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.

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.

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.

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.

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.

<i>In Vitro</i>Clonal Propagation from Juvenile and Different Explant Types of Two Edible <i>Annonaceae</i>Species: <i>Annona muricata</i>L. and <i>Annona squamosa</i>L.

Annona muricata L. and Annona squamosa L. are tropical species whose fleshy fruit is edible. They offer real possibilities for socio-economic use, particularly in the fields of medicine, nutrition, ecosystem conservation and the poverty alleviation. This study was set up to evaluate different methods of micropropagation from juvenile material for the regeneration of these species. Thus, MS medium supplemented with [BAP 2 mg·L-1] i.e. M2 produced 2.87 newly formed shoots from the cotyledonary nodes of A. muricata. For the terminal apices of A. squamosa, it was MMS medium supplemented with [BAP 2 mg·L-1] i.e. MM2 that was most conducive to new shoot formation (3.12). The addition of 0.1 and 0.2 mg·L-1 of NAA in the M2 medium, made it possible to have the best elongations and average number of nodes for the new shoots from cotyledonary nodes of A. muricata (9.11 cm for 5.62 nodes). With A. squamosa, MM7 medium [MMS + BAP 1 mg·L-1 + KIN 1 mg·L-1] resulted in an average length of 9.05 cm with 5.62 nodes on average for the apical shoots. A 3-day rhizogenic induction period in the dark with [IBA 50 mg·L-1] and 2 g·L-1 of activated charcoal gave a rooting rate of 66.67% for shoots originating from the cotyledonary nodes of A. squamosa;while with vitroplants from cotyledonary nodes of A. muricata, a better rooting rate (83.33%) was obtained following a 5-day rhizogenic induction. After 30 days of acclimatization, the survival rate reached 83.33% for plants from the tips of A. muricata, whereas for A. squamosa, it was plants grown from cotyledonary nodes that had the same survival rate.

Micropropagation from Juvenile Material of Annona senegalensis Pers.

Annona senegalensis is a savannah species present in the wild in tropical and sub-tropical African regions. It is used by the local populations for its many economic, pharmacological, nutritional and ecological interests. However, the strong pressure exerted on this species as well as the increasing degradation of the ecosystems, in which it evolves, seriously compromise its sustainability and development. Conventional methods of vegetative propagation are not sufficient to ensure its durable regeneration and the renewal of endemic populations in Senegal. In this context, it was undertaken in vitro propagation, from 30 days old juvenile seedlings. Thus, axillary, cotyledonary and apical nodes were in vitro cultured in Murashige and Skoog (1962) medium (MS), with different concentrations of cytokinins and/or auxins. BAP used alone at 2 mg•L−1 proved to be more beneficial for micropropagation of different types of explants compared to Kinetin used alone or in combination with BAP, especially for axillary and cotyledonary nodes. The BAP-NAA combination allowed to have a better elongation of newly formed shoots. For shoots of apical and cotyledonary nodes origin, a rhizogenic induction of 5 days, with IBA at 25 mg•L−1, resulted in a better rooting rate with, respectively, 75% (for 2.22 roots) and 66.67% (for 4.17 roots). For newly formed shoots of axillary origin, a 24-hour rooting induction with IBA at 50 mg•L−1 gave a rooting rate of 58.33% (for 2.4 roots). After 30 days of acclimatization, the survival rate was 75% for the young plants from the apices and 83.33% for those originating from the cotyledonary and axillary nodes. This protocol can therefore be adopted for the in vitro clonal propagation of A. senegalensis juvenile material.