Endogenous nitric oxide and hydrogen peroxide detection in indole-3-butyric acid-induced adventitious root formation in Camellia sinensis

Nitric oxide（NO）and hydrogen peroxide（H2O2）are essential signaling molecules with key roles in auxin induced adventitious root formation in many plants.However,whether they are the sole determinants for adventitious root formation is worth further study.In this study,endogenous NO and H2O2 were monitored in tea cutting with or without indole-3-butyric acid（IBA）treatment by using the fluorescent probes diaminofluorescein diacetate（DAF-2DA）and 2＇,7＇-dichlorodihydrofluorescein diacetate（DCF-DA）,respectively.The overproduction of NO and H2O2 was detected in the rooting parts of tea cuttings treated with or without IBA.But little NO and H2O2 was detected before the initiation phase of tea cuttings even with IBA treatment indicating that they might be not directly induced by IBA.Further carbon and nitrogen analysis found that the overproduction of NO and H2O2 were coincident with the consumption of soluble sugars and the assimilation of nitrogen.These results suggest that rooting phases should be taken into consideration with the hypothesis that auxin induces adventitious root formation via NO-and H2O2-dependent pathways and sink establishment might be a prerequisite for NO and H2O2 mediated adventitious root formation.

Transport and Metabolism of the Endogenous Auxin Precursor Indole-3-Butyric Acid

Plant growth and morphogenesis depend on the levels and distribution of the plant hormone auxin. Plants tightly regulate cellular levels of the active auxin indole-3-acetic acid （IAA） through synthesis, inactivation, and transport. Although the transporters that move IAA into and out of cells are well characterized and play important roles in devel- opment, little is known about the transport of IAA precursors. In this review, we discuss the accumulating evidence sug- gesting that the IAA precursor indole-3-butyric acid （IBA） is transported independently of the characterized IAA transport machinery along with the recent identification of specific IBA efflux carriers and enzymes suggested to metabolize IBA. These studies have revealed important roles for IBA in maintaining IAA levels and distribution within the plant to support normal development.

Multi-walled carbon nanotube as a solid phase extraction adsorbent for analysis of indole-3-butyric acid and 1-naphthylacetic acid in plant samples

In this work, a new sample pretreatment method prior to HPLC separations was developed for the determination of auxins in plant samples, Owing to its large surface area and high adsorption capacity, multi-walled carbon nanotube （MWCNT） was chosen as the adsorbent for the extraction of auxins from plant samples. In this study, two important auxins were selected as model analytes, namely indole-3- butyric acid （IBA） and 1-naphthylacetic acid （NAA）. They could be extracted and concentrated due to their π-π stacking interactions with MWCNT. Then HPLC-UV was introduced to detect IBA and NAA after sample pretreatment. Factors that may affect the enrichment efficiency were investigated and optimized. Comparative studies showed that MWCNT was superior to CI 8 for the extraction of the two analytes. Validation experiments showed that the optimized method had good linearity （0.9998 and 0.9960）, high recovery （81.4%-85.4%）, and low detection limits （0.0030 mg/L and 0.0012 mg/L）. The results indicated that the novel method had advantages of convenience, good sensitivity, high efficiency, and it was feasible for the determination of auxins in plant samples.

Effect of Indole 3-Butyric Acid and Media Type on Adventitious Root Formation in Sheanut Tree (<i>Vitellaria paradoxa</i>C. F. Gaertn.) Stem Cuttings

Shea nuts play an important role in food security for rural folks within sub-Sahara Africa, serving as the main source of income for many people living in Northern Ghana. Unfortunately, the full economic potential of the Sheanut tree has not been fully realized due to the difficulty involved in its domestication. This difficulty in vegetatively propagating sheanut trees has greatly hindered its cultivation and the realization of its true economic potential. Two experiments were conducted to investigate the effects of rooting media and varying indole 3-butyric acid (IBA) concentrations on adventitious root formation in cuttings taken from coppiced sheanut trees. Results indicated that 3000 ppm produced significantly (p 0.05) better rooting (57.5%) than 5000 ppm (30%), 7000 ppm (45.0%) and the control (7.5%). Although the levels of soluble sugars (SS) and total free phenols (TFP) in the cutting were significantly (p 0.05) higher at the end of the experiment (after IBA treatment) compared to the start (prior to IBA treatment), the SS and TFP trends observed did not clearly explain the rooting differences found between the IBA levels investigated. Callus formation was significantly (p 0.05) higher (35.0%) in the control (no IBA). Generally, callus formation decreased with increasing IBA concentration. In the rooting media experiment, rooting was significantly (p 0.05) higher in the rice husk medium (35.0%) compared to that in the palm fiber (18.3%), saw dust (14.1%) and top soil (16.7%) media.

