Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions.

In this study, the tolerance of tissue balls (TBs, 100-300 µm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me2SO) at concentrations between 1.0 and 4.5M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in µm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.

Effect of shooting medium and source of material on grapevine (Vitis vinifera L.) shoot tip recovery after cryopreservation.

BACKGROUND: Selecting experimental material at the optimal physiological stage is of paramount importance for successful cryopreservation. OBJECTIVE: The study was to investigate the effect of the physiological state of grapevine buds on their regrowth after liquid nitrogen exposure. METHODS: In a first set of experiments, we tested the regrowth of cryopreserved buds sampled from microcuttings cultured on shooting medium containing benzylaminopurine or zeatin riboside for various durations. In a second set of experiments, we studied the regrowth after liquid nitrogen exposure of buds sampled from different positions on the stem of in vitro plantlets. RESULTS: Regrowth of cryopreserved buds sampled from microcuttings was higher (30%), compared to buds sampled directly from in vitro plantlets (23%), for all culture durations of microcuttings on shooting medium tested (2-6 weeks). Addition of cytokinin in the shooting medium improved regrowth of cryopreserved buds compared to buds sampled from microcuttings cultured on medium devoid of growth regulators; however similar results were obtained with the two cytokinins tested. Buds sampled on nodes 3-4 and 6-7 (from the top of the stem) displayed higher regrowth compared to shoot tips. No significant differences were noted in regrowth after cryopreservation between buds sampled from microcuttings produced from the terminal node, or nodes 3-4 and 6-7. CONCLUSION: The physiological state of the plant material is important for cryopreservation success. Actively growing buds sampled from microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets.

Tolerance of apexes of coral Pocillopora damicornis L. to cryoprotectant solutions.

In this study, we investigated the tolerance of Pocillopora damicornis apexes to treatments with solutions containing penetrating and non-penetrating cryoprotective agents (CPAs). CPAs were employed individually or in binary, tertiary or quaternary solutions. In some experiments apexes were treated successively with two CPA solutions with increasing total concentration. P. damicornis apexes withstood exposure for up to 30 min to solutions containing 0.6-0.8 M sucrose (Suc) or trehalose (Tre). When apexes were treated with binary cryoprotectant solutions containing Suc and ethylene glycol (EG), methanol (Meth), dimethyl sulfoxide (Me(2)SO) or glycerol (Gly), the CPAs employed in combination with Suc could be ranked in the following order of decreasing tolerance: EG>Meth>Me(2)SO>Gly. P. damicornis apexes tolerated exposure to complex CPA solutions containing Suc, Me(2)SO, EG and/or Meth with a total molarity of 2.45 M. In experiments where two successive CPA solutions were employed, apexes withstood treatment with the second, more concentrated solution at 0°C for up to 10 min. These preliminary results pave the way to the development of a cryopreservation protocol for P. damicornis apexes.

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation-dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

Homoleptic zinc(II) complexes with first and second coordination shells of 5-tert-butylpyrazole.

Reaction of hydrated Zn[NO3]2 or Zn[BF4]2 with four or more equivalents of 3{5}-tert-butylpyrazole (L(tBu)) yields [Zn(L(tBu))4]X2 (X- = NO3- or BF4-). The nitrate complex contains C2-symmetric four-coordinate zinc(II) centers with a slightly flattened tetrahedral geometry, and each nitrate anion hydrogen bonds to two pyrazole N-H groups. Similar reactions with Zn[ClO4]2 or ZnCl2 in the presence of 2 equiv of AgPF6 or AgSbF6 yield instead [{Zn(L(tBu))4}(L(tBu))4][ClO4]2 and [{Zn(L(tBu))4}(L(tBu))2]Y2 (Y- = PF6- or SbF6-). Crystals of [{Zn(L(tBu))4}(L(tBu))4][ClO4]2 are composed of discrete [{Zn(L(tBu))4}(L(tBu))4]2+ supramolecules that are formed from N-H...N hydrogen bonding between zinc-bound and uncoordinated pyrazole rings. The [{Zn(L(tBu))4}(L(tBu))4]2+ moieties are linked into planar 4(4) nets by hydrogen bonding to bridging ClO4- anions. The ClO4- ions are almost perfectly encapsulated in near-spherical cavities of approximate dimensions 5.0 x 5.0 x 4.5 A that are formed by two interlocked supramolecular dications. Similarly, [{Zn(L(tBu))4}(L(tBu))2][PF6]2 crystallizes as discrete supramolecules in the crystal with the PF6- anions occupying a shallow bowl-shaped cavity on the surface of the complex that is formed by two zinc-bound and one uncoordinated pyrazole ligands. (1)H NMR and IR studies of [{Zn(L(tBu))4}(L(tBu))4][ClO4]2 in CD2Cl2 imply that the second-sphere L(tBu) ligands dissociate from the [Zn(L(tBu))4]2+ center in this solvent and that free and metal-bound L(tBu) are in rapid chemical exchange.

