Plant growth regulators as putative physiological markers of developmental stage in Prunus persica

Prunus persica plants of different ages and statesof maturation were analysed to compare their phytohormonal status. Endogenouslevels of indole-3-acetic acid (IAA), abscisic acid (ABA), and severalcytokinins (Cks): zeatin (Z), zeatin riboside ([9R]Z), dihydrozeatin ((diH)Z),dihydrozeatin riboside ((diH)Z[9R]), isopentenyl adenine (iP) and isopentenyladenosine ([9R]iP), were measured in order to determine their possible use asphysiological indices of phase change and maturation. A decrease in Ck levels(Z, [9R]Z, (diH)Z, (diH)[9R]Z, and iP, [9RiP) was found from the embryonic tojuvenile stage as well as a decrease in the ratio of iP-type (iP and[9R]iP)/Z-type Cks paralleling the increase in tree age. ABA levels increasedduring maturation in Prunus persica and the ratio ofCks/IAA decreased with tree age. From our results, we propose that the balancesof Cks/IAA and iP-type/Z-type Cks are good indices of different developmentalstates in Prunus persica.     

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.     

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.     

Phytohormone contents in Corylus avellana and their relationship to age and other developmental processes

Endogenous levels of indole-3-acetic acid, abscisic acid, and cytokinins (Z-type: dihydrozeatin, dihydrozeatin riboside, zeatin, and zeatin riboside; iP-type: N ⁶-isopentenyl adenine and N ⁶-isopentenyl adenosine), were determined in leaves of hazelnut (Corylus avellana L.) (adult material from spring, autumn and forced outgrowth, and juvenile material). Our results showed high levels of indole-3-acetic acid, abscisic acid and total cytokinins in spring samples and low levels of the same hormones in autumn and forced outgrowth materials. The ratios of iP-type/Z-type cytokinins were low in autumn and spring leaves, while they were high in the juvenile and forced outgrowth samples. Both juvenile and forced-outgrowth hazel tissues also showed a high morphogenetic potential, suggesting that the ratio of iP-type/Z-type cytokinins may be a good index of in vitro potential of hazelnut materials.     

Abscisic acid and 3-indolacetic acid levels during the reinvigoration process of <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don adult material

The maturation and phase change processes in woody species have not been completely clarified, specially the role of growth regulators during the maturation phase. Understanding their role better will allow to comprehend the physiological aspects of the species, which would allow to chose the optimal management conditions in forestry programs and make them more useful. In the present study, the endogenous content of abscisic acid (ABA) and 3-indolacetic acid (IAA) were quantified by high performance liquid chromatography, in the reinvigoration process of elite Pinus radiata material, during the development of homomicrografts (P. radiata/P. radiata) and heteromicrografts (P. radiata/Pinus caribaea), developed under in vitro conditions. The results showed an increase in the endogenous content of ABA through time and a decrease in the content of IAA. Significant differences were found in the endogenous content of ABA, where the maximum values were detected at 120 days of micrograft culture, being similar to the values obtained in juvenile buds. As well, significant differences were also detected in the endogenous content of IAA in the micrografted buds during all the development phases, compared with adult buds. However, no significant differences were detected in the endogenous content of ABA and IAA, regarding the type of rootstock employed. Considering the endogenous content of ABA and IAA, the micrografted plant material would be equal to the juvenile buds, since statistically they were equal to these buds, considering that the values obtained in this case are independent from the type of rootstock used.     

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.     

Cytokinins in the hemiparasiteMelampyrum arvense L. before and after attachment to the host

Concentration of cytokinins (CKs) of zeatin-type (Z) and isopentyladenine-type (iP) were determined using an enzyme-linked immunosorbent assay (ELISA) technique in xylem sap and bulk leaf extract of the hemiparasiteMelampyrum arvense before and after attachment to the host (Capsella bursa pastoris). In all the samples Z-type were dominant, though the iP-type was also frequent. The results also demonstrate that in comparison with the unattached hemiparasite after attachment to the host the concentration of Z-type in xylem sap increased about 92 and 182 times in the light and dark phase, respectively, and that of the iP-type about 34 times in the two phases. The concentration of Z-type and iP-type in leaves expressed per dm³ of cell water was at the level of 10^-7 M and 10^-8 M, respectively in the unattached hemiparasite. After attachment, the concentration of CKs increased to 10^-5 M and 10^-6 M for Z-type and iP-type, respectively. In xylem sap the concentration of Z-type was at the level of 10^-9 M and 10^-7 M and in the unattached and attached hemiparasite, respectively. The concentrations of iP-type were 10^-10 M and 10^-8 M for the unattached and attached hemiparasite, respectively. The diurnal oscillation of CK concentration was evident in the unattached hemiparasite, but after attachment their level was nearly constant and independent of the diurnal cycle.     