Effect of Different Indole-3-Buteric Acid(IBA)Concentrations and Cutting Types on Plane Trees(Platanus orientalis L.)

Effects of different Indole-3-Buteric Acid （IBA） concentrations （0, 1 000, 1 500 and 2 500 ppm） and curing types （soft wood, semi hard and hard wood） of plane tree were investigated with Randomized Complete Block Design （RCBD） at the Agricultural research station, Charsadda. The maximum survival percentage, plant height （cm）, root length （era）, numbers of roots and root weight per cutting were significantly higher in hard wood cuttings. IBA concentration had no speculative effect on plane tree cuttings. Thus, hard wood cutting was the best choice for plan tree propagation.

Lateral root elongation in maize is related to auxin synthesis and transportation mediated by N metabolism under a mixed NO_(3)^(–) and NH_(4)^(+) supply

A mixed nitrate (NO_(3)^(–)) and ammonium (NH_(4)^(+)) supply can promote root growth in maize (Zea mays),however,the changes in root morphology and the related physiological mechanism under different N forms are still unclear.Here,maize seedlings were grown hydroponically with three N supplied in three different forms (NO_(3)^(–)only,75/25 NO_(3)^(–)/NH_(4)^(+)and NH_(4)^(+)only).Compared with sole NO_(3)^(–)or NH_(4)^(+),the mixed N supply increased the total root length of maize but did not affect the number of axial roots.The main reason was the increased total lateral root length,while the average lateral root (LR) length in each axle was only slightly increased.In addition,the average LR density of 2nd whorl crown root under mixed N was also increased.Compared with sole nitrate,mixed N could improve the N metabolism of roots (such as the N influx rate,nitrate reductase (NR) and glutamine synthase (GS)enzyme activities and total amino content of the roots).Experiments with exogenously added NR and GS inhibitors suggested that the increase in the average LR length under mixed N was related to the process of N assimilation,and whether the NR mediated NO synthesis participates in this process needs further exploration.Meanwhile,an investigation of the changes in root-shoot ratio and carbon (C) concentration showed that C transportation from shoots to roots may not be the key factor in mediating lateral root elongation,and the changes in the sugar concentration in roots further proved this conclusion.Furthermore,the synthesis and transportation of auxin in axial roots may play a key role in lateral root elongation,in which the expression of ZmPIN1B and ZmPIN9 may be involved in this pathway.This study preliminarily clarified the changes in root morphology and explored the possible physiological mechanism under a mixed N supply in maize,which may provide some theoretical basis for the cultivation of crop varieties with high N efficiency.

Synthesis,Spectroscopic Characterization,Thermogravimetric and Biological Activity Evaluation of Te(Ⅳ),Se(Ⅳ),V(Ⅲ),Nb(Ⅴ),Ta(Ⅴ)Complexes With Indole-3-Acetic Acid Plant Hormone Ligand

