Search

Phase change refers to the process of maturation and transition from the juvenile to the adult stage. In response to this shift, certain species like chestnut lose the ability to form adventitious roots, thereby hindering the successful micropropagation of adult plants. While auxin is the main hormone involved in adventitious root formation, other hormones, such as ethylene, are also thought to play a role in its induction and development. In this study, experiments were carried out to determine the effects of ethylene on the induction and growth of adventitious roots. The analysis was performed in two types of chestnut microshoots derived from the same tree, a juvenile-like line with a high rooting ability derived from basal shoots (P2BS) and a line derived from crown branches (P2CR) with low rooting responses. By means of the application of compounds to modify ethylene content or inhibit its signalling, the potential involvement of this hormone in the induction of adventitious roots was analysed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterise the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. The findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modulation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour. [ABSTRACT FROM AUTHOR]

Wounding and exogenous auxin are needed to induce adventitious roots in chestnut microshoots. However, the specific inductive role of wounding has not been characterized in this species. In the present work, two main goals were established: First, we prompted to optimize exogenous auxin treatments to improve the overall health status of the shoots at the end of the rooting cycle. Second, we developed a time-series transcriptomic analysis to compare gene expression in response to wounding alone and wounding plus auxin, focusing on the early events within the first days after treatments. Results suggest that the expression of many genes involved in the rooting process is under direct or indirect control of both stimuli. However, specific levels of expression of relevant genes are only attained when both treatments are applied simultaneously, leading to the successful development of roots. In this sense, we have identified four transcription factors upregulated by auxin (CsLBD16, CsERF113, Cs22D and CsIAA6), with some of them also being induced by wounding. The highest expression levels of these genes occurred when wounding and auxin treatments were applied simultaneously, correlating with the rooting response of the shoots. The results of this work clarify the genetic nature of the wounding response in chestnut, its relation to adventitious rooting, and might be helpful in the development of more specific protocols for the vegetative propagation of this species. [ABSTRACT FROM AUTHOR]

Micropropagation has enormous potential in the forestry industry for the mass production of elite genotypes. A limiting factor that hinders a wider adoption of this technique is its high associated cost, dissuading small producers and laboratories from its use. Thus, it is important to develop low-cost protocols to increase the competitiveness of micropropagation. The goal of the present study was to elaborate a low-cost culture medium that would allow plant growth without diminishing plant quality and production. The replacement of laboratory sucrose with commercial sugar as a carbon source, and laboratory agar with locally produced agar as a gelling agent was tested. To validate the effects of the medium for woody species micropropagation, two relevant forest species, chestnut and birch, were used and several phenotypic characteristics were recorded. Our data indicated that the substitutions made in the media composition did not alter growth or rooting parameters. Substitution of laboratory sucrose and Bacto agar by table sugar and local commercial agar provides a valuable alternative for forest species micropropagation and for increasing their potential profitability. [ABSTRACT FROM AUTHOR]

The mechanisms underlying the de novo regeneration of adventitious roots are still poorly understood, particularly in trees. We developed a system for studying adventitious rooting (AR) at physiological and molecular levels using leaves excised from chestnut microshoots of the same genotype but with two distinct ontogenetic origins that differ in rooting competence. Leaves were treated with auxin and N-1-naphthyl-phthalamic acid (NPA), an inhibitor of auxin polar transport (PAT). The physiological effects were investigated by recording rooting rates and the number and quality of the roots. Molecular responses were examined by localizing and monitoring the changes in the expression of CsSCL1, an auxin-inducible gene in juvenile and mature shoots during AR. The rooting response of leaves was ontogenetic-stage dependent and similar to that of the donor microshoots. Initiation of root primordia and root development were inhibited by application of NPA, although its effect depended on the timing of application. CsSCL1 was upregulated by auxin only in rooting-competent leaves during the novo root organogenesis, and the expression was reduced by NPA. The inhibitory effect on gene expression was detected during the reprograming of rooting competent cells towards root initials in response to auxin, indicating that PAT-mediated upregulation of CsSCL1 is required in the initial steps of AR in chestnut leaves. The localized expression of CsSCL1 in the quiescent center (QC) also suggests a role for this gene in the maintenance of meristematic competence and root radial patterning. [ABSTRACT FROM AUTHOR]

Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants. [ABSTRACT FROM AUTHOR]

Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids. [ABSTRACT FROM AUTHOR]