Molecular and electronic structures of oxo-bis(benzene-1,2-dithiolato)chromate(V) monoanions. A combined experimental and density functional study.

Two oxo-bis(benzene-1,2-dithiolato)chromate(V) complexes, namely, [CrO(L(Bu))2]1- and [CrO(L(Me))2]1-, have been synthesized and studied by UV-vis, EPR, magnetic circular dichroism (MCD), and X-ray absorption spectroscopy and by X-ray crystallography; their electro- and magnetochemistries are reported. H2L(Bu) represents the pro-ligand 3,5-di-tert-butylbenzene-1,2-dithiol, and H2L(Me) is the corresponding 4-methyl-benzene-1,2-dithiol. A structural feature of interest for both the complexes is the folding of the dithiolate ligands about the S-S vector providing Cs symmetry to the complexes. Geometry optimizations using all-electron density functional theory with scalar relativistic corrections at the second-order Douglas-Kroll-Hess (DKH2) and zeroth-order regular approximation (ZORA) levels result in excellent agreement with the experimentally determined structures and electronic and S K-edge X-ray absorption spectra. From DFT calculations, the Cs instead of C2v symmetry for the complexes is attributed to the strong S(3p) --> Cr(3d(x2-y2)) pi-donation in Cs geometry providing additional stability to the complexes.

Copper(II) complexes of thioether-substituted salcyen and salcyan derivatives and their silver(I) adducts.

New syntheses are reported of 5-tert-butyl-2-hydroxy-3-methylsulfanylbenzaldehyde, 5-tert-butyl-2-hydroxy-3-phenylsulfanyl-benzaldehyde, and salcyen (H(2)L(1)-H(2)L(3)) and salcyan (H(2)L(4)-H(2)L(6))-type ligands derived from these aldehydes and from 5-tert-butyl-2-hydroxybenzaldehyde. The complexes [CuL](L(2-)=[L(1)](2-)-[L(6)](2-)) bearing sulfanyl substituents each show two distinct voltammetric ligand-based oxidations under the same conditions, the first of which is chemically reversible. The first oxidation product is much longer lived by coulometry for the salcyen than for the salcyan ligand complexes, despite the latter having a substantially lower oxidation potential. The lifetimes of all the ligand oxidation products in this system are substantially smaller than for similar compounds derived from 3,5-di(tert-butyl)-2-hydroxybenzaldehyde (Dalton Trans., 2004, 2662). Attempted chemical oxidation of the Schiff base compounds using AgBF(4) yielded instead stable silver(i) adducts. A crystal structure of one such compound showed that the Ag atom was coordinated in a slightly bent geometry by the two ligand sulfanyl groups, with two additional long-range Ag...O interactions to the phenoxide donors. EPR spectra showed that some of these silver adducts dimerise in CH(2)Cl(2), probably through basal, apical intermolecular Cu-O...Cu bridging. In contrast the parent copper(ii) complexes are all monomeric in this solvent by EPR.

2-[Bis(pyrazol-1-yl)methyl]-4-tert-butyl-6-(phenylsulfanyl)phenol.

The title compound, C23H24N4OS, contains a highly asymmetric bifurcated intramolecular hydrogen bond between the hydroxy group and two pyrazole N atoms. The compound associates into centrosymmetric dimers in the crystal through two unique C-H...pi interactions, which are in turn linked into a (6,3)-network through an additional intermolecular C-H...N hydrogen bond.