Te(Ⅳ),Se(Ⅳ),V(Ⅲ),Nb(Ⅴ)and Ta(Ⅴ)complexes of indole-3-acetic acid(IAAH)ligand were synthesized,characterized by elemental analysis and various spectroscopic techniques like,IR,1H-NMR,X-ray powder diffraction,UV-Visible,thermogravimetry analysis,magnetic measurements,molar conductance and surface morphology using SEM.All the synthesized complexes of IAAH ligand have 1∶2 stoichiometry of the types[Te(IAA)2(NH 3)2]·2Cl(Ⅰ),[Se(IAA)2(NH 3)2]·2Cl(Ⅱ),[V(IAA)2(NH 3)(Cl)](Ⅲ),[Nb(IAA)2(Cl)3](Ⅳ),and[Ta(IAA)2(Cl)3](Ⅴ).Spectral analysis indicates octahedral geometry for the Te(Ⅳ),Se(Ⅳ)and V(Ⅲ)complexes,whereas both Nb(Ⅴ)and Ta(Ⅴ)have a seven-coordination.The bonding sites are the oxygen atoms of carboxylate group for the deprotonated indole-3-acetic acid(IAA)ligand.The thermogravimetry analysis studies gave evidence for the presence of other coordinated molecules(Cl or NH 3)in the composition of IAA complexes,which were further supported by IR and micro analytical measurements.The higher molar conductance data of tellurium and selenium(Ⅳ)complexes reveal that these chelates are electrolytes,while low conductivity values for the vanadium(Ⅲ),niobium and tantalum(Ⅴ)chelates indicated a non-electrolytes.To test the antibacterial property of the five complexes in this study,four bacterial strains Klebsiella(G-),Escherichia coli(G-),Staphylococcus aureus(G+)and Staphylococcus epidermidis(G+)were used in the investigation.The effects of the five complexes in the cytotoxicity of Caco-2 and Mcf-7 human cancer cell lines were studied Neutral red uptake assay for the estimation of cell viability/cytotoxicity protocol.

The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

The difference of sensitivity to indole- 3-acetic acid （ IAA ） combined with horseradish peroxidase （HRP） in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTF assay. The cell cycle and apoptosis of K562 and BXPC-3 cells were examined by a fluorescence flow cytometer （FCM） and terminal deoxynacleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC-3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

Amperometric Determination of lndole-3-acetic Acid Based on Platinum Nanowires and Carbon Nanotubes

Platinum nanowire （PtNW） can be grown by electrodeposition in polycarbonate membrane, with the average diameter of the nanowires about 250 nm. The PtNW and multiwalled carbon nanotubes （CNT） are then dispersed into chitosan （CHIT） solution. The resulting PtNW-CNT-CHIT material brings new capabilities for electrochemical devices by using the synergistic action of the electrocatalytic activity of PtNW and CNT. By dropping the PtNW-CNT-CHIT film onto the glassy carbon （GO） electrode surface, and after evaporation an amperometric sensor for the determination of indole-3-acetic acid （IAA） was developed. The oxidation current of IAA increased significantly at the PtNW-CNT-CHIT film coated GC electrode, in contrast to that at the CNT-CHIT modified GC. The linear response of the sensor is from 50 ng/ml to 50 μg/ml with a detection limit of 25 ng/mL.

Rooting of Stem Cuttings with Different Indole 3 Butyric Acid (IBA) Treatments and Development of Micropropagation Protocol for <i>Piper betle</i>L. Node Culture

The present study, conducted during 2016 and 2017 seasons, aimed to investigate the effect of IBA on rooting of Piper betle L. stem cuttings (softwood and semi-hardwood). The experiment was undertaken in misting house field 2 UPM using the sand media to determine the adventitious roots initiation and development using the histological method. The cuttings were treated with different IBA concentrations (0, 500, 1000, 1500 and 2000 mg/L). The nodes explants were used in the development of a protocol for in vitro propagation of P. betle L., with different concentrations of Clorox with different times of immersion (20% Clorox 10 minutes, 30% Clorox 10 minutes, 20% Clorox 20 minutes, and 30% 20 minutes). In multiplication of the plantlets, Murashige and Skoog (MS) medium with different concentrations of BAP (0, 0.5, 1.0, 2.0 mg/L) were used to investigate the rooting of the explants. The results indicated that the types of the cuttings were different in the rooting capacity and the length of the roots. Moreover, it was found that in comparison with the control treatment, by a rise in the concentrations of the IBA, there was a significant upsurge in the rooting percentage, the root diameter, and the number of the roots. The results indicated that the types of cutting with 1000, 1500 and 2000 mg/L IBA perform better in the root percentage (100%) in the semi hardwood cuttings. The best results, however, were 2000 mg/L IBA in the semi hardwood cuttings, with the number of the roots to be 35.05, and the fresh weight of the roots to be 3.94 g, the dry weight of the roots to be 0.33 g, the length of the roots to be 391.88 cm, the roots diameter to be 1.21 mm, the surface area of the roots to be 121.83 cm2, and the root volume to be 2.99 cm3. Nonetheless, the optimal concentration of Clorox with the time immersion was 20% with the 20-minute immersion time, which produced a shoot induction percentage of 30% dead explants and a mean number of 70.00 shoots per explant and the optimal concentration of benzylaminopurine (BAP) at 1.0 mg/L. It is of note that a shoot induction percentage of 22.29% and a mean number of 4.1% number of auxiliary bud per treatment. P. betle shoots in MS medium without PGR MS (0.0) yielded a good rooting.

