Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N⁶‐benzyladenine (BA), meta‐topolin (mT) and trans‐zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole‐3‐acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O‐glucosides in non‐treated P. radiata explants from the field and remark the importance of O‐glucosides as storage forms.     

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACT

Pinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.     

Hormonal changes throughout maturation and ageing in Pinus pinea.

Phytohormones, which are responsible for certain age-related changes in plants, play a major role throughout maturation and ageing. Previous results dealing with this topic allowed us to describe an ageing and vigour index in Pinus radiata based on a ratio between different forms of cytokinins (Cks). The aim of the present study was to extend the studies on the changes in the hormonal status throughout maturation and ageing to Stone pine (Pinus pinea L.). With this aim in mind, a number of Cks were analysed in addition to indole-3-acetic acid (IAA) and abscisic acid (ABA) in terminal buds, axillary buds and in the apical portion of needles collected from trees at different stages of development. The results showed an increasing pattern in the levels of various Cks similar to that found in previous studies on P. radiata. Although the maintenance of the same ratio as an ageing and vigour index was not ratified, these results seem to point to Cks as major hormones throughout maturation and related processes in conifers. The distribution of hormones between the two parts of the needle is also discussed.     

Changes in abscisic acid and indole-3-acetic acid in axillary buds of Elytrigia repens released from apical dominance

Growth of axillary buds on the rhizomes of Elytrigia repens (L) Nevski is strongly dominated by the rhizome apex, by mechanisms which may involve endogenous hormones. We determined the distribution of indole-3-acetic acid (IAA) and abscisic acid (ABA) in rhizomes and measured (by gas-chromatography-mass spectrometry) their content in axillary buds after rhizomes were decapitated. The same measurements were also made in buds induced to sprout by removing their subtending scale leaves. The ABA content tended to be higher in the apical bud and in the axillary buds than in the adjacent internodes, and tended to decline basipetally in the internodes and scale leaves. IAA was similary distributed, except that there was less difference between the buds and other rhizome parts. After rhizomes were decapitated, the ABA content of the first axillary bud declined to 20% of that of control values within 24 h, while the IAA content showed no marked tendency to change. The ABA content also declined within 12 h in the first axillary bud after rhizomes were denuded, while the content of IAA tended to increase after 6 h. These changes occurred before the length of the first axillary bud increased 24–48 h after rhizomes were decapitated or denuded. We conclude that the release of axillary buds from apical dominance in E. repens does not require IAA content to be reduced, but is associated with reduced ABA content.     

Could plant hormones be the basis of maturation indices in Pinus radiata?

Maturation and phase change in woody plants are developmental processes at present poorly understood, especially from a physiological point of view. Maturation indices, such as phytohormones, could be used to understand more about the events that accompany the increase in the developmental state and to choose optimal conditions in order to improve forest programs and make them more profitable. In order to determine this putative index, the contents in abscisic acid-like substances (ABA-like), indole-3-acetic acid (IAA) and several cytokinins (Cks) were analysed in terminal and axillary buds of Pinus radiata trees with different developmental states to compare their phytohormonal status. An increase in the content of some zeatin-type (Z-type) Cks, and a decrease in isopentenyladenine-type (iP-type) Cks levels throughout maturation were shown both in terminal and axillary buds. This fact allowed us to consider the ratio iP-type/Z-type Cks, which decreased in parallel with the increasing developmental state of the tree, as a maturation index. According to the results, ABA-like compounds and IAA contents could not be considered as a maturation index in Pinus radiata, even though similar patterns were in fact found between the two kinds of buds.     

Effect of exogenous ABA on embryo maturation and quantification of endogenous levels of ABA and IAA in <Emphasis Type="Italic">Quercus suber</Emphasis> somatic embryos

Knowledge of the relationship between indole-3-acetic acid (IAA) and abscisic acid (ABA) is relevant to control the development and the maturation of cork oak (Quercus suber L.) somatic embryos. The addition of 1 M ABA to the culture medium significantly promoted somatic embryo maturation and increased both fresh and dry matter without affecting the relative water content. This effect was parallel to the pattern of variation observed in the endogenous ABA level, which increased from the immature to the mature stage. Endogenous ABA content during the occurrence of secondary embryogenesis was similar to that of the immature stage, showing that embryos with lower ABA levels produced secondary embryos. In contrast, IAA showed the highest concentration during early embryo development and decreased afterwards. Only in somatic embryos subjected to 1-week desiccation followed by stratification at 4 °C for 2 weeks, was a moderate increment of endogenous IAA content observed. IAA and ABA showed opposite levels during the development and maturation of cork oak somatic embryos and characterised specific stages of the embryonic development.     

