Optimized somatic embryogenesis in Pinus strobus L.

Summary Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.     

Ultrastructure of Sieve Elements in Secondary Phloem of Dalbegia odorifera During Leaf-Bearing and Leaf-Absent Period

Ultrastructures of sieve elements of secondary phloem of 1–2 year old branchlet of tropical deciduous tree Dalbegia odorifera T. Chen growing on Hainan Island were studied under transmission electron microscope and a comparation was made between the sieve elements in leaf-bearing and leaf-absent period. During the leaf-bearing period, there was a tailed spindleshaped P-protein body in each mature sieve element. The main part of the P-protein body con sisted of a disordered fine fiber mass with two crystalline tails. The sieve elements had horizontal end walls with simple sieve plate. The inner layers of the wall near the sieve plate appeared intumescent, protruding into the sieve element lumen. During the leaf-absent period, a functional phloem remained about the same thickness as that during the leaf-bearing period. The sieve elements in the leaf-absent period contained normal protoplasts and the P-protein and the sieve plate pores had the same structures as those during the leaf-bearing period. More starch grains and vesicles were found in sieve elements in the leaf-absent period.     

Plant regeneration from cotyledon-hypocotyl explants of Pinus strobus L.

Rooted adventitious shoots were obtained from cotyledon-hypocotyl explants of aseptically grown seedlings of Pinus strobus L. The explants consisted of the top 2 to 3 mm of the hypocotyl attached to the whorl of cotyledons which had been trimmed to about half of their original length. The explants were cultured on a modified Murashige and Skoog's medium containing 0.2 mg/L NAA and 2 mg/L BA for 2–3 weeks and then on the medium of Litvay et al. without any growth hormones for 8 to 10 weeks to obtain shoot induction and shoot growth. Root induction and root growth were achieved by culturing the shoots on half strength Gresshoff and Doy's medium supplemented with 0.5 mg/L NAA for 2 weeks and then transferring them to the same medium without any growth hormones. The rooted shoots could be grown outside the culture tube in a peat/perlite/vermiculite mixture.Abbreviations BA 6-Benzyl-aminopurine - GD Gresshoff and Doy's medium - LM conifer cell culture medium of Litvay et al. - MS modified Murashige and Skoog's medium - NAA -naphthaleneacetic acid     

Anatomical Studies on Secondary Phloem of Pinus bungeana

This paper deals with structures of the secondary phloem of Pinus bungeana Zucc. The sieve cells lived for only one growing season. Most of them formed in spring and summer and then died in the end of winter. However, some of them formed in autumn and died eventually in the end of next spring. Two types of albuminous cells: type A and type B were seen in radial plates and rays, which possessed the following common characteristics, there were unilateral sieve area connections between these and the sieve cells. These cells had larger nuclei, denser cytoplasm with abundant mitochondria and rich RNA-protein. Their death closely followed that of the sieve cells. Type A albuminous cells differed from type B in that the former collapsed before the contents of sieve cells accompanied with it dissppeared. But, type B did not collapse until complete disappearance of the contents of sieve cells. The cytological characteristics of albuminous cells, the relationship between radial plates and rays, and possible physiological significance are also disscussed.     

Junction Complexes between Sieve Tubes in the Secondary Phloem of Myrtaceae

Junction complexes of unusual structure form between neighbouringsieve tubes in the secondary phloem of Eucalyptus species. Thick-walledribs support thin-walled ‘sieve areas’. In longitudinalsections the structures have a ‘concertina’- likeappearance. They are relatively large, up to 0.2 mm in length.Electron micrographs confirmed that the structures consistedof thin-walled areas perforated with pores, supported by muchthicker ribs. The structures provide a vast surface area fortransfer of metabolites between sieve tubes compared with thatof lateral wall sieve areas of other plants. Hydrolysis of parenchymacell walls occurs during the development of the junction complexes.The structures are only found when sieve tubes are in closeproximity and it is the redifferentiation and partitioning ofintervening parenchyma cells which result in junction complexformation. A survey for the presence of the structures in thephloem of other genera in the family Myrtaceae was made andthey were found in Tristania and Angophora but were not observedin Acmena and Metrosideros. Eucalyptus, sieve tubes, lateral walls, ultrastructure     

Structural Changes in the Vascular Cambium of Pinus strobus L. during an Annual Cycle

The changes in the ultrastructure of cambial cells of easternwhite pine (Pinus strobus L.) during an annual cycle are observedand recorded as are relationships of cambial cells during dormancyand at resumption of cambial activity. Cambial activity wasresumed late in March or early in April, when a few cells dividedpericlinally. Cambial activity reached a maximum during thelatter part of May with 15 to 20 undifferentiated cells present.In July it declined markedly, and the number of undifferentiatedcells equalled that of the dormant period. The xylem and phloemtissue cells produced late in the annual cycle overwinteredat varying developmental stages. In October cambial cells structurallyresembled dormant cells. The number of dormant cells in easternwhite pine cambium varied from 6 to 10. Active cells were characterizedby a large central vacuole, by an abundance of all cell organelles,and by thin cell walls. Dormant cells were characterized bynumerous small vacuoles, by structurally and quantitativelymodified cell organelles, and by relatively thick cell walls.     