Maturation imposes several changes in plants, which are particularly drastic in the case of trees. In recalcitrant woody species, such as chestnut (Castanea sativa Mill.), one of the major maturation-related shifts is the loss of the ability to form adventitious roots in response to auxin treatment as the plant ages. To analyze the molecular mechanisms underlying this phenomenon, an in vitro model system of two different lines of microshoots derived from the same field-grown tree was established. While juvenile-like shoots root readily when treated with exogenous auxin, microshoots established from the crown of the tree rarely form roots. In the present study, a transcriptomic analysis was developed to compare the gene expression patterns in both types of shoots 24 h after hormone and wounding treatment, matching the induction phase of the process. Our results support the hypothesis that the inability of adult chestnut tissues to respond to the inductive treatment relies in a deep change of gene expression imposed by maturation that results in a significant transcriptome modification. Differences in phytohormone signaling seem to be the main cause for the recalcitrant behavior of mature shoots, with abscisic acid and ethylene negatively influencing the rooting ability of the chestnut plants. We have identified a set of related MADS-box genes whose expression is modified but not suppressed by the inductive treatment in mature shoots, suggesting a putative link of their activity with the rooting-recalcitrant behavior of this material. Overall, distinct maturation-derived auxin sensibility and homeostasis, and the related modifications in the balance with other phytohormones, seem to govern the outcome of the process in each type of shoots. [ABSTRACT FROM AUTHOR]

Gibberellins have been classically related to a few key developmental processes, thus being essential for the accurate unfolding of plant genetic programs. After more than a century of research, over one hundred different gibberellins have been described. There is a continuously increasing interest in gibberellins research because of their relevant role in the so-called "Green Revolution", as well as their current and possible applications in crop improvement. The functions attributed to gibberellins have been traditionally restricted to the regulation of plant stature, seed germination, and flowering. Nonetheless, research in the last years has shown that these functions extend to many other relevant processes. In this review, the current knowledge on gibberellins homeostasis and mode of action is briefly outlined, while specific attention is focused on the many different responses in which gibberellins take part. Thus, those genes and proteins identified as being involved in the regulation of gibberellin responses in model and non-model species are highlighted. The present review aims to provide a comprehensive picture of the state-of-the-art perception of gibberellins molecular biology and its effects on plant development. This picture might be helpful to enhance our current understanding of gibberellins biology and provide the know-how for the development of more accurate research and breeding programs. [ABSTRACT FROM AUTHOR]

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, β -A and β -H, all supplemented with 2-μM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in β -A medium supplemented with 2-μM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species. [ABSTRACT FROM AUTHOR]

In this study, we propagated two old Galician plum varieties in liquid medium using a temporary immersion system with RITA© bioreactors. Environmental variables including culture system, light intensity, CO2 enrichment, immersion frequency and sucrose supplementation were evaluated in relation to in vitro proliferation, physiological status and ex vitro performance. Bioreactors were superior to jars for culturing shoots in photomixotrophic conditions, producing up to 2 times more shoot numbers and up to 1.7 times more shoot length (depending on the genotype) using shoot clusters. The number and quality of shoots were positively influenced by the sucrose concentration in the medium, plus by the light and gaseous environment. For individual apical sections the best response occurred with 3% sucrose, 150 µmol m−2 s−1 photosynthetic photon flux density and 2000 ppm CO2, averaging 2.5 shoots per explant, 26 mm shoot length and 240 mm2 leaf area, while with 50 µmol m−2 s−1 light and ambient CO2 (400 ppm) values decreased to 1.2 shoots per explant, 14 mm of shoot length and 160 mm2 of leaf area. Shoots cultured photoautotrophically (without sucrose) were successfully rooted and acclimated despite of showing limited growth, low photosynthetic pigments, carbohydrate, phenolic and antioxidant contents during the multiplication phase. [ABSTRACT FROM AUTHOR]

Current breeding programs aim to increase the number of ink-tolerant chestnut trees using vegetative propagation of selected genotypes. However, the commercial vegetative propagation of chestnut species is still a bottleneck for the forest industry, mainly due to problems in the rooting and acclimation of propagules. This study aimed to explore the potential benefits of decreasing sucrose supplementation during chestnut micropropagation. Explants were cultured with high light intensity and CO2-enriched air in temporary or continuous immersion bioreactors and with different sucrose supplementation to evaluate the impact of these treatments on growth, rooting and physiological status (monosaccharide content, soluble phenolics and antioxidant activity). The proliferation and rooting performance of shoots cultured by continuous immersion decreased sharply with sucrose concentrations lower than 1%, whereas shoots cultured by temporary immersion grew and rooted successfully with 0.5% sucrose. These results suggest this system is appropriate to culture chestnut with low sucrose concentration and to explore photoautotrophic propagation of this species. [ABSTRACT FROM AUTHOR]