罗汉果花芽分化过程中形态及其激素水平变化特征

采用石蜡切片和酶联免疫法(ELISA)对罗汉果雄性、雌性、两性花芽分化过程的形态和激素水平变化进行观测,为罗汉果开花调控和品种选育提供科学依据。结果表明:(1)罗汉果雄性、雌性、两性花的花芽分化过程均可分为花芽未分化期、花芽分化初期、花序分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期7个阶段。雄蕊原基分化期前,3种花芽分化过程无明显差异,各时期形态特征均依次为:茎端呈圆锥状(花芽未分化期)→茎端经半球形变成扁平状(花芽分化初期)→距茎端5~7节位处分化出穗状花序(花序分化期)→小花原基周围形成5个萼片原基(萼片原基分化期)→萼片原基内侧形成5个花瓣原基(花瓣原基分化期)。雄蕊和雌蕊原基分化期,3种花芽分化过程存在明显差异,雄蕊原基内侧出现雌蕊原基后,雄花芽雄蕊原基继续发育成雄蕊,雌蕊原基停滞生长,退为一个小突起;雌花芽雌蕊原基继续发育成雌蕊,雄蕊原基生长缓慢,退化为小花丝;两性花芽雌蕊和雄蕊原基均继续发育,形成外观正常的雌蕊和雄蕊。(2)内源激素脱落酸(ABA)、赤霉素(GAs)和玉米素核苷(ZR)含量在3种花芽分化过程中变化规律相似,即ABA含量在花芽生理分化期降低,花芽形态分化期升高,而GAs和ZR含量则基本保持不变;吲哚乙酸(IAA)含量在3种花芽分化过程中变化存在明显差异,雌花芽IAA含量在花芽生理分化期升高,花芽形态分化期逐渐降低,而雄性和两性花芽的IAA含量则基本保持不变。ABA/GAs、ABA/IAA、ZR/IAA和ZR/GAs激素含量比值在3种花芽分化过程中变化规律相似,ABA/GAs在花芽生理分化期降低,花芽形态分化期升高,而BA/IAA、ZR/IAA和ZR/GAs则基本保持不变。研究认为,罗汉果花芽分化过程经历一个"两性期",高ABA含量和ABA/GAs比值有利于罗汉果花芽分化,IAA可能对罗汉果花性分化具有重要作用。     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

QTL associations for density and diameter in<Emphasis Type="Italic"> Pinus radiata</Emphasis> and the potential for marker-aided selection

A large full-sib family of radiata pine (Pinus radiata Donn. ex D. Don) was used for quantitative trait locus (QTL) detection and independent verification. QTL detection experiments were carried out for juvenile wood density (JWD) and stem diameter at breast height (DBH) using selective genotyping. Evenly spaced RFLP and microsatellite markers were selected from an existing linkage map. QTLs were verified in an independent set of progeny from the same family. Based on map location, at least eight QTL positions for JWD and two for DBH were detected and verified. The percent variance accounted for by the markers ranged from 0.78% to 3.58%, suggesting a genomic architecture of many genes with small effect. Two unrelated bridging families were chosen as candidates for marker-aided selection (MAS), and six microsatellite markers showing an association with JWD or DBH were tested in these families. Of these, four markers showed a consistent association with JWD in one or both of the bridging families. Results from this study provide a basis for MAS in P. radiata.Communicated by D.B. Neale     

Aridity promotes differences in proline and phytohormone levels in <Emphasis Type="Italic">Pinus pinaster</Emphasis> populations from contrasting environments