Studies on Cytochemistry of Albuminous Ceils in Secondary Phloem of Stem in Pinus bungeana Zucc.

Mature albuminous cells which are connected with differentiated and functional sieve cells show rather high activity of acid phosphatase at pH 5.0. No increased activity could be detected in ordinary ray cells, cambium cells and young albuminous cells contiguous with developing sieve cells. Collapsed albuminous cells show little or none of the activity. With ATP as substrate, enzyme localization is the same as with β-glycerolphosphate. When adenosine triphosphatase activity is visualized at pH 7.2 with ATP as substrate, the highest activity still localized in mature albuminous cells. With the development of young albuminous cells, the enzyme activity is being gradually exhibited, but disappearing when the maturecells begin collapsing. On the other hand, in ordinary ray cells the enzyme activity is ahnost not detectable.     

The Cytological Studies on Albuminous Cells in Secondary Phloem of Pinus bungeana

This paper emphatically deals with the ultrastructure of albuminous cells in different stages of development in the secondary phloem of Pinus bungeana. The secondary phloem of Pinus bungeana is composed of sieve cells, axial parenchyma cells, radial plates and rays. Among the constituents, most of upright ray cells and radial plate parenchyma cells are albuminous cells. Although the shape and distribution of this kind of albuminous cells may be different, they possess the following common cytological characteristics. These cells have dense cytoplasm with abundant mitochondria, ribosomes, rough endoplasmic reticula and a large nucleus, the nuclei of some albuminous cells are lobed in shape which increases the outer surface of the nuclei. Usually the albuminous cell contains some starch granules, the quantity of the starch granules in albuminous cells is less than the other parenchyma cells of the secondary phloem. All these cytological characteristics suggest that albuminous cells are active physiologically. The distinguishing characteristics of albuminous cells from other parenchyma ceils are that the albuminous cells are associated with sieve cells through unilateral sieve area and they died together with the sieve cells.     

Somatic stability of microsatellite loci in Eastern white pine,Pinus strobus L

Variation at nuclear- and chloroplast-encoded microsatellite loci was studied among and within clonally propagated individuals of Eastern white pine. Total DNA was extracted and assayed from gamete-bearing tissue (megagametophytes) located on six different branch positions on each of 12 individual genets. No within-individual variation was observed among 12 loci studied. Estimates of numbers of mitotic cell divisions required to produce the tissue used as the source of genomic DNA were obtained by combining tree growth and anatomical data. This allowed for the calculation of upper bound estimates of numbers of mutations per locus per somatic cell division. The estimated somatic mutation rate was found to be substantially lower than those published for genomic microsatellite mutation rates in other plant species.     

Physiology of Pinus strobus L. Seedlings Grown under High or Low Soil Moisture Conditions

White pine seedlings (Pinus strobus L.) were grown under highor low soil-moisture levels. The increase in the length andin the fresh weight of seedlings, respiration, photosynthesis,transpiration, translocation of photosynthate from shoots toroots, and bio-electric potentials between the tip and the baseof a stem were measured throughout the growing season from Aprilto October 1964. At both moisture levels the lowest translocation of recent photosynthatefrom shoots to roots occurred during early summer, or at thetime when the rate of root growth was the lowest and that ofthe shoot the highest. The specific activity of ¹⁴CO₂, respiredby the shoots of such plants remained high throughout the 8h of the experiment, indicating a continuous utilization ofrecent photosynthate as a respiratory substrate. On the otherhand, early and late in the growing season, when translocationof recent photosynthate from shoots to roots and the rate ofroot growth were high, the specific activity of ¹⁴CO₂, respiredby the shoots rapidly decreased during the 8 h of the experiment,indicating a drop in the utilization of recent photosynthateas respiratory substrate. The highest positive values for thepotential difference between the top and the base of the mainshoot also occurred in early summer or during the period ofhigh rates of transpiration per needle stomatal surface area.     

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements

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Sieve Elements of Minor Veins in the Leaves of Beta vulgaris L.

