Wesley P. Hackett

Differential dihydroflavonol reductase transcription and anthocyanin pigmentation in the juvenile and mature phases of ivy (Hedera helix L.)

Juvenile-phase English ivy (Hedera helix L.) accumulates anthocyanin pigment in the hypodermis of stems and petioles, whereas mature-phase ivy does not. Lamina tissue of both phases of ivy accumulate flavonols, another class of the flavonoids, in response to sucrose and light treatment in vitro. However, juvenile- but not mature-phase lamina tissue accumulates anthocyanin in response to sucrose and light. The lack of anthocyanin accumulation in mature phase tissue is due to a lack of dihydroflavonol reductase (DFR) activity, which catalyzes a reaction late in the anthocyanin biosynthetic pathway. The objective of this work was to determine the level of regulation of gene expression that limits DFR activity in mature phase tissue. There was an induction of DFR transcription and accumulation of DFR mRNA in juvenile-phase lamina tissue treated with sucrose and light. In contrast, transcription and mRNA accumulation of DFR was not detectable in treated mature-phase lamina tissue. The induction of DFR transcription in juvenile tissue required the combination of sucrose and light. There was an induction of transcription of chalcone synthase, which catalyzes the first committed reaction of flavonoid biosynthesis, in both juvenile- and maturephase lamina tissue, indicating that mature-phase tissue is responsive to sucrose and light treatment.

Maturation and rejuvenation in woody species

In the development of all woody plants from seed there is a so-called juvenile phase lasting up to 30–40 years in centain forest trees, during which flowering does not occur and cannot be induced by the normal flower-initiating treatment or conditions. In time, however, the ability to flower is achieved and maintained under natural conditions; at this stage, the tree is usually considered to have attained the adult or mature condition. The length of the juvenile period can be influenced by environmental and genetic factors [20]. Maturation is distinct from aging as used by Wareing [47] to describe changes such as reduced growth rate and type of branching due to increased size and complexity of the tree, which disappear when a scion is grafted onto a young rootstock or when a stem cutting is rooted.

Gradients of adventitious bud formation on excised epicotyl and root sections of citrus

Abstract Epicotyl and root sections from ‘Valencia’ orange ( Citrus sinensis (L.) Osb.) seedlings were cultured in vitro on a Murashige-Tucker medium containing benzylaminopurine (BAP) and varying naphthaleneacetic acid (NAA) concentrations. A gradient of bud-forming potential was observed in both epicotyls and roots. NAA influenced the expression of the gradient with a concentration of 0.02 mg/l enhancing the expression and a concentration of 2 mg/l diminishing it. The presence or absence of the shoot and/or root apices did not influence the bud-forming capacity of cultured sections of epicotyls or roots. However, the presence of cotyledons did increase the bud-forming capacity of epicotyl sections 1 cm from the cotyledonary node. These data support the hypothesis that the bud-forming gradient in ‘Valencia’ epicotyls may be due to the presence of promoting substance(s) emanating from the cotyledons. However, the data do not support this hypothesis for explaining the bud-forming gradient in roots.

Influence of regeneration method and tissue source on the frequency of somatic variation in Populus to infection by Septoria musiva

Septoria leaf spot and canker are serious diseases of many hybrid poplar clones in plantations established for biomass production. Developing resistant clones through breeding is the best long-term strategy to minimize tree damage caused by this disease. Tissue culture and somaclonal selection techniques may reduce the time needed to develop disease resistance in poplars. We used a single source clone of a hybrid Populus to determine the influences of explant tissue source and regeneration method on the frequency of somatic variation in disease resistance. Plants were regenerated adventitiously via shoots and somatic embryos from callus derived from several tissue sources and from axillary buds. The resulting plants expressed somatic variation in disease resistance in different frequencies, except for the plants regenerated from hardwood cuttings. Several regenerated plants from various tissue sources exhibited variant morphological phenotypes, providing further evidence of the instability of this clone when cultured in vitro. Although mutagenic effects of the culture regimes alone cannot be ruled out, results of this study suggest that the explant source and culture method influenced how frequently variant plants were recovered.

The formation of adventitious buds in vitro on micro-cross sections of hybrid Populus leaf midveins

Abstract Micro-cross sections (MCS) of midveins from hybrid Populus leaves were1used to study adventitious bud formation in vitro. MCS of 100 μm, 200 μm, 300 μm, 400 μm, and 500 μm in length were made with a Lancer Vibratome and cultured in Woody Plant Medium (WPM). In the presence of naphthaleneacetic acid (NAA), 400 and 500-μm sections had equal bud forming capacity and produced as many adventitious buds (average four shoots/MCS) as a 1-cm explant under the same culture conditions. This means that MCS of midveins are approximately 25 times more efficient in shoot regeneration than a 1-cm explant. The conditions used were suboptimal for the 200-μm and 100-μm sections. Shoot numbers from the 200-μm MCS were considerably reduced and the 100-μm MCS did not form buds at all. The size of lamina remnants was critical with 1–2 mm on each side of the midvein being optimal for bud formation. The MCS (300 and 400 μm) were not only an effective system for production of adventitious shoots, but also allowed direct microscopic observations of early morphogenetic events in vitro. These direct observations indicate that the callus and buds are initiated from a group of small chlorophyllous cells located in areas of the midvein that are adjacent to the mesophyll cells.