The auxin transporter OsAUX1 regulates tillering in rice(Oryza sativa)

Tillering is an important agronomic trait of rice(Oryza sativa)that affects the number of effective panicles,thereby affecting yields.The phytohormone auxin plays a key role in tillering.Here we identified the high tillering and semi-dwarf 1(htsd1)mutant with auxin-deficiency root characteristics,such as shortened lateral roots,reduced lateral root density,and enlarged root angles.htsd1 showed reduced sensitivity to auxin,but the external application of indole-3-acetic acid(IAA)inhibited its tillering.We identified the mutated gene in htsd1 as AUXIN1(OsAUX1,LOC_Os01g63770),which encodes an auxin influx transporter.The promoter sequence of OsAUX1 contains many SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)binding sites,and we demonstrated that SPL7 binds to the OsAUX1 promoter.TEOSINTE BRANCHED1(OsTB1),a key gene that negatively regulates tillering,was significantly downregulated in htsd1.Tillering was enhanced in the OsTB1 knockout mutant,and the external application of IAA inhibited tiller elongation in this mutant.Overexpressing OsTB1 restored the multi-tiller phenotype of htsd1.These results suggest that SPL7 directly binds to the OsAUX1 promoter and regulates tillering in rice by altering OsTB1 expression to modulate auxin signaling.

Differences of Plasma Levels of Tryptophan, Serotonin, 5-Hydroxyindole Acetic Acid, and Kynurenine between Healthy People and Patients of Major Monopolar Depression at Various Age and Gender

Background: It is not well analyzed whether there are differences in plasma levels of tryptophan (TRP) metabolites between healthy control people (HC) and patients of major monopolar depression (MMD). Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: There are no significant differences between plasma levels of TRP between HC and MMD. Plasma levels of TRP of HC are higher in young men, young women, old men, and old women in this order. Serotonin (5-HT) levels are higher in MMD than HC. Plasma levels of 5-HIAA of HC are also higher than those of patients of MMD. Plasma levels of kynurenine (KYN) of healthy old men and old women are higher than those of young men and old women. Plasma levels of KYN are higher in old women and young men of MMD than those of HC. Conclusion: Plasma levels of 5-HT are higher in patients of MMD than those of HC, which may suggest that use of drugs inhibiting the 5-HT transportation may increase plasma levels of 5-HT in MMD.

Synthesis, Reactions and Antimicrobial Activity of Some New 3-Substituted Indole Derivatives

Reaction of indole-3-carboxaldehydes 4 with hydrazine derivatives and different substituted acid hydrazides afforded the corresponding hydrazine derivatives 5a-c and acid hydrazide derivatives 7-11 respectively. Condensation of indole-3-carboxaldehydes 4 with phenacyl bromide and thiourea gives 1,3-thiazol-2-amine derivative 18. On the other hand, reaction 4 with 3-acetylchromene-2-one afforded chalcone derivative 19. Compound 4 undergoing Knoevenagel condensation with cyanoacetamide, ethyl cyanoacetate, benzimidazol-2-ylacetonitrile, rhodanine-3-acetic acid, 2,3-dihydropyrimidin-4-one derivative and 2,4-dihydropyrazol-3-one afforded the compounds 20a,b, 22, 23, 27 and 28 respectively. The structure of the newly synthesized compounds has been confirmed by elemental analysis and spectra data. The antimicrobial activities of the some newly synthesized compounds were measured and showed that most of them have high

Effects of Zinc Oxide Particles with Different Sizes on Root Development in Oryza sativa