This analysis allowed the authors to track specific gradients of these two phytohormones in the different developing zones of the root, while also detecting the presence of amide-linked indole-acetic acid metabolites as the predominant form of auxin catabolite, rather than the oxidized by-products found in other species [[8]]. In root development, the plastic behaviour shown by plants is a consequence of the channelling effect of phytohormones on the potential responses encoded by the genome and the epigenome that drives towards the most favourable responses to ensure plant survival and optimal growth. ELISA-based analysis of the phytohormone content in response to spermidine revealed that it modulated the levels of auxins, gibberellins, and jasmonic acid, which resulted in changes in expression of several rooting-related genes [[14]]. The authors found that melatonin altered the profiles of several phytohormones, increasing auxin, cytokinin, and gibberellins and lowering abscisic acid two days after the beginning of the treatment. [Extracted from the article]

Gibberellins are amongst the main plant growth regulators. Discovered over a century ago, the interest in gibberellins research is growing due to their current and potential applications in crop production and their role in the responses to environmental stresses. In the present review, the current knowledge on gibberellins' homeostasis and modes of action is outlined. Besides this, the complex interrelations between gibberellins and other plant growth regulators are also described, providing an intricate network of interactions that ultimately drives towards precise and specific gene expression. Thus, genes and proteins identified as being involved in gibberellin responses in model and non-model species are highlighted. Furthermore, the molecular mechanisms governing the gibberellins' relation to stress responses are also depicted. This review aims to provide a comprehensive picture of the state-of-the-art of the current perceptions of the interactions of gibberellins with other phytohormones, and their responses to plant stresses, thus allowing for the identification of the specific mechanisms involved. This knowledge will help us to improve our understanding of gibberellins' biology, and might help increase the biotechnological toolbox needed to refine plant resilience, particularly under a climate change scenario. [ABSTRACT FROM AUTHOR]

Plant biotechnology can be used to conserve the germplasm of natural forests, and to increase the productivity and sustainability of plantations. Both goals imply working with mature trees, which are often recalcitrant to micropropagation. Conventional in vitro culture uses closed containers and gelled medium with sugar supplementation. Bioreactor culture uses liquid medium and usually incorporates aeration. The increased absorption of nutrients via the liquid medium together with the renewal of the air inside the bioreactors may improve the physiological state of the explants. In this review, we will explore the feasibility of using bioreactors to overcome the recalcitrance of many trees to micropropagation and/or to decrease the cost of large‐scale propagation. We will focus on the recent use of bioreactors during the multiplication, rooting (plant conversion in the case of somatic embryos), and acclimation stages of the micropropagation of axillary shoots and somatic embryos of forest trees (including some shrubs of commercial interest), in both temporary and continuous immersion systems. We will discuss the advantages and the main obstacles limiting the widespread implementation of bioreactor systems in woody plant culture, considering published scientific reports and contributions from the business sector. [ABSTRACT FROM AUTHOR]

Key message: Culturing axillary shoot explants of willow in liquid medium in plantform™ and RITA®vessels increased the multiplication coefficient relative to that obtained by culturing the explants in semisolid medium in jars.Abstract: A protocol for micropropagating willow by multiplying axillary shoots in liquid medium was developed, and the effects of the type of explant, medium composition, bioreactor type and frequency of immersion were investigated. The type of explant influenced shoot quality and the multiplication coefficient, with basal segments showing a higher capacity for proliferation than apical and mid-shoot segments. Hyperhydricity was not observed in shoots cultured with Murashige and Skoog medium with half-strength nitrates and supplemented with 0.22 µM BA and 3% sucrose. The willow shoots cultured in plantform™ and RITA® vessels yielded higher multiplication coefficients than shoots grown in semisolid medium. High proliferation rates were obtained by immersion of shoots for 1 min three or six times a day, with additional aeration of 1 min per hour in the plantform™ bioreactors. Transfer to medium with 1% sucrose after 4 weeks of culture in medium with 3% sucrose increased the multiplication coefficient of basal segments cultured in RITA® vessels, suggesting that the species could be micropropagated under photoautotrophic conditions. The willow shoots rooted spontaneously and were successfully acclimatized to greenhouse conditions. This is the first report of the use of a temporary immersion system to culture Salix viminalis. The study findings indicate the feasibility of the system as a method for the large-scale propagation of this species. [ABSTRACT FROM AUTHOR]

