under mild Cd treatment was higher compared to controls. Cd2+ influx showed an obvious spatial distribution, with the highest Cd2+ influx measured 300 µm from the root tips, and a gradually decrease above and below this site. The cultivar Liaodou32 had a lower Cd uptake (3.40 pmol cm-2 s-1), while Liaodou23 had a relatively higher Cd uptake (66.37 pmol cm-2 s-1). Cluster analysis showed that the order of the average Cd2+ influx of the cultivars at a distance of 300 µm from the root tips was as follows: high-uptake cultivars (61.80 pmol cm-2 s-1)>medium-high-uptake cultivars (33.92 pmol cm-2 s-1)>medium-low-uptake cultivars (19.78 pmol cm-2 s-1)>low-uptake cultivars (4.84 pmol cm-2 s-1). We also analyzed physiological responses of different soybean cultivars to mild Cd stress. The results indicated that mild Cd stress could inhibit soluble protein production and root vigor among individual soybean cultivars. Moreover, stress increased SOD, CAT and POD activities and MDA content in root tissues. It should be noted that the physio-biochemical indicators of low-uptake cultivars did not change significantly after exposure to mild Cd stress compared to controls. Pearson's correlation analyses showed that all physio-biochemical indicators were significantly positively associated with influx, except of root SP and biomass. PCA analysis demonstrated that root vigor was a dominant factor causing the differences in Cd tolerance among different soybean seedling cultivars. NMT is of great significance for safe utilization of contaminated soil to distinguish the cultivars with different enrichment capacity for heavy metals from different crop cultivars.

Using single cell type proteomics to identify Al-induced proteomes in outer layer cells and interior tissues in the apical meristem/cell division regions of tomato root-tips.

Aluminum (Al) toxicity primarily targets the root tips, inhibiting root growth and function and leading to crop yield losses on acidic soils. Previously we reported using laser capture microdissection (LCM) proteomics to identify Al-induced proteins in the outer layer cells in the transitional zone of tomato root-tips. This study aims to further characterize Al-induced proteomic dynamics from the outer to interior tissues, thus providing a panoramic view reflecting Al resistance in the root tip as a whole in tomatoes. Three types of cells were isolated via LCM from the basal 350-400 µm (below cell elongation regions) of root tips using tomato (Solanum lycopersicum) 'Micro-Tom' plants. Type I and Type II were from Al-treated plants. Type I included cells of the outer three layers, i.e., the epidermis and cortex initials and the quiescent center (QC) in root apical meristem (RAM), and Type II possessed the interior tissues of the same region. Type III contained cells from the non-Al-treated root tips collected in the same region as Type I. Two tandem mass tag (TMT) proteomics analyses with three biological replicates for each sample type were conducted. The TMTexp1 (comparing Type I and Type II) identified 6575 quantifiable proteins and 178 different abundance proteins (DAPs). The TMTexp2 (comparing Type I and Type III) identified 7197 quantifiable proteins and 162 DAPs. Among all quantified proteins (7685) from the two TMT experiments, 6088 (79%) proteins, including 313 DAPs (92% of the 340 total), were identified in all tissues. A model reflecting the tissue-specific Al-resistance mechanism was proposed, in which the level of the citrate transporter MATE protein, involved in Al exclusion, accumulated to the highest level in the outer-layer cells but decreased toward the interior of root-tips (which concurs with the tissue-specific importance in Al resistance). Proteins for biosynthesis of ethylene and jasmonic acid, proteolytic enzymes, stress-responsive proteins, and cell wall modeling were affected by Al treatment, some in a cell type-specific manner. The KEGG metabolite pathways enriched with these DAPs changed depending on the cell types. This study demonstrated the advantage of using the tissue/cell-specific analysis for identifying proteins and their dynamic changes directly associated with Al resistance in the root-tip region. The proteomics datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/) with the dataset identifier as PXD021994 under project title: Proteomics studies of outer and inner cellular layers of tomato root-tips for Al stress, Project DOI: 10.6019/ PXD021994; and PXD018234 under Project title: Al-induced root proteomics changes in stress-acclimated tomato plant, Project DOI: https://doi.org/10.6019/PXD018234. SIGNIFICANCE: This paper presents the method of using laser capture microdissection (LCM) to collect homogenous cell-type specific tissue samples from the outer layers and inner central regions of tomato root-tips. The tandem mass tag-proteomics analysis showed that the outer-layer cells expressed proteomes that were different from the inner tissues of Al-treated root-tips; proteins related to resistance/tolerance to Al toxicity were highly accumulated in the outer-layer cells. Furthermore, the Al-treated outer-layer cells expressed proteomes which were different from the non-Al treated counterpart cells. This study has provided the first dataset of proteins differentiating from the outer to inner layers of cells in Al-treated root-tips. It provided convincing experimental evidences demonstrating the single-cell type proteomics as a powerful analytical approach to identify Al tolerance mechanisms in plants. The analytical procedure of LCM-tandem mass tag-quantitative proteomics analysis has a broad application for proteomics analysis of spatially separated cells in complex tissues.