Given the consistent release of zinc oxide(ZnO)nanoparticles into the environment,it is urgent to study their impact on plants in depth.In this study,grains of rice were treated with two different concentrations of ZnO nanoparticles(NP-ZnO,10 and 100 mg/L),and their bulk counterpart(B-ZnO)were used to evaluate whether ZnO action could depend on particle size.To test this hypothesis,root growth and development assessment,oxidative stress parameters,indole-3-acetic acid(IAA)content and molecules/enzymes involved in IAA metabolism were analyzed.In situ localization of Zn in control and treated roots was also performed.Though Zn was visible inside root cells only following nanoparticle treatment,both materials(NP-ZnO and B-ZnO)were able to affect seedling growth and root morphology,with alteration in the concentration/pattern of localization of oxidative stress markers and with a different action depending on particle size.In addition,only ZnO supplied as bulk material induced a significant increase in both IAA concentration and lateral root density,supporting our hypothesis that bulk particles might enhance lateral root development through the rise of IAA concentration.Apparently,IAA concentration was influenced more by the activity of the catabolic peroxidases than by the protective action of phenols.

Indole-3-propionic Acid-aggravated CCl_(4)-induced Liver Fibrosis via the TGF-β1/Smads Signaling Pathway

Background and Aims:The pathogenesis of liver fibrosis involves liver damage,inflammation,oxidative stress,and intestinal dysfunction.Indole-3-propionic acid(IPA)has been demonstrated to have antioxidant,anti-inflammatory and anticancer activities,and a role in maintaining gut homeostasis.The current study aimed to investigate the role of IPA in carbon tetrachloride(CCl4)-induced liver fibrosis and explore the underlying mechanisms.Methods:The liver fibrosis model was established in male C57BL/6 mice by intraperitoneal injection of CCl4 twice weekly.IPA intervention was made orally(20 mg/kg daily).The degree of liver injury and fibrosis were assessed by serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and histopathology.Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction(qPCR)were used to detect the inflammatory cytokines.The malondialdehyde(MDA),glutathione,glutathione peroxidase,superoxide dismutase,and catalase were determined via commercial kits.Hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.The expression of mRNA and protein was assayed by qPCR,Western blotting,or immunohistochemical staining.Results:After IPA treatment,the ALT and AST,apoptotic cells,and pro-inflammatory factor levels were enhanced significantly.Moreover,IPA intervention up-regulated the expression of collagen I,α-smooth muscle actin,tissue inhibitor of matrix metalloproteinase-1,matrix metalloproteinase-2,transforming growth factor-β1(TGF-β1),Smad3,and phosphorylated-Smad2/3.Additionally,IPA intervention did not affect the MDA level.Attractively,the administration of IPA remodeled the gut flora structure.Conclusions:IPA aggravated CCl_(4)-induced liver damage and fibrosis by activating HSCs via the TGF-β1/Smads signaling pathway.

Arabidopsis Acetyl-Amido Synthetase GH3.5 Involvement in Camalexin Biosynthesis through Conjugation of Indole-3-Carboxylic Acid and Cysteine and Upregulation of Camalexin Biosynthesis Genes

Camalexin （3-thiazol-2＇-yl-indole） is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile （IAN） is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Ara- bidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid （ICA） and cysteine （Cys） and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate （ICA（Cys）） in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid （DHCA）, the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1DIpad3-1, suggesting that ICA（Cys） could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA（Cys）, and upregulation of the major biosynthetic pathway genes.

Sulfur nutrient availability regulates root elongation by affecting root indole-3-acetic acid levels and the stem cell niche

Sulfur is an essential macronutrient for plants with numerous biological functions. However, the influence of sulfur nutrient availability on the regulation of root development remains largely unknown. Here, we report the response of Arabidopsis thaliana L. root development and growth to different levels of sulfate, demonstrating that low sulfate levels promote the primary root elongation. By using various reporter lines, we examined in vivo IAA level and distribution, cell division,and root meristem in response to different sulfate levels.Meanwhile the dynamic changes of in vivo cysteine, glutathione,and IAA levels were measured. Root cysteine, glutathione, and IAA levels are positively correlated with external sulfate levels in the physiological range, which eventually affect root system architecture. Low sulfate levels also downregulate the genes involved in auxin biosynthesis and transport, and elevate the accumulation of PLT1 and PLT2. This study suggests that sulfate level affects the primary root elongation by regulating the endogenous auxin level and root stem cell niche maintenance.

