Proteomic Analysis of Immature Fraxinus mandshurica Cotyledon Tissues during Somatic Embryogenesis: Effects of Explant Browning on Somatic Embryogenesis.

Manchurian ash (Fraxinus mandshurica Rupr.) is a valuable hardwood species in Northeast China. In cultures of F. mandshurica, somatic embryos were produced mainly on browned explants. Therefore, we studied the mechanism of explant browning and its relationship with somatic embryogenesis (SE). We used explants derived from F. mandshurica immature zygotic embryo cotyledons as materials. Proteins were extracted from browned embryogenic explants, browned non-embryogenic explants, and non-brown explants, and then separated by 2-dimensional electrophoresis. Differentially and specifically expressed proteins were analyzed by mass spectrometry to identify proteins involved in the browning of explants and SE. Some stress response and defense proteins such as chitinases, peroxidases, aspartic proteinases, and an osmotin-like protein played important roles during SE of F. mandshurica. Our results indicated that explant browning might not be caused by the accumulation and oxidation of polyphenols only, but also by some stress-related processes, which were involved in programmed cell death (PCD), and then induced SE.

Establishment of the technology of cambial meristematic cells (CMCs) culture from shoots and high expression of FmPHV (PHAVOLUTA) functions in identification and differentiation of CMCs and promoting the shoot regeneration by hypocotyl in Fraxinus mandshurica.

In Fraxinus mandshurica, we successfully isolated and identified the loose, uniform and creamy-white cambial meristematic cells (CMCs) from newborn shoots, and established a culture technology for induction, proliferation and differentiation of CMCs. In this technology, higher induction rate (83.0%, 0.57-fold to the control) was obtained by an effective pretreatment after 28-day induction culture, CMCs can be better proliferation cultured than common calli and maintain same growth states after several times of cultures and 3.3% CMCs primarily realized differentiation. Gene expressions in the differentiated CMCs revealed that, low expression of FmWOX5 (regulator in establishment of competence for shoot formation, 0.09-fold to the control) and high expressions of FmWOX4 (cambium stem cell regulator, 16.7-fold to the control) and 9 key genes in shoot regeneration (2.4-fold-72.1-fold to the control) function in CMCs differentiation. In addition to the function of high expression of PHAVOLUTA (FmPHV) in CMCs differentiation (5.4-fold-157.3-fold to undifferentiated CMCs), functions of high expression of FmPHV in CMCs identification (22.4-fold to common calli) and generating more shoots (2.3-fold to the control) by significantly changing expressions of key regulators in HD-Zip Class III related shoot regeneration networks in positive transgenic plants through the hypocotyl transforming system in F. mandshurica, were further revealed. These works were of profound significance in providing the culture technology of CMCs from newborn shoots in F. mandshurica for the first time and revealing the positive functions of FmPHV in CMCs identification and differentiation in F. mandshurica and promoting the shoot regeneration by hypocotyls.

Ephemeral root modules in Fraxinus mandshurica.

Historically, ephemeral roots have been equated with 'fine roots' (i.e. all roots of less than an arbitrary diameter, such as 2 mm), but evidence shows that 'fine roots' in woody species are complex branching systems with both rapid-cycling and slow-cycling components. A precise definition of ephemeral roots is therefore needed. Using a branch-order classification, a rhizotron method and sequential sampling of a root cohort, we tested the hypothesis that ephemeral root modules exist within the branching Fraxinus mandshurica (Manchurian ash) root system as distal nonwoody lateral branches, which show anatomical, nutritional and physiological patterns distinct from their woody mother roots. Our results showed that in F. mandshurica, distal nonwoody root branch orders die rapidly as intact lateral branches (or modules). These nonwoody branch orders exhibited highly synchronous changes in tissue nitrogen concentrations and respiration, dominated root turnover, nutrient flux and root respiration, and never underwent secondary development. The ephemeral root modules proposed here may provide a functional basis for differentiating and sampling short-lived absorptive roots in woody plants, and represent a conceptual leap over the traditional coarse-fine root dichotomies based on arbitrary size classes.

Cryopreservation of Fraxinus excelsior L. embryogenic callus by one-step freezing and slow cooling techniques.