Differential Gene Expression in Response to Auxin Treatment in the Wild Type and rac, an Adventitious Rooting-Incompetent Mutant of Tobacco

Histological analyses of auxin-treated cuttings from the wild type and the rac mutant of tobacco (Nicotiana tabacum cv Xanthii) previously revealed that some rac phloem parenchyma or inner cortical parenchyma cells form callus in response to exogenous auxin treatment but these cells never undergo the organized divisions associated with adventitious root initiation in the wild type. Here we report the effect of the rac mutation on the temporal and spatial expression patterns of three genes previously shown to be associated with adventitious root meristems, HRGPnt3, iaa4/5, and gh3. Using histochemical staining analyses of HRGPnt3-GUS transformant cuttings, we determined that the rac mutation blocks auxin activation of the HRGPnt3 promoter. Thus, activation of the HRGPnt3 promoter occurs specifically during adventitious root initiation in tobacco cuttings. Histochemical staining analyses of iaa4/5-GUS and gh3-GUS transformant cuttings revealed that the rac mutation does not repress the auxin activation of the iaa4/5 and gh3 promoters. Based on our histochemical staining analyses, we conclude that differential gene expression occurs in response to auxin treatment during adventitious root initiation in the wild type compared with callus formation in rac cuttings. We also determined that HRGPnt3 mRNA accumulation occurs in response to components of our root-induction protocol other than auxin, indicating that HRGPnt3 expression is regulated both developmentally and environmentally.

Gibberellin-induced reorganization of spatial relationships of emerging leaf primordia at the shoot apical meristem in Hedera helix L.

The transition from spiral to distichous leaf arrangement during gibberellic-acid (GA3)-induced rejuvenation in Hedera was studied in detail by scanning electron microscopy of the shoot apical meristem. The transition, which involves the initiation of about 14 new leaf primordia, is accomplished by progressive increments in the divergence angle between the leaf primordia from an initial average value of 138.9 ° until it approaches 180 °. This process is preceded, as well as accompanied, by an increased radial displacement of young leaf primordia away from the apical meristem. Although the width of the leaf primordia also increases, this is unlikely to be a causal factor since it occurs only late in the transition. The size of the primordium-free area of the apical meristem is also unlikely to be involved. Quantitative analysis shows that the divergence angle of consecutive leaf primordia commonly fluctuates between relatively large and small values. Thus the transitional stages form a spirodistichous arrangement in which the divergence angle within each pair of leaves is large relative to that between leaf pairs. The stimulation of the radial displacement of the leaf primordia and the associated phyllotactic transition may involve GA3-induced modification in the spatial organization of cortical microtubules in the apical meristem and related changes in directional cell expansion.

Plantlet multiplication from white pine (Pinus strobus L.) embryos in vitro: bud induction and rooting

White pine embryos were grown on 4 different media with 6 different benzyladenine (BA) concentrations. Maximum adventitious shoot initiation and growth were obtained on a modified Lepoivre medium with 20 μM BA. Modified Schenk and Hildebrandt, Murashige and Skoog, and Gresshoff and Doy media were also tested. Shoot elongation was achieved on half-strength basal medium lacking growth regulators. Three rooting experiments involving indole-3-butyric acid (IBA), sucrose concentration, shoot orientation, IBA pulse length, and light or dark were carried out. Treatment of shoots in an upright position with 50 μM IBA for eight days followed by culture in a medium with 3% sucrose in the light produced the most rooting (50% at 3 months). Rooted shoots were transplanted to the greenhouse for further growth.

Cellular, Biochemical and Molecular Characteristics Related To Maturation and Rejuvenation In Woody Species

In the development of all woody plants from seed there is a so-called juvenile phase lasting up to 30–40 years in certain forest trees, during which flowering does not occur and cannot be induced by the normal flower-initiating treatment or conditions. In time, however, the ability to flower is achieved and maintained under natural conditions; at this stage, the tree is usually considered to have attained the adult or mature condition. The transition from the juvenile to the mature phase has been referred to as phase change by Brink (1962), ontogenetic aging by Fortanier and Jonkers (1976), or meristem aging (cyclophysis) by Seeliger (1924) and Oleson (1978). Associated with this transition are progressive changes in morphological, developmental, and physiological characteristics. Changes in such characteristics during development vary from species to species. Most change gradually during the period preceding the mature phase, resulting in transitional forms. Usually no distinct change in any one characteristic is apparent at the time the ability to flower is attained.

Differential Competence for Adventitious Root Formation in Histologically Similar Cell Types

Shoot tissue formed during the protracted juvenile phase of woody perennials lacks the ability to flower. With the transition to the mature phase, shoot apices or axillary meristems of newly formed tissue gain the ability to flower and this ability is maintained in subsequently formed shoot tissue (Zimmerman, 1973, 1976). In addition to this phase-dependent difference in ability to form flowers, other persistent phenotypic differences exist between shoot tissue of the basal (i.e., juvenile) and apical (i.e., mature) portions of a plant (Hackett, 1985; Poethig, 1990). Due to the protracted nature of both the juvenile and mature phases of woody plants, phase-dependent phenotypic characters are stably expressed through a large number of cell divisions over years of growth within a phase. It is presumed that phase-dependent characters do not result from a genetic change, but result from an epigenetic difference in the capacity to express genes that permit or prevent expression of a phenotype.