We analysed proline, abscisic acid, (ABA), jasmonic acid (JA), indole acetic acid (IAA) and salicylic acid (SA) accumulation after summer drought at two Pinus pinaster provenance-progeny trial sites. The aim of the study was to evaluate P. pinaster phenotypic plasticity and intraspecific variation in the endogenous concentrations of these metabolites and to determine the best stress indicators for family and population discrimination. The environmental effect was remarkable, as striking differences between the sites were obtained for all indicators except for SA, which was unaffected by the environmental conditions. The levels of proline, ABA and IAA were higher in the xeric than in the mesic site. In contrast, JA was higher in the mesic site. The higher variation displayed at the family level led to family differences for all parameters and sites. Differences in proline and ABA between populations were exclusively found in the xeric site, where the population from the wet climate showed higher accumulation. This study provides evidence for differentiation among P. pinaster populations and families in their plastic responses to drought and highlights the importance of considering intraspecific variability when evaluating biochemical stress indicators in environmental studies.     

Cytokinins and bud morphology in Pinus radiata

Cytokinins (CKs) play essential roles in the regulation of plant growth and development. In the previous paper (Zhang et al. 2001), we reported the detection and identification of a wide spectrum of CKs, including several novel forms, in the buds of Pinus radiata D. Don. In this paper we examine the relationship between the CKs and buds from juvenile and adult trees of P. radiata. During development the morphology of buds alters significantly, from buds bearing primary needles during their juvenile phase to buds sealed in scales at the adult phase. The morphology of adult buds is a very stable character, as fascicle meristems released from apical dominance, or cultured in vitro, produced only secondary needles. However, exogenous CK causes the adult buds to revert to juvenile bud development in vitro . Analyses of the endogenous CKs revealed that juvenile buds had a relatively higher level of isopentenyladenine and isopentenyladenosine, extremely low levels of phosphorylated CKs and a relatively low level of novel CK glycosides. The adult buds contained lower levels of free base and riboside CKs but very high levels of phosphorylated CKs and novel CK glycosides. Possible roles for CKs in the regulation of bud development are discussed.     

Abscisic acid, indole-3-acetic acid and cytokinin changes in buds of Pseudotsuga menziesii during bud quiescence release

Bud quiescence release, considered as the ultimate dormancy breaking phase, was achieved in Pseudotsuga menziesii (Mirb.) Franco by a 9-week cold (5°C) treatment, under short daylength (9 h) followed by a transfer to mild temperature (22°C) under long daylength (16 h). Indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin-type (Z) and isopentenyladenine-type (iPA) cytokinin (CK) levels were measured by means of an ELISA technique performed on HPLC-fractionated extracts of terminal and axillary buds. During the cold period, all hormones except IP-type CK levels decreased, whereas the opposite observation was made after transfer to mild temperature and long daylength, when buds started to grow. Some other immunoreactive compounds were also detected and quantified. The ABA-glucosyl ester (ABA-GE) level pattern was similar to that of ABA, but no accumulation occurred at mild temperatures. A putative IAA conjugate, more polar than IAA, was also detected. Its level increased transiently like IAA in terminal buds and, to a lesser extent, in axillary buds during the 10th week of the experiment. In terminal buds, isopentenyladenosine ([9R]-iP) was released by alkaline hydrolysis of a polar immunoreactive compound detected with anti-[9R]iP antibodies. This compound accumulated during the cold period and quickly dropped at 22°C. Relationships between environmental conditions and endogenous hormones are discussed.     

Alterations in endogenous levels of cytokinins following grafting of Pinus radiata support ratio of cytokinins as an index of ageing and vigour

Markers for ageing or maturation in woody plants provides the possibility of identifying genotypes with a prolonged juvenile phase, or to evaluate reinvigoration procedures, such as grafting of adult material. This study focuses specifically on cytokinins (Cks) as markers, due to their role in the transition process from juvenile to adult states. Previous reports from our group disclosed that the ratio of isopentenyladenine-type (iP-type) to zeatin-type (Z-type) Cks decreases during tree maturation, a maturation and ageing index being likely in Pinus radiata. Based on these results, Cks were tested in terminal buds of adult and grafted adult P. radiata material to corroborate the importance of the aforementioned balance as an index in the reinvigoration process of adult trees associated with grafting procedures. Results revealed the reversion of this index pattern parallel to the reactivation of some juvenile traits, namely, increasing values of the ratio throughout the reinvigoration process. According to previous results, however, the opposite was the case during the reverse process. This fact allowed us to validate the iP-type/Z-type Ck ratio as an ageing and vigour index.     

