Selection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the between-sample transcriptionally more stable genes, and subsequent ...choosing of the most suitable genes as internal controls. Both steps can be carried out through generally accepted strategies, each having different strengths and weaknesses. The present study proposes reinforcement of the normalization of gene expression data by integrating analytical revision at critical steps of those accepted procedures. In vitro olive adventitious rooting was used as an experimental system. Candidate RGs were ranked according to transcriptional stability according to several methods. An algorithm of one of these programs (GeNorm) was adapted to allow for partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to choose the more appropriate set of RGs, the achieved results were analytically revised, with special emphasis on biasing effects such as co-regulation. The obtained putative RG sets were also tested for cases restricted to fewer variables. The set formed by the genes H2B, OUB and ACT is valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays. Such internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.
•Olive may become a model species for studies on adventitious rooting.•Fundamental research on adventitious root formation is scarce.•Analytical methods are difficult to establish in woody ...perennials.•Studies must be redirected to specific tissues and cells.•Biochemical analyses are inefficient without concomitant genetic evaluation.
Olive (Olea europaea) is one of the most important fruit species in the Mediterranean basin, where 95% of the world’s olive orchards are planted, and it has become an economically valuable crop worldwide, due to an increasing interest in olive oil for human consumption. New olive orchards are being planted outside the Mediterranean, calling for an effort to identify the genotypes best adapted to the new conditions. However, some olive cultivars remain difficult to propagate, which significantly reduces the capacity to use the full genetic diversity of the species. Improving rooting ability in cuttings from recalcitrant olive cultivars has become a critical topic, which implies fundamental research on the anatomy, physiology, biochemistry and genetics of the adventitious root formation process. Besides, the existence of different rooting behaviors among olive cultivars also makes the species a candidate model plant for these studies. Olive propagation techniques evolved through time from field- or nursery-planted hardwood cuttings, to semi-hardwood cuttings in greenhouses under mist, and, more recently, to in vitro culture techniques. Nevertheless, research about adventitious root formation carried on each propagation method was mostly based on trial and error approaches. Researchers have mainly investigated different factors involved in the process of adventitious rooting by testing their effect in the rooting capacity of different cultivars, leading to a high dispersion and fragmentation of the available information. The goal of this review is to present the most relevant results achieved on adventitious root formation in olive cuttings, aiming to provide an integrated perspective of the current knowledge.
Exogenous auxins supplementation plays a central role in the formation of adventitious roots (AR) for several plant species. However, the molecular mechanisms underlying the process of adventitious ...rooting are still not completely understood and many plants with economic value, including several olive cultivars, exhibit a recalcitrant behavior towards cutting propagation, which limits its availability in plant nurseries. PIN-formed proteins are auxin efflux transporters that have been widely characterized in several plant species due to their involvement in many developmental processes including root formation. The present study profiled the expression of the
,
,
,
, and
gene members during indole-3-butyric acid (IBA)-induced in vitro adventitious rooting using the olive cultivar 'Galega vulgar'. Gene expression analysis by quantitative real time PCR (RT-qPCR) showed drastic downregulation of most transcripts, just a few hours after explant inoculation, in both nontreated and IBA-treated microcuttings, albeit gene downregulation was less pronounced in IBA-treated stems. In contrast,
showed a distinct expression pattern being upregulated in both conditions, and
was highly upregulated in IBA-induced stems. All transcripts, except
, showed different expression profiles between nontreated and IBA-treated explants throughout the rooting experiment. Additionally, high levels of reactive oxygen species (ROS) were observed soon after explant preparation, decreasing a few hours after inoculation. Altogether, the results suggest that wounding-related ROS production, associated with explant preparation for rooting, may have an impact on auxin transport and distribution via changes in
gene expression. Moreover, the application of exogenous auxin may modulate auxin homeostasis through regulation of those genes, leading to auxin redistribution throughout the stem-base tissue, which may ultimately play an important role in AR formation.
