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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.

[Resumen] El estudio realizado durante el crecimiento y desarrollo de plantas de Moringa incluyó la caracterización bioquímica de sus órganos a partir de extractos metanólicos y acuosos, observándose que las hojas de plantas en fase vegetativa y floral son ricas en nitrógeno, contenido de fenoles solubles y actividad antioxidante. Para evaluar la actividad fortificante de esta planta se prepararon diferentes concentraciones de extractos acuosos procedentes de fases vegetativa y floral y se aplicaron a vitroplantas de banano clon Williams que habían sido aclimatadas durante seis semanas. A los 21 días de la aplicación se obtuvieron plantas fortificadas con órganos de mayor crecimiento morfológico, contenido de fenoles solubles y actividad antioxidante. En las hojas se obtuvo mayor contenido de clorofila a, clorofila b, así como una menor fuga de electrolitos de membranas celulares. Para la optimización del cultivo in vitro de banano se utilizaron plantas de banano locales brotadas en invernadero, lo que facilitó el establecimiento de cultivos asépticos e incrementó la capacidad de proliferación. El uso de sistemas de inmersión temporal RITA® aumentó la eficiencia de la multiplicación in vitro. Este sistema se utilizó para crear un prototipo local con contenedores y tapa de polipropileno que propició la producción de vitroplantas vigorosas cuando se añadió al medio de cultivo extracto acuoso de hojas de Moringa en fase floral., [Resumo] O estudo realizado durante o crecemento e desenvolvemento das plantas de Moringa incluíu a caracterización bioquímica dos seus órganos a partir de extractos metanólicos e acuosos, observandose que as follas das plantas en fase vexetativa e floral son ricas en nitróxeno, e teñen un elevado contido de fenois solubles, así como unha elevada actividade antioxidante. Para avaliar a actividade fortificante desta planta, preparáronse diferentes concentracións de extractos acuosos de follas procedentes de plantas en fases vexetativa e floral que se aplicaron a vitroplantas de banano clon Williams que levaban seis semanas aclimatadas. Vinte e un días despois da aplicación, obtivéronse plantas fortificadas, con órganos de crecemento morfolóxico máis elevados, altos contidos de fenois solubles soluble e alta actividade antioxidante. Nas follas obtívose un maior contido de clorofilas, así como unha menor fuga de electrólitos das membranas celulares. Para a optimización do cultivo in vitro de banano utilizáronse plantas de banano locais brotadas en invernadoiros, o que facilitou o establecemento de cultivos asépticos e aumentou a súa posterior capacidade de proliferación. O uso dos sistemas de inmersión temporal RITA® aumentou a eficiencia da multiplicación in vitro. Este sistema utilizouse para crear un prototipo local con recipientes e tapa de polipropileno que favoreceu a produción de vitroplantas vigorosas ao engadir ao medio de cultivo extracto acuoso de follas de Moringa en fase floral., [Abstract] The study carried out during the growth and development of Moringa plants included the biochemical characterization of their organs using methanolic and aqueous extracts. It was observed that leaves in the vegetative and floral phase are rich in nitrogen and have high soluble phenol content and antioxidant activity. To evaluate the fortifying activity of Moringa plants, different concentrations of aqueous extracts from the vegetative and floral phases were applied to banana vitro plants of the “Williams” clone that had been acclimated for six weeks. Twenty-one days after the extract application, treated plants showed more growth, higher soluble phenol content and higher antioxidant activity than controls, whereas leaves had higher content of chlorophylls and lower leakage of electrolytes from cell membranes. For the optimization of in vitro banana cultivation, local banana plant material was sprouted in a greenhouse, this facilitated the establishment of aseptic cultures and increased the proliferation capacity. The use of RITA® temporary immersion systems increased the efficiency of in vitro multiplication. This system was used to design and assemble a local prototype using polypropylene containers. Banana vitroplants cultured in this prototype showed more vigour when the culture medium was supplemented with aqueous Moringa extracts obtained from leaves of plants in the floral phase.

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] 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] 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] 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.

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 x 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.

A protocol has been developed for the propagation of Alnus glutinosa axillary shoots in liquid medium. The explants were cultured in Woody Plant Medium supplemented with 0.1 mg l− 1 benzyladenine and 0.5 mg l− 1 indole acetic acid. The effect of the bioreactor type (RITA® and Plantform™), frequency of the immersions, regulator concentrations, and volume of medium per explant was investigated. All the treatments with the temporal immersion systems (TIS) increased the proliferation rates. The best results were obtained on using the Plantform™ vessels and, unlike the shoots cultured in RITA® vessels, the culture in Plantform™ had very low hyperhydricity percentages. Shoots originating from the culture in semi-solid medium and in Plantform™ rooted in semi-solid medium with 0.1 mg/l indole butyric acid for 7 days. No significant differences were observed in the rooting or acclimatization percentages, with survival percentages greater than 85% being achieved. This is the first work on the use of TIS systems in alder propagation, with the results of this study providing new perspectives for its mass propagation.

The present study investigated how auxin concentration and the method of application affected the formation of adventitious roots in microshoots of chestnut (Castanea sativa) and oak (Quercus robur). The activity of two urea derivatives (2, 3-MDPU and 3, 4-MDPU) was also evaluated. Microshoots were derived from basal sprouts of two mature chestnut trees (P1 and P2) and one adult oak genotype (Sainza). In chestnut, rooting percentage was positively affected by auxin in a dose -dependent manner, particularly in shoots treated with the hormone for 24 h. The effect of auxin on rooting also differed depending on the application method. In shoots treated for 24 h, the highest concentration of auxin produced the healthiest rooted plantlets, in terms of the root system and shoot quality. By contrast, in shoots treated by the basal quick-dip method, the shoot quality was best at the lowest auxin concentration. The effect of urea derivatives on the root system depended on the species as well as on the auxin concentration and application period. Use of the MDPUs improved the root system architecture of auxintreated shoots by promoting lateral root development and triggering the synchronous initiation of root primordia at the base of the shoot. Shoot quality was also improved by MDPUs, which promoted resumption of growth and reduced shoot-tip necrosis.