Cadmium (II)-Induced Oxidative Stress Results in Replication Stress and Epigenetic Modifications in Root Meristem Cell Nuclei of Vicia faba.

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

Awareness and integrated information theory identify plant meristems as sites of conscious activity.

Lacking an anatomical brain/nervous system, it is assumed plants are not conscious. The biological function of consciousness is an input to behaviour; it is adaptive (subject to selection) and based on information. Complex language makes human consciousness unique. Consciousness is equated to awareness. All organisms are aware of their surroundings, modifying their behaviour to improve survival. Awareness requires assessment too. The mechanisms of animal assessment are neural while molecular and electrical in plants. Awareness of plants being also consciousness may resolve controversy. The integrated information theory (IIT), a leading theory of consciousness, is also blind to brains, nerves and synapses. The integrated information theory indicates plant awareness involves information of two kinds: (1) communicative, extrinsic information as a result of the perception of environmental changes and (2) integrated intrinsic information located in the shoot and root meristems and possibly cambium. The combination of information constructs an information nexus in the meristems leading to assessment and behaviour. The interpretation of integrated information in meristems probably involves the complex networks built around [Ca2+]i that also enable plant learning, memory and intelligent activities. A mature plant contains a large number of conjoined, conscious or aware, meristems possibly unique in the living kingdom.

Mentha piperita L. Micropropagation and the Potential Influence of Plant Growth Regulators on Volatile Organic Compound Composition.

Due to the industrial use of Mentha piperita L. (peppermint), it is important to develop an optimal method to obtain standardized plant material with specific quality parameters. In vitro cultures may allow the production of desirable odor-active compounds (OACs) and improve their share in the plant aroma profile. There are two types of explants that are commonly used, apical meristems and nodal segments. In this study, the best overall effects were shown to be produced by the combination of MS medium with the addition of 0.5 mg·dm-3 indolyl-3-butyric acid. In this case, a very high degree of rooting was found (97% for apical meristems, 100% for nodal meristems), lateral shoots were induced in 83% of both types of explant, and the content of OACs in the plant aroma profile increased significantly, especially menthofurolactone and cis-carvone oxide, responsible in this case for a characteristic mint-like aroma. The comparison of the volatile organic compounds (VOCs) obtained from plants of different origin by GC-MS showed no significant differences in their qualitative composition. Moreover, in-vitro-cultivated peppermint on a medium containing 0.5 mg·dm-3 2-isopentinloadenine and 0.1 mg·dm-3 indolyl-3-acetic acid showed significant amounts of menthofurolactone in its VOC composition.

Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea.

KEY MESSAGE: Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

A laser ablation technique maps differences in elemental composition in roots of two barley cultivars subjected to salinity stress.

In saline soils, high levels of sodium (Na+ ) and chloride (Cl- ) ions reduce root growth by inhibiting cell division and elongation, thereby impacting on crop yield. Soil salinity can lead to Na+ toxicity of plant cells, influencing the uptake and retention of other important ions [i.e. potassium (K+ )] required for growth. However, measuring and quantifying soluble ions in their native, cellular environment is inherently difficult. Technologies that allow in situ profiling of plant tissues are fundamental for our understanding of abiotic stress responses and the development of tolerant crops. Here, we employ laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to quantify Na, K and other elements [calcium (Ca), magnesium (Mg), sulphur (S), phosphorus (P), iron (Fe)] at high spatial resolution in the root growth zone of two genotypes of barley (Hordeum vulgare) that differ in salt-tolerance, cv. Clipper (tolerant) and Sahara (sensitive). The data show that Na+ was excluded from the meristem and cell division zone, indicating that Na+ toxicity is not directly reducing cell division in the salt-sensitive genotype, Sahara. Interestingly, in both genotypes, K+ was strongly correlated with Na+ concentration, in response to salt stress. In addition, we also show important genetic differences and salt-specific changes in elemental composition in the root growth zone. These results show that LA-ICP-MS can be used for fine mapping of soluble ions (i.e. Na+ and K+ ) in plant tissues, providing insight into the link between Na+ toxicity and root growth responses to salt stress.

