Carolina Michels Ruedell

When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings.

Adventitious rooting (AR) is a multifactorial response leading to new roots at the base of stem cuttings, and the establishment of a complete and autonomous plant. AR has two main phases: (a) induction, with a requirement for higher auxin concentration; (b) formation, inhibited by high auxin and in which anatomical changes take place. The first stages of this process in severed organs necessarily include wounding and water stress responses which may trigger hormonal changes that contribute to reprogram target cells that are competent to respond to rooting stimuli. At severance, the roles of jasmonate and abscisic acid are critical for wound response and perhaps sink strength establishment, although their negative roles on the cell cycle may inhibit root induction. Strigolactones may also inhibit AR. A reduced concentration of cytokinins in cuttings results from the separation of the root system, whose tips are a relevant source of these root induction inhibitors. The combined increased accumulation of basipetally transported auxins from the shoot apex at the cutting base is often sufficient for AR in easy-to-root species. The role of peroxidases and phenolic compounds in auxin catabolism may be critical at these early stages right after wounding. The events leading to AR strongly depend on mother plant nutritional status, both in terms of minerals and carbohydrates, as well as on sink establishment at cutting bases. Auxins play a central role in AR. Auxin transporters control auxin canalization to target cells. There, auxins act primarily through selective proteolysis and cell wall loosening, via their receptor proteins TIR1 (transport inhibitor response 1) and ABP1 (Auxin-Binding Protein 1). A complex microRNA circuitry is involved in the control of auxin response factors essential for gene expression in AR. After root establishment, new hormonal controls take place, with auxins being required at lower concentrations for root meristem maintenance and cytokinins needed for root tissue differentiation.

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

BackgroundEucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings.ResultsBy the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.ConclusionOur study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

Pre and post-severance effects of light quality on carbohydrate dynamics and microcutting adventitious rooting of two Eucalyptus species of contrasting recalcitrance

Adventitious rooting is a complex developmental response affected by genetic and environmental factors. Radiation quality effects on adventitious rooting depend on characteristics such as species, growth stage, irradiance, spectral quality, and time of exposure. Eucalyptus is an essential genus for the paper industry, and high yield plantations depend on adventitious rooting of selected genotypes. This work addressed two hypotheses: (1) radiation quality equally affects adventitious rooting in Eucalyptus species of different recalcitrance; (2) adventitious rooting outcome depends on both donor plant and cutting radiation quality treatments. To that end, the easy-to-root Eucalyptus grandis and the recalcitrant Eucalyptus globulus were evaluated. The effect of white, blue, red and far-red radiation enrichment on microcuttings and donor plants of both species was evaluated in relation to rooting. There was no effect of radiation quality on adventitious rooting of E. grandis or when radiation treatments were applied to E. globulus microcuttings. In contrast, donor plants of E. globulus, grown in medium devoid of sucrose and exposed to far-red radiation, yielded microcuttings showing higher rooting percentage, even in the absence of exogenous auxin in the rooting medium. Sucrose in donor plant medium abolished the positive effect of far-red radiation. An increase in endogenous soluble sugars and starch contents in basal microcuttings was associated with far-red radiation treatment of donor plants. These results underline the importance of appropriate carbohydrate partitioning in donor plants for adventitious rooting of cuttings and provide a basis for understanding and overcoming rooting recalcitrance in E. globulus clones.

Environmental control of adventitious rooting in Eucalyptus and Populus cuttings

Key messageConcerted control of irradiance, temperature, water availability, mineral nutrition and beneficial root-associated microorganisms significantly improves adventitious rooting in eucalypts and poplars, essentially by modulating auxin and carbohydrate metabolism.AbstractEucalyptus and Populus are among the most economically relevant tree genera. Clonal propagation allows fast genetic gain obtained using elite genotypes. Adventitious rooting (AR), a complex and multifactorial process, is often the main limiting factor for tree cloning. Herein, practical and basic approaches to optimize clonal propagation of eucalypts and poplars, focusing on the main environmental control factors affecting it, are explored. Auxin homeostasis and function are central to AR. Irradiance quality and quantity, as well as temperature, can effectively modulate auxin availability, transport and activity. The interaction of carbohydrates, irradiance and temperature is also at the core of AR. Root architecture may be effectively modified by different N sources. Several macro and micronutrients impact on central factors of rhizogenesis, including energy metabolism, gene expression and enzymatic activities regulating auxin and other phytohormonal steady-states driving AR. Appropriate mineral nutrition is often determinant for successful AR and survival. Microbial associations with the root system and the rhizosphere, both bacterial and fungal, can have a role in auxin availability to cuttings, as well as improve disease resistance, nutrition and water relations. Significant cost reduction in clonal propagation systems of eucalypt and poplar are attainable with an adequate control of environmental factors, particularly for donor plants. Future studies should extend the molecular and physiological findings of basic research to the commercial propagation systems, and these, by their turn, should be explored to provide further advances in the basic understanding of this crucial developmental process for human economy.

Expression of auxin carrier genes during adventitious rooting in Eucalyptus globulus

Background Eucalyptus globulus and its hybrids are important for the cellulose and paper industry, mainly due to their relatively low lignin content. However, rooting of cuttings of this species is often recalcitrant and exogenous auxin application is necessary for adventitious root development. Auxin plays a central role in rooting capacity, which is particularly affected by its endogenous content and transport rate. The shoot apex is a major source of endogenous auxin, which is mainly transported by both influx (AUX1) and efflux (PIN) carriers in a specific basipetal active transport through the vascular parenchyma in stems. As part of a larger study to investigate the causes of low rooting in E. globulus microcuttings without exogenous auxin, we evaluated the expression profiles of AUX1 andPIN1 during the process of adventitious rooting using qPCR.

Nitrogen Sources and Adventitious Root Development in Eucalyptus globulus Microcuttings

In previous studies, it has been shown that nitrate supply may favor adventitious rooting in the rooting recalcitrant Eucalyptus globulus. Herein, the impact of various N sources on adventitious rooting and root branching in microcuttings of E. globulus was investigated. The positive effect of nitrate on adventitious root development was confirmed and extended to root branching. Urea yielded a rooting response comparable to that observed in presence of nitrate. Urease activity was observed, displaying two peaks: one at the root induction and another at the root formation step. The use of glutamic acid, glutamine or asparagine promoted higher root number, but yielded shorter roots. Rooted microcuttings derived from all nitrogen (N) sources were successfully acclimated to ex vitro conditions. The manipulation of N sources in adventitious rooting media can be a tool for improving new root density, length and branching in this species.