Micropropagation of Salix pseudolasiogyne from nodal explants

The effect of phytohormones on the breaking of dormancy of axillary buds in Salix pseudolasiogyne and their subsequent proliferation from nodal explants were examined. Nodal explants obtained from a 20–year-old S. pseudolasiogyne tree were cultured either on woody plant basal medium (WPM) or WPM supplemented with benzyladenine (BA, 2.2/4.4 μM), zeatin (1.1/2.2 μM), gibberillic acid (GA₃, 2.9 and 14.5 μM), and GA₃ + BA (2.9 + 4.4 μM). Although axillary shoots developed in all the media, a higher percentage bud break occurred on BA supplemented media. To corroborate the results, endogenous levels of cytokinins [Cks, N ⁶-isopentenyladenine (iP), zeatin riboside (t-ZR), dihydrozeatinriboside (DHZR)] and abscisic acid (ABA) were determined. On BA supplemented media, the levels of zeatin type (Z-type) of Cks were higher than those of isopentenyladenine type of Ck in the explants, while the ABA level was low. Axillary shoots did not grow well and became necrotic upon subculture to fresh basal WPM. In order to improve shoot growth, they were subcultured twice at a 4-week interval on to WPM supplemented with BA (2.2/4.4 μM), GA₃ (1.4 μM), or GA₃ + BA (1.4 + 4.4/2.9 + 4.4 μM). Maximal shoot growth (93%) was achieved on WPM supplemented with 2.2 μM BA. Comparative analyses of endogenous Cks revealed that higher Cks (Z-type Cks) were present in actively growing shoots. Rooting was readily achieved when the shoots were subcultured to WPM without phytohormones. The rooted plants were acclimatized well upon transplantation.     

Comparison of endogenous cytokinins, ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine

In lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.), cone bud initiation within long-shoot buds varies according to genotype. We chose to study hormone profiles of two genotypes that differed significantly in cone yield. Phytohormone profiles were established by high performance liquid chromatography–electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode with samples collected from genotypes 299 and 233, the typically high and low cone producers. Generally, concentrations of trans-zeatin-O-glucoside were higher in genotype 299, whereas dihydrozeatin concentrations were higher in genotype 233. Both isopentenyl adenine and isopentenyl adenosine were present at higher concentrations in genotype 233. The ratio of total quantifiable zeatin (Z)-type cytokinins to isopentenyl (iP)-type cytokinins was approximately threefold higher in genotype 299 during female cone bud differentiation. In genotype 299, ABA concentration was significantly lower than in genotype 233 on the first sampling date, while the phaseic acid concentration was lower consistently throughout the period investigated. Dihydrophaseic acid was present in low concentrations in most samples of genotype 233, but was not quantifiable in genotype 299. Our study reveals that long-shoot buds of the high cone-producing genotype had higher ratios of Z-type cytokinins to iP-type cytokinins than were found in the low cone-producing genotype. High cone-producing buds also contained less ABA, phaseic acid and dihydrophaseic acid during female cone bud differentiation.     

Growth and development of Pinus radiata D. Don: the effect of light quality

Abstract. Plants of Pinus radiata of two physiological ages, juvenile (seedlings and micropropagated plantlets) and adult (rooted cuttings from mature trees), were grown under lighting fromthree combinations of metal halide (MH) and tungsten halogen (TH) lamps for up to 10 months in controlled environment rooms. The three lamp combinations, MH alone, 50: 50 MH: TH and 25:75 MH: TH by wattage, produced red: farred ratios of 4.59, 1.51, and 1.15, respectively. Photosynthetic photon flux density was 700 μmol m⁻² s⁻¹. An increase in proportion of TH lamps markedly increased shoot elongation and internode length, decreased numbers of fascicles per unit stem length and increased the proportion of stem weight in both juvenile and adult material. In addition, in adult material, it increased the number of fascicle initials and expanded fascicles per growth flush, reduced the duration of the 'rest' phase between growth flushes, accelerated the rate of elongation growth during each flush, and increased apical dominance. Tracheid length, but not diameter or wall thickness, was significantly affected by light quality and found to be associated with longer internodes. Any treatment effects on needle weight or length, stem diameter or root weight were non-significant or very small. Different clones from either the juvenile micro-propagated material or the mature rooted cuttings each showed similar patterns of response, although they often differed in the degree of response to light quality. The main response could be related solely to the red: far-red ratio and the calculated phytochrome photoequilibrium. This is the first report of phytochrome-controlled photomorphogenesis in older specimens of a woody perennial. Recommendations for artificial light sources for growing P. radiata and some ecological implications of the results are presented.     

Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Müll. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

Identification and quantification of the major endogenous cytokinins in pistachio seedlings

The major endogenous cytokinins, Z, ZR, DHZ, DHZR, iP and iPR in pistachio seedlings (Pistacia vera L. cv. Ohadi) were purified by HPLC and their identities confirmed using GC-MS. The aerial parts of two-year old pistachio seedlings including mature leaves, young leaves, lateral buds, debarked stems and bark were subjected to analysis. All of the above mentioned cytokinins were identified in the aerial parts except DHZ which was only present in mature leaves. Z-type cytokinins contributed almost 43% of the total cytokinins. ZR and DHZR were identified as the major ribosides and iP as the main base. The greatest concentration of ZR was detected in the bark, amounting to about 48%. DHZR and ZR constituted the major portion of the total cytokinins detected in both young and mature leaves while Z was detected as a minor cytokinin in leaves. The sharp increase of iP concentration during leaf maturation indicates that mature leaves are probably capable of de novo biosynthesis of cytokinins. The absence of DHZ (except in mature leaves) and the presence of considerable concentrations of DHZR in pistachio stems suggest that these tissues are able to metabolize DHZ to DHZR. The large amount of ZR in pistachio leaves suggests that root-derived ZR is transported into the leaves after loading into the xylem. The presence of high amounts of iP in pistachio lateral buds indicates that iP has been accumulated in these parts. The occurrence of a totally different cytokinin distribution pattern in buds, as compared with the other aerial parts, possibly results from their different metabolism.     

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.     

Phase-change related epigenetic and physiological changes in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.     

Maturation and rejuvenation of Coriandrum sativum shoot clones during micropropagation

Three clones of Coriandrum sativum L. shoots were obtained from three seedlings and micropropagated alternately on modified MS media containing kinetin only and kinetin plus indolyl-3-acetic acid (IAA). During the first 9 months of culture the shoots possessed the juvenile phenotype after which a sharp transition to mature phenotype occurred. In 15–17 months this was followed by shoot necrosis and decrease in number of shoots in the clones, leading to death of the clones.Conditions of in vitro culture tripled the length of the juvenile period. Mature phase of the shoots was stable in that no reversion to the juvenile phase was observed. Partial rejuvenation of mature shoots took place owing to formation of adventitious shoots in the callus formed at the shoot base. However maturation of such rejuvenated adventitious shoots took place much more rapidly in comparison with micropropagated juvenile shoots derived from seedlings. Reduction of the morphogenic potential of the mature shoots after 15–17 months of subculturing, an increase in number of abnormal shoots and shoot necrosis indicated physiological ageing of the clones.Data presented in the paper provide evidence of the clone ageing phenomenon during prolonged subculture in vitro.     

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.     

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

Uptake and metabolism of N6-benzyladenine and 1-naphthaleneacetic acid and dynamics of indole-3-acetic acid and cytokinins in two callus lines of Actinidia deliciosa differing in growth and shoot organogenesis

Uptake, metabolism and accumulation of N⁶‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) as well as changes in endogenous indole‐3‐acetic acid (IAA) and several isoprenoid‐type cytokinins (Cks) were characterized in two callus lines of Actinidia deliciosa cv. Hayward showing different growth and shoot organogenic responses to exogenously applied 2.7 µM NAA and 4.4 µM BA. Studies were carried out in callus 0, 1, 12, 24 and 48 h after the onset of their fifth subculture on a medium containing [³H]NAA or 8‐[¹⁴C]BA. Kiwifruit callus of line A presented high caulogenic response and lower growth that was positively associated with faster BA uptake, with transient accumulation of BA and isoprenoid‐type Cks, mainly zeatin, exceeding three‐ and four‐fold that of the non‐caulogenic callus, and with values of the BA/NAA ratio exceeding 1, in fact higher than the BA/NAA ratio in the culture medium. The accumulation of BA took place in both callus lines during the first 24 h of subculture and before the re‐initiating of callus proliferation. The higher growth and the low or null caulogenic response shown by line B callus were correlated with faster NAA uptake, with endogenous NAA levels two‐fold higher than in A calli, with higher IAA amounts and with values of the BA/NAA ratio below 1. Moreover, at 48 h free NAA in both kinds of callus reached levels close to those found after 35 days of subculture. Results suggest that temporal accumulation of BA and endogenous Cks is involved in the initiation of cell division leading to callus growth, whereas the maintenance of high NAA and IAA levels are related to the support of long‐term callus development. It also appears that for callus cells to become committed to shoot organogenesis, they must have a concentration of active Cks higher than a threshold value during the first 2 days of culture on fresh medium, while at the same time the concentration of auxins must not exceed a certain maximum.