The PIN-FORMED (PIN) proteins represent the most important polar auxin transporters in plants. Here, we characterized the PIN gene family in two olive genotypes, the Olea europaea subsp. europaea ...var. sylvestris and the var. europaea (cv. ‘Farga’). Twelve and 17 PIN genes were identified for vars. sylvestris and europaea, respectively, being distributed across 6 subfamilies. Genes encoding canonical OePINs consist of six exons, while genes encoding non-canonical OePINs are composed of five exons, with implications at protein specificities and functionality. A copia-LTR retrotransposon located in intron 4 of OePIN2b of var. europaea and the exaptation of partial sequences of that element as exons of the OePIN2b of var. sylvestris reveals such kind of event as a driving force in the olive PIN evolution. RNA-seq data showed that members from the subfamilies 1, 2, and 3 responded to abiotic and biotic stress factors. Co-expression of OePINs with genes involved in stress signaling and oxidative stress homeostasis were identified. This study highlights the importance of PIN genes on stress responses, contributing for a holistic understanding of the role of auxins in plants.
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•Rooting ability is likely related with IBA-IAA conversion and oxidative enzymes activity.•Olive microcuttings and semi-hardwood cuttings present similar rooting behavior.•Molecular ...pathways potentially involved in olive adventitious rooting are described.•The potential involvement of AOX genes in rooting is exploited.•SHAM may be a good choice to obtain negative controls in rooting assays.
The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro-cultured microshoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid (SHAM)+IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the same time-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase (IAAox) was evaluated in the microshoots. Microshoots treated with SHAM+IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. In contrast, free IBA levels were higher in microshoots treated with SHAM+IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase (AOX), leading to a downregulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.
Several olive cultivars, characterized by high-quality olive oil show agronomical issues such as excessive vigor, high susceptibility to biotic and abiotic stresses, and low propagation ability. They ...are strong candidates for breeding based on new technologies to improve their performance in a short period of time. For this reason, the first step is developing efficient somatic embryogenesis (SE) protocols. Somatic embryogenesis in olive is highly genotype-dependent for both adult tissues and mature embryos as initial explants, requiring the development of specific protocols for each genotype. Trials using cotyledons and radicles as initial explants, isolated from ripe seeds from the Portuguese olive cv. ‘Galega vulgar’, gave more than 95% calli development. Radicles proved to be the most responsive tissue for SE induction, with an average of 2 embryos per callus after callus transfer to expression medium, and 14 embryos per callus after subculture on the olive cyclic embryogenesis medium (ECO). Embryogenic competence could be recovered after several subcultures on ECO medium that maintained cyclic embryogenesis for an indeterminate period of time. Embryo conversion and plant acclimatization were also attained with high success rates. Media management for cyclic embryogenesis maintenance is of general importance for SE protocols in any olive genotype. Somatic embryogenesis was thus attained for the first time in embryo-derived explants of cv. ‘Galega vulgar’.
Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative ...effects on olive oil quality. The work presented here describes the development and application of a new SYBR
Green-based real-time quantitative PCR (qPCR) analysis for specific and reliable quantification of highly spread olive tree viruses:
(OLV-1)
(TNV-D),
(OMMV), and
(OLYaV). qPCR methodology revealed high specificity and sensitivity, estimated in the range of 0.8-8 copies of the virus genome, for the studied viruses. For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. "Galega vulgar" from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR
Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.
In vitro micropropagation is already a current multiplication tool for walnut self-rooted cultivars and rootstocks, but walnut grafting is still performed in the field or in greenhouses, mainly using ...seedlings as rootstocks. The present work describes a new approach to obtain clonal walnut-grafted plants, involving in vitro shoot production of ‘Paradox’ (Juglans hindsi × Juglans regia) cl. ’Vlach’, to be used as rootstock, and J. regia cv. ‘Chandler’, to be used as scion. After completing the in vitro multiplication phase and a seven-day root induction treatment, ‘Vlach’ explants are transferred to ex vitro conditions for root expression while being simultaneously grafted using the in vitro produced ‘Chandler’ scions. The importance of the presence of leaves on both the scion and the rootstock for the success rate of the technique was evaluated. Under optimal conditions, average success rates of 82% for rootstock rooting, 72% for micrografting survival, and 84% for grafted plant acclimatization were achieved. This rooting/grafting combination technique seems able to compete with the traditional techniques of nursery grafting, allowing obtaining high-quality walnut-grafted plants independently of the external weather conditions in a significantly shorter time.
Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar "Galega vulgar". ...The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.
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