Phytochemical analysis and effect of the essential oil of Psidium L. species on the initial development and mitotic activity of plants.

The use of allelopathic compounds is an alternative for weeds control, since they present low toxicity when compared with the synthetic herbicides, that may cause several damages, as the contamination of the environment. Our objective was to determine the chemical composition and allelopathic properties of the essential oils of Psidium cattleianum, P. myrtoides, P. friedrichsthalianum, and P. gaudichaudianum on the germination and root growth of Lactuca sativa and Sorghum bicolor, and to evaluate their action on the cell cycle of root meristematic cells of L. sativa. The main compound found in all the studied species was (E)-caryophyllene (P. cattleianum-23.4 %; P. myrtoides-19.3%; P. friedrichsthalianum-24.6% and P. gaudichaudianum-17.0%). The different essential oils were tested at different concentrations on L. sativa and S. bicolor, reducing germination, germination speed index, and root and shoot growth of lettuce and sorghum seedlings. The cytotoxicity and aneugenic potential of these oils were evidenced by the reduction of the mitotic index and increase of the frequency of chromosomal alterations in L. sativa. The essential oils of the species of Psidium studied have potential to be used in weeds control.

Growth and biomechanics of shoot organs.

Plant organs arise through complex interactions between biological and physical factors that control morphogenesis. While there has been tremendous progress in the understanding of the genetics behind development, we know much less about how mechanical forces control growth in plants. In recent years, new multidisciplinary research combining genetics, live-imaging, physics, and computational modeling has begun to fill this gap by revealing the crucial role of biomechanics in the establishment of plant organs. In this review, we provide an overview of our current understanding of growth during initiation, patterning, and expansion of shoot lateral organs. We discuss how growth is controlled by physical forces, and how mechanical stresses generated during growth can control morphogenesis at the level of both cells and tissues. Understanding the mechanical basis of growth and morphogenesis in plants is in its early days, and many puzzling facts are yet to be deciphered.

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species.

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

Connected through the force: mechanical signals in plant development.

As multicellular organisms, plants acquire characteristic shapes through a complex set of biological processes known as morphogenesis. Biochemical signalling underlies much of development, as it allows cells to acquire specific identities based on their position within tissues and organs. However, as growing physical structures, plants, and their constituent cells, also experience internal and external physical forces that can be perceived and can influence key processes such as growth, polarity, and gene expression. This process, which adds another layer of control to growth and development, has important implications for plant morphogenesis. This review provides an overview of recent research into the role of mechanical signals in plant development and aims to show how mechanical signalling can be used, in concert with biochemical signals, as a cue allowing cells and tissues to coordinate their behaviour and to add robustness to developmental processes.

Global Topological Order Emerges through Local Mechanical Control of Cell Divisions in the Arabidopsis Shoot Apical Meristem.

The control of cell position and division act in concert to dictate multicellular organization in tissues and organs. How these processes shape global order and molecular movement across organs is an outstanding problem in biology. Using live 3D imaging and computational analyses, we extracted networks capturing cellular connectivity dynamics across the Arabidopsis shoot apical meristem (SAM) and topologically analyzed the local and global properties of cellular architecture. Locally generated cell division rules lead to the emergence of global tissue-scale organization of the SAM, facilitating robust global communication. Cells that lie upon more shorter paths have an increased propensity to divide, with division plane placement acting to limit the number of shortest paths their daughter cells lie upon. Cell shape heterogeneity and global cellular organization requires KATANIN, providing a multiscale link between cell geometry, mechanical cell-cell interactions, and global tissue order.

Supraoptimal Cytokinin Content Inhibits Rice Seminal Root Growth by Reducing Root Meristem Size and Cell Length via Increased Ethylene Content.