An efficient cryopreservation protocol for the safe storage of Fraxinus excelsior L. embryogenic callus cultures is reported. The cryopreservation methods tested included one-step freezing by means of (i) encapsulation-vitrification; or (ii) encapsulation-dehydration; and (iii) slow cooling using the Nalgene Freezing container, Mr Frosty, which produces a temperature decrease of about 1 masculineC min-1 when placed in a -70 degree C freezer. None of the one-step freezing techniques was effective for cryopreservation of encapsulated callus masses, irrespective of the cryoprotective treatment applied, i.e., treatment with the PVS2 vitrification solution or physical dehydration with silica gel before direct immersion in liquid nitrogen. On the contrary, when a slow cooling protocol was applied to embryogenic callus which had been pretreated for 60 min with a 210 g per liter (0.61 M) sucrose-7.5 percent DMSO cryoprotective solution, up to about 1.3 g per Petri dish of proliferating callus was observed 42 days after recovery from liquid nitrogen, and cultures were able to produce somatic embryos 8 weeks after transfer to semi-solid medium. TTC staining of callus cultures provided a fast evaluation of culture viability.

Ethylene evolution changes in tilted Fraxinus mandshurica Rupr. var. japonica maxim. seedlings in relation to tension wood formation.

The effects of ethylene on tension wood formation were studied in 3-year-old Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings in two separate experiments. In experiment 1, ethylene evolution of buds and stems was measured using gas chromatography after 0, 2, 4, 7, 14, and 21 d of treatment; in experiment 2, both aminoethoxyvinylglycine (AVG) and AgNO3 were applied to the horizontally-placed stems, and the cell numbers on sites of applications were measured after 40 d. Ethylene evolution from buds was found to be much greater in tilted seedlings than in upright ones. The cell numbers of wood fibers in shoots and 1-year-old stems were reduced in treatments with 12.5 x 10(-7)micromol/L AVG, 12.5 x 10(-8)micromol/L AVG, and 11.8 x 10(-8)micromol/L AgNO3; whereas the horizontal and vertical diameters were reduced by treatment of 12.5 x 10(-7)micromol/L AVG. Ethylene evolutions of shoots and 1-year-old stems were inhibited greatly in comparison with the control by applying 12.5 x 10(-7)micromol/L AVG. The formation of a gelatinous layer of wood fibers was affected by neither AVG nor AgNO3 application. These results suggest that ethylene regulates the quantity of wood production, but does not affect G-layer formation in F. mandshurica Rupr. var. japonica Maxim. seedlings.

In vitro propagation of ash (Fraxinus excelsior L.) by somatic embryogenesis.

Induction of somatic embryogenesis is described in common ash (Fraxinus excelsior L.). Embryogenic tissues are obtained from immature zygotic embryos and cultured on a modified Murashige and Skoog (MS) medium containing 8.8 µM 2,4-dichlorophenoxyacetic acid and 4.4 µM benzyl-adenine. Embryogenic tissue is subcultured and multiplied on medium supplemented with reduced concentration of plant growth hormones. Somatic embryos develop and mature by transfer to hormone-free medium and subsequent culture on medium containing low amount of benzyladenine. Somatic embryo germination and conversion are enhanced by cold storage at 4°C and successive transfer onto Woody Plant Medium (WPM). Fully developed plantlets are then transferred to pots and acclimatized in the greenhouse equipped with a mist system.

Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age?

Sapwood respiration often declines towards the sapwood/heartwood boundary, but it is not known if parenchyma metabolic activity declines with cell age. We measured sapwood respiration in five temperate species (sapwood age range of 5-64 years) and expressed respiration on a live cell basis by quantifying living parenchyma. We found no effect of parenchyma age on respiration in two conifers (Pinus strobus, Tsuga canadensis), both of which had significant amounts of dead parenchyma in the sapwood. In angiosperms (Acer rubrum, Fraxinus americana, Quercus rubra), both bulk tissue and live cell respiration were reduced by about one-half in the oldest relative to the youngest sapwood, and all sapwood parenchyma remained alive. Conifers and angiosperms had similar bulk tissue respiration despite a smaller proportion of parenchyma in conifers (5% versus 15-25% in angiosperms), such that conifer parenchyma respired at rates about three times those of angiosperms. The fact that 5-year-old parenchyma cells respired at the same rate as 25-year-old cells in conifers suggests that there is no inherent or intrinsic decline in respiration as a result of cellular ageing. In contrast, it is not known whether differences observed in cellular respiration rates of angiosperms are a function of age per se, or whether active regulation of metabolic rate or positional effects (e.g. proximity to resources and/or hormones) could be the cause of reduced respiration in older sapwood.