One-step co-electrodeposition of graphene oxide doped poly(hydroxymethylated-3,4-ethylenedioxythiophene) film and its electrochemical studies of indole-3-acetic acid

A novel graphene oxide （GO） doped poly（hydroxymethylated-3,4-ethylenedioxythiophene） （PEDOTM） film has been achieved via one-step co-electrodeposition and utilized for electrochemical studies of indole-3-acetic acid （IAA）. The incorporation of GO into PEDOTM film facilitated the electrocatalytic activity and exhibited a favorable interaction between the PEDOTM/GO film and the phytohormone during the oxidation of IAA. Under optimized conditions, differential pulse voltammetry and square wave voltammetry were used for the quantitative analysis of IAA, respectively, each exhibiting a wide linearity range from 0.6 μmol L-1 to 10 μmol L-1 and 0.05 μmol L-1 to 40 μmol L-1, good sensitivity with a low detection Iimit of 0.087 μmol L-1 and 0.033μmol L T, respectively, as well as good stability. With the notable advantages of a green, sensitive method, expeditious response and facile operation, the as-prepared PEDOTM/GO organic-inorganic composite film provides a promising platform for electrochemical studies of IAA.

Low temperature,IBA concentrations and optimal time for adventitious rooting of Eucalyptus benthamii mini-cuttings

Eucalyptus benthamii is a forest species of economic interest that has difficulty with seed production and also is considered to have difficulty with adventitious rooting using propagation techniques, such as cutting or mini-cutting. We aimed to assess the adventitious rooting percentage under different storage times in low temperatures and at various IBA （indole-3-butyric acid） concentrations to determine the optimal time of permanence for rooting Eucalyptus benthamii minicuttings in a greenhouse. Shoots collected from mini-stumps cultivated in a semi-hydroponic system were used to obtain the mini-cuttings. For the first experiment, the mini-cuttings were stored at 4℃ for 0 （immediate planting）, 24, 48, 72, 96 and 120 h. The second experiment evaluated the rooting dynamic to determine the optimal time of permanence for minicuttings in a greenhouse. The basal region of the mini-cutting was treated with various IBA solutions： 0 （free of IBA）, 1,000, 2,000, 3,000 and 4,000 mg.L^-1. Every seven days （0 （immediate planting）, 7, 14, 21 and 28 days）, destructive sampling of the mini-cuttings was performed to evaluate the histology of the adventitious rooting. Eucalyptus benthamii minicuttings should be rooted immediately after the collection of the shoots. The 2,000 mg.L^-1 IBA concentration induced a greater speed and percentage of adventitious rooting, and an interval of 35 to 42 days was indicated for permanence of the mini-cuttings in the greenhouse. Expo- sure to low temperature induced adventitious root formation with diffuse vascular connections.

In <i>Vitro</i>Clonal Propagation from Adult Material of a Savannah Species of Socio-Economic Importance: <i>Annona senegalensis</i>Pers.

Annona senegalensis (Pers.) or Annone from Senegal is a species found in the West African savannah and para-littoral sands. It offers real possibilities of socio-economic use, particularly in the medical, nutritional, ecosystem conservation and poverty alleviation fields. However, this wild species remained not fully exploited despite its potentialities. As a result, there is a need to regenerate this species;therefore, the in vitro propagation from adult material was undertaken. For this purpose, axillary nodes from plant regrowth taken from elderly subjects were aseptically introduced into different media enriched or not with phytohormones. Among the 3 culture media tested, that of Murashige and Skoog (MS, 1962) was the most reactive and made it possible to develop a micropropagation protocol for adult material of A. senegalensis. Thus, the introduction of these explants in media containing cytokinins (BAP and Kinetin) significantly increased the reactivity compared to media without hormones. If the best average numbers of shoots (2.3) and nodes (5.3) are obtained in MS + BAP 2 mg·L−1, with a reactivity of 91.66%, the addition of 0.1 mg·L−1 NAA gave the best average length (8.25 cm) of vitroplants. An induction time of 3 days into darkness with IBA at 50 mg·L−1, followed by a transfer to hormone-free expression medium (MS/2) under light, resulted in a rooting rate of 58.33%. After the gradual weaning of the young plants in a mini-greenhouse, under shade for 3 weeks, 50% of the plants survived. This in vitro regeneration protocol can therefore be adopted for the clonal propagation of adult material of A. senegalensis. However, the rooting rate can be improved by trying other hormonal combinations based on auxins or by considering ex-vitro rooting during shade weaning.