Cytokinins (CKs), a class of phytohormone, regulate root growth in a dose-dependent manner. A certain threshold content of CK is required for rapid root growth, but supraoptimal CK content inhibits root growth, and the mechanism of this inhibition remains unclear in rice. In this study, treatments of lovastatin (an inhibitor of CK biosynthesis) and kinetin (KT; a synthetic CK) were found to inhibit rice seminal root growth in a dose-dependent manner, suggesting that endogenous CK content is optimal for rapid growth of the seminal root in rice. KT treatment strongly increased ethylene level by upregulating the transcription of ethylene biosynthesis genes. Ethylene produced in response to exogenous KT inhibited rice seminal root growth by reducing meristem size via upregulation of OsIAA3 transcription and reduced cell length by downregulating transcription of cell elongation-related genes. Moreover, the effects of KT treatment on rice seminal root growth, root meristem size and cell length were rescued by treatment with aminoethoxyvinylglycine (an inhibitor of ethylene biosynthesis), which restored ethylene level and transcription levels of OsIAA3 and cell elongation-related genes. Supraoptimal CK content increases ethylene level by promoting ethylene biosynthesis, which in turn inhibits rice seminal root growth by reducing root meristem size and cell length.

Cytological Effects of Bleaching Agent (Quneex) on Plant Cells and Plant DNA.

BACKGROUND AND OBJECTIVE: There have been a number of reported drawbacks and efficacy issues regarding the use of bleaching agents in the plant industry. This study was conducted to determine the cytological effects of the bleaching agent (Quneex) on the plant cells and plant DNA using the Allium cepa assay. MATERIALS AND METHODS: It was subjected sixteen root meristems of A. cepa to different concentrations of the bleaching agent (0.1, 0.2, 0.3, 0.4 and 0.5%) with different periods of time (6, 12 and 24 h). Recovery was done for 6, 12 and 24 h after exposure. RESULTS: The mitotic index significantly decreased with time and also decreased with increase in the concentration of the bleaching agent. Abnormal chromosomal changes reflecting mutagenesis including stickiness, laggards, bridges, C-metaphase, star-metaphase, binucleation, polyploidy, disturbance and multinucleation were observed in the different concentrations and periods of time. After recovery, a slow increase in the mitotic index was observed. All treatments with or without recovery for 12 and 24 h resulted in reduction in the amount of DNA. CONCLUSION: Bleaching agents similar to Quneex containing sodium hypochlorite have mutagenic properties that can be potentially hazardous to the environment and also to humans. Thus, there is a need to regulate the use and disposal of such chemicals into the environment particularly to the sewers, to prevent contamination of potable water, plant and biodiverse aquatic animals.

Polyphenolic profile and antioxidant activity of meristem and leaves from "chagual" (Puya chilensis Mol.), a salad from central Chile.

The Bromeliaceae Puya chilensis Mol. is a native monocotyledonous food plant that can be found in central Chile. It is traditionally known as chagual. The tender basal part of the leaves, just starting from the meristem, are consumed as a salad. The aim of this work was to describe the phenolic content and composition of the meristem and leaves of chagual, as well as their antioxidant capacity and inhibitory activity against metabolic syndrome-associated enzymes. Samples of chagual, including two cultivated and three wild growing plants, were analyzed and compared for composition and bioactivity. From the phenolic enriched extract of the plant (PEE), 26 compounds were tentatively identified by HPLC-DAD-ESI-MSn, including 12 hydroxycinnamic acids and 14 flavonoids. The main compounds were identified as diferuloyl hexaric acid isomers and 5-p-Coumaroylquinic acid. The compounds were quantified in both meristem and leaves. The PEE content was up to ten times higher in the meristem than in the leaves, ranging from 0.18 to 124.08 mg/g PEE. The samples inhibited α-glucosidase, but did not show effect on α-amylase and pancreatic lipase. This is the first report on the polyphenol composition and bioactivity of the edible components of the chagual food plant.

A phosphoinositide map at the shoot apical meristem in Arabidopsis thaliana.