Background Adventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown. Results Here, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata's SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or nonexistent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation. Conclusions The GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells. [ABSTRACT FROM AUTHOR]

We characterized SCARECROW-LIKE genes induced by auxin in rooting-competent cuttings of two distantly related forest species (Pinus radiata D. Don and Castanea sativa Mill.) before the activation of cell division that results in adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family and show a strong similarity to the SCARECROW-LIKE proteins, indicating conserved functions of these proteins. Quantitative RT-PCR analysis showed that these genes are expressed at relatively high levels in roots. Induction of increased mRNA levels in rooting-competent cuttings of both species in response to exogenous auxin was observed within the first 24 h of the root induction process, a time when cell reorganization takes place, but before the resumption of cell division and the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes play a role during the earliest stages of adventitious root formation. [ABSTRACT FROM AUTHOR]

The effect of sucrose concentration on the micropropagation of axillary shoots of willow was investigated. The following factors were examined: the culture system (semisolid medium in glass jars versus liquid medium in temporary immersion bioreactors), the type of explant (apical and basal sections), the frequency of immersion, and CO2 enrichment. Shoots and leaf growth were significantly higher in RITA® bioreactors than in the jars for all the sucrose treatments. Apical or basal sections of willow cultured in bioreactors under high light intensity (150 µmol m−2 s−1) and ventilated six times a day with CO2-enriched air were successfully proliferated without sucrose, whereas shoots cultured in jars did not proliferate well if sucrose concentration was 0.5% or lower. More roots were formed when sucrose was added to the medium. Shoots cultured in bioreactors were successfully acclimatized irrespective of the sucrose treatment and the root biomass when transferred to ex vitro conditions. This is the first report of photoautotrophic willow micropropagation, our results confirm the importance of proper gaseous exchange to attain autotrophy during in vitro propagation. [ABSTRACT FROM AUTHOR]

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development. [ABSTRACT FROM AUTHOR]

The genus Castanea includes several tree species that are relevant because of their geographical extension and their multipurpose character, that includes nut and timber production. However, commercial exploitation of the trees is hindered by several factors, particularly by their limited regeneration ability. Regardless of recent advances, there exists a serious limitation for the propagation of elite genotypes of chestnut due to decline of rooting ability as the tree ages. In the present review, we summarize the research developed in this genus during the last three decades concerning the formation of adventitious roots (ARs). Focusing on cuttings and in vitro microshoots, we gather the information available on several species, particularly C. sativa, C. dentata and the hybrid C.sativa × C. crenata, and analyze the influence of several factors on the achievements of the applied protocols, including genotype, auxin treatment, light regime and rooting media. We also pay attention to the acclimation phase, as well as compile the information available about biochemical and molecular related aspects. Furthermore, we considerate promising biotechnological approaches that might enable the improvement of the current protocols. [ABSTRACT FROM AUTHOR]

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA ® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA ® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants.

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

A full-length cDNA clone named QRCPE (Quercus robur crown preferentially expressed) that is differentially expressed during in vitro culture of mature and juvenile-like shoots of Quercus robur L. was identified by differential display. The deduced amino acid sequence showed that the encoded protein is small, contains a predicted N-terminal hydrophobic signal peptide that targets the protein to the cell wall, and is rich in glycine and histidine residues. Accumulation of QRCPE mRNA was higher in oak microshoots derived from crown branch shoot cultures than in oak microshoots derived from basal shoot cultures at the end of the multiplication and rooting period. Among organs, the highest accumulation of QRCPE transcripts was detected in roots, followed by stems and leaves, with preferential accumulation in specific organs of ontogenetically older shoots. Although QRCPE mRNA was abundant in oak zygotic and somatic embryos, almost no QRCPE mRNA accumulation was detected in nodular callus cells, suggesting a possible role of this gene in embryonic development. In proliferating shoot cultures of two chestnut (Castanea sativa Mill.) clones, the QRCPE homolog was preferentially expressed in crown-derived shoots. On the other hand, expression analysis of QRCPE in juvenile and mature material from soil-grown oak plants indicated that this gene is expressed from the embryonic to mature phases, but is progressively down-regulated during plant maturation. In vitro culture conditions induced changes in QRCPE transcript abundance in both basal and crown shoots in a phase-dependent manner. We conclude that QRCPE expression in oak is correlated with the ontogenetic stage of shoots, and thus this gene may be useful as a potential molecular marker for maturation-related characteristics.