Novel cytokinins: The predominant forms in mature buds of Pinus radiata

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O - or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.     

Gene silencing studies in the gymnosperm species Pinus radiata

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.     

2种石蒜生长发育期鳞茎内源激素的动态变化

应用酶联免疫吸附测定法（ELISA）研究了石蒜与中国石蒜不同生长发育期鳞茎内源激素质量摩尔浓度的动态变化,分析了内源激素与2种石蒜生长发育的关系。结果表明,花芽分化前期2种石蒜鳞茎IAA浓度较低,而iPAs、ABA与GAs浓度较高;临近抽葶IAA浓度均有所增加,而iPAs与Zrs、ABA与GAs浓度均降到极值。花期ABA、GAs与ZRs浓度均有所回升,说明高浓度的ABA与GAs对2种石蒜的开花可能起正向调控的作用,但对叶生长的协同规律不明显。内源激素比例分析表明,ZRs/IAA、GAs/IAA、ABA/GAs三者比例高有利于2种石蒜的花原基分化,三者比例低则有助于2种石蒜的叶生长。     

The influence of water deficits on needle conductance, assimilation rate and abscisic acid concentration of seedlings of Pinus radiata D. Don

Abstract Seedlings of Pinus radiata, 10–20 weeks old and hitherto fully watered, responded rapidly when water was withheld. Wilting occurred 9d later, at which time soil matric water potential at dawn (Ψ_m) was –1.06MPa and shoot water potential (Ψ) was –1.9 MPa. Small reductions in Ψ_m elicited large responses in assimilation rate (A) and leaf conductance to water vapour (g). Seedlings appear to be more sensitive to small water deficits than are older Plants of P. radiata. After rewatering, significant increases of A and g occurred within one day, but neither regained the values measured prior to the imposition of a single drying cycle. This residual effect of drought on A, after one or six drying cycles, was partially caused by a decrease in photosynthetic capacity. In plants wilted for the first time, the concentration of abscisic acid (ABA) in the bulk foliage increased 3.4 times as Ψ decreased to –1.77 MPa. In comparison, pretreatment with six drying cycles significantly reduced Ψ to –2.13 MPa (indicating some osmotic adjustment) and induced only a doubling of ABA concentration. However, these differences in Ψ and ABA concentration did not Persist after the plants of all pretreatments had been watered for 7 d, although g of drought-pretreatment Plants remained approximately half that of continuously-watered plants.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Changes in cell ultrastructure and endogenous abscisic acid and indole-3-actic acid concentrations in Fatsia japonica leaves under polyethylene glycol-induced water stress

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA = abscisic acid - IAA = indole-3-acetic acid - PEG = polyethylene glycol - = leaf water potential - TEM = transmission electron microscope     

Hormonal Regulation of Pedicel Abscission in Begonia Flower Buds

In order to analyse the hormonal regulation of flower bud shedding in Begonia, levels of indoleacetic acid (IAA), abscisic acid (ABA) and ethylene were determined in buds and pedicels. The translocation and metabolism of ¹⁴C-labeled IAA in pedicel segments were also studied. In a monoecious Begonia fuchsioides hybrid, abscising male flower buds contain about 1% of the IAA present in non-abscising female flowers. In a male Begonia davisii hybrid, the seasonal variation in bud drop coincides with changes in the IAA content of the buds, while also the release of IAA from the bud to the pedicel is hampered. Abscission zones of these pedicels always contain abscission promoting ethylene concentrations. The tissue is prevented from responding with abscission by IAA from the flower buds. The buds also contain ABA but without influencing abscission considerably. Pretreatment with ethylene or ABA does not affect IAA transport in pedicel segments. The rate of this transport is 4–6 mm × h^–1:; the capacity increases with the transverse area. In young segments, IAA is decarboxylated and also otherwise metabolized.