End walls of sieve elements of minor veins of the leaves ofBeta vulgaris L. do not contain the multi-perforate sieve plateswhich typically occur on the end walls of sieve-tube membersof major veins. Instead, both end and side walls of the sieveelements of minor veins contain scattered pores which may occursingly or in small numbers. These pores are similar to thosewhich are grouped in sieve plates of major veins in size, possessionof callose and plugs of filaments. In addition to these pores,there are tubular connections 0.1 µ in diameter throughcharacteristically thickened parts of the cell wall betweensieve cells and companion cells. Sieve elements of minor veinsdiffer from those of major veins in structure as well as infunction.     

Parenchyma cells of secondary phloem in Pinus strobus

Summary Parenchyma cells of the secondary phloem in Pinus strobus have all the cellular organelles common in other plant cells. They have mitochondria, endoplasmic reticulum, ribosomes, dictyosomes, and plastids. Parenchyma cells are very conspicuous because of their organic inclusions, starch and lipids. Plasmodesmata in transverse and tangential walls of axial parenchyma cells and in end walls of ray parenchyma cells are regularly distributed and of uniform size, about 500 Å in diameter. In radial walls of axial parenchyma cells and horizontal walls of ray parenchyma cells plasmodesmata are located in primary pit-fields; there they are of variable size and often divided into several branches. The branches are confluent into a median nodule. Perforation of the transverse wall between two axial parenchyma cells and the resultant union of the cellular material of the two connected cells is reported.This research has been supported by NSF Grant GB 3193.     

Linkage mapping and genome length in eastern white pine (Pinus strobus L.)

 Haploid linkage analysis of eastern white pine, Pinus strobus L., was carried out using mainly RAPD markers and microsatellite, or simple-sequence-repeat, markers. Ninety one loci mapped to 12 linkage groups of three or more markers. The resulting framework genome map, the first for a soft pine species, contained 69 markers. The map covered 58% of the estimated genome length of 2071 cM(K), with a 95% confidence interval of 1828–2242 cM(K). A systematic comparison of linkage data from eastern white pine, longleaf pine (P. palustris Mill.) and maritime pine (P. pinaster Ait.), gave genome-length estimates for all three species very close to either 2000 cM(K) or 2600 cM(H), depending on whether the Kosambi(K) or Haldane(H) map functions, respectively, were employed. Differences among previous pine genome-length estimates were attributed to the divergent criteria used in the methods of estimation, and indicate the need for the adoption of uniform criteria when performing genome-length estimates. Current data suggest that members of the two pine subgenera, which diverged during the late Mesozoic era, have highly conserved rates of recombination. Received: 5 January 1997/Accepted: 24 January 1997     

Restricted lateral gas movement in Pinus strobus branches

Argon gas was incorporated into the sap flowing through xylem of cut branches, or added to the air in a sealed cuvette surrounding a needle-free portion of the branch to investigate lateral movement of gases in Pinus strobus. Microdialysis was used to sample air in the xylem and evacuated vials were used to collect samples of air from cuvettes attached to branches. Argon concentrations of samples of air were measured by GC-MS. When argon was added through the sap, concentrations of argon in the xylem and in air of chambers enclosing needle stumps was greater than that of controls, but argon concentrations of air in cuvettes enclosing a needle-free portion of the branch were not greater than controls. When argon was added to cuvettes enclosing a needle-free portion of the branch, the argon did not enter the xylem and it was not emitted by needle stumps.     

Eine Triebspitzenerkrankung der Weymouthskiefer (Pinus strobus L.), verursacht durch Scleroderris lagerhergii Gremmen

Inhalt: Einleitung. – 1. Krankheitsbild.—2. Der Krankheitserreger und seine Begleit-pilze.—3. Regenerationserscheinungen.—Zusammenfassung.—Summary.—Literatur- verzeichnis.     

Antifungal metabolites from fungal endophytes of Pinus strobus

The extracts of five foliar fungal endophytes isolated from Pinus strobus (eastern white pine) that showed antifungal activity in disc diffusion assays were selected for further study. From these strains, the aliphatic polyketide compound 1 and three related sesquiterpenes 2-4 were isolated and characterized. Compound 2 is reported for the first time as a natural product and the E/Z conformational isomers 3 and 4 were hitherto unknown. Additionally, the three known macrolides; pyrenophorol (5), dihydropyrenophorin (6), and pyrenophorin (7) were isolated and identified. Their structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data where available. The isolated compounds 1, 2, and 5 were antifungal against both the rust Microbotryum violaceum and Saccharomyces cerevisae.