BACKGROUND: In plants, the shoot apical meristem (SAM) has two main functions, involving the production of all aerial organs on the one hand and self-maintenance on the other, allowing the production of organs during the entire post-embryonic life of the plant. Transcription factors, microRNA, hormones, peptides and forces have been involved in meristem function. Whereas phosphatidylinositol phosphates (PIPs) have been involved in almost all biological functions, including stem cell maintenance and organogenesis in animals, the processes in meristem biology to which PIPs contribute still need to be delineated. RESULTS: Using biosensors for PI4P and PI(4,5)P2, the two most abundant PIPs at the plasma membrane, we reveal that meristem functions are associated with a stereotypical PIP tissue-scale pattern, with PI(4,5)P2 always displaying a more clear-cut pattern than PI4P. Using clavata3 and pin-formed1 mutants, we show that stem cell maintenance is associated with reduced levels of PIPs. In contrast, high PIP levels are signatures for organ-meristem boundaries. Interestingly, this pattern echoes that of cortical microtubules and stress anisotropy at the meristem. Using ablations and pharmacological approaches, we further show that PIP levels can be increased when the tensile stress pattern is altered. Conversely, we find that katanin mutant meristems, with increased isotropy of microtubule arrays and slower response to mechanical perturbations, exhibit reduced PIP gradients within the SAM. Comparable PIP pattern defects were observed in phospholipase A3ß overexpressor lines, which largely phenocopy katanin mutants at the whole plant level. CONCLUSIONS: Using phospholipid biosensors, we identified a stereotypical PIP accumulation pattern in the SAM that negatively correlates with stem cell maintenance and positively correlates with organ-boundary establishment. While other cues are very likely to contribute to the final PIP pattern, we provide evidence that the patterns of PIP, cortical microtubules and mechanical stress are positively correlated, suggesting that the PIP pattern, and its reproducibility, relies at least in part on the mechanical status of the SAM.

Spatial imaging and speciation of Cu in rice (Oryza sativa L.) roots using synchrotron-based X-ray microfluorescence and X-ray absorption spectroscopy.

Knowledge of elemental localization and speciation in rice (Oryza sativa L.) roots is crucial for elucidating the mechanisms of Cu accumulation so as to facilitate the development of strategies to inhibit Cu accumulation in rice grain grown in contaminated soils. Using synchrotron-based X-ray microfluorescence and X-ray absorption spectroscopy, we investigated the distribution patterns and speciation of Cu in rice roots treated with 50 µM Cu for 7 days. A clear preferential localization of Cu in the meristematic zone was observed in root tips as compared with the elongation zone. Investigation of Cu in the root cross sections revealed that the intensity of Cu in the vascular bundles was more than 10-fold higher than that in the other scanned sites (epidermis and cortex) in rice roots. The dominant chemical form of Cu (79.1%) in rice roots was similar to that in the Cu-cell wall compounds. These results suggest that although Cu can be easily transported into the vascular tissues in rice roots, most of the metal absorbed by plants is retained in the roots owing to its high binding to the cell wall compounds, thus preventing metal translocation to the aerial parts of the plants.

Isolation of Nuclei and Nucleoli.

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Comparative proteomic analysis of the shoot apical meristem in maize between a ZmCCT-associated near-isogenic line and its recurrent parent.

The ZmCCT, one of the most important genes affecting photoperiod response, delays flowering under long-day conditions in maize (Zea mays). In this study we used the isobaric tags for relative and absolute quantification (iTRAQ) technique-based proteomics approach to identify differentially expressed proteins between a near-isogenic line (NIL) and its recurrent parent, contrasting in alleles of ZmCCT. A total of 5,259 distinct proteins were identified. Among them, 386 proteins were differentially expressed between NIL-cml line (ZmCCT-positive) and H4 line (ZmCCT-negative). Functional categorization showed that the differentially proteins were mainly involved in energy production, photosynthesis, signal transduction, and cell organization and biogenesis. Our results showed that during shoot apical meristem (SAM) development cell division proteins, carbohydrate metabolism-related proteins, and flower inhibition-related proteins were more abundant in the ZmCCT-positive line than the ZmCCT-negative line. These results, taken together with morphological observations, showed that the effect of ZmCCT on flowering might be caused by its effect on one or all of these biological processes. Although the exact roles of these putative related proteins remain to be examined, our results obtained using the proteomics approach lead to a better understanding of the photoperiodicity mechanism in maize plants.

Shoot Tip Meristem Cryopreservation of Hypericum Species.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 µM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.