Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%–62.5%) compared to free IAA or IBA applications (50.0%–33.3%), except for 2.0 mg L^–1 IBA (66.7%). However, the application of 2 mg L^–1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.     

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.Abbreviations IAA Indole-3-acetic acid - IBA Indole-butyric acid - NAA A-naphthalene acetic acid     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

Interaction of paclobutrazol and indole-3-butyric acid in relation to rooting of mung bean (Vigna radiata) cuttings

Paclobutrazol (PB) only slightly stimulated the rooting of mung bean cuttings but, interestingly, the number of adventitious roots formed was dramatically increased when PB was used together with indole-3-butyric acid (IBA). Application of PB in the first phase of root formation, when root initials are induced, caused the greatest enhancement of the promotive effect of IBA on rooting. Investigation of the effect of PB on uptake, transport and metabolism of [5^-3H]-IBA in mung bean cuttings revealed some changes in the rate of metabolism of IBA in comparison with control cuttings. PB was found to be involved in the partitioning of carbohydrates along the cuttings. Application of sucrose, like PB to the base of IBA-treated cuttings enhanced the effect of IBA. The patterns of the effects of PB and IBA, separately and together, on rooting were similar in defoliated and intact cuttings, however the number of roots was much lower in the defoliated cuttings, which lacked a source of assimilates. PB counteracted the effect of GA₃ in the upper regions of the cuttings and seemed to increase the sink capacity at the base of the cuttings. The results of the present study clearly demonstrated the enhancing influence of PB on IBA stimulation of the rooting of mung bean cuttings. It is suggested that PB may affect the rate of metabolism of IBA during rooting and the status of the local sink, in the base of the cuttings, thus partially contributing to the enhancement of the rooting-promotive effect of IBA.     

Transport and metabolism of indole-3-butyric acid in cuttings of Leucadendron discolor

Indole-3-butyric acid (IBA) greatly enhanced the rooting of an early-flowering variety of protea, Leucadendron discolor, but had very little effect on a late-flowering variety. IBA transport and metabolism were studied in both varieties after incubating the cuttings in ³H-IBA. More of the radio-label was transported to the leaves of the easy-to-root variety than the difficult-to-root (35–45% and 10%, respectively). IBA was metabolized rapidly by the cuttings of both varieties and after 24 h most of the label was in the new metabolite. However, free IBA (about 10%) was present in the cuttings during the whole period up to the time of root emergence (4 weeks). More free IBA was accumulated in the base of easy-to-root cuttings, while in the difficult-to-root variety most of the IBA was found in the leaves. The metabolite was identified tentatively as an ester conjugate with a glucose. It is possible that IBA-glucose serves as a source for free IBA, and the difference between the varieties is a consequence of the free IBA which is released, transported and accumulated in the site of a root formation.     

NAA处理桉树插条后IAAO活性与生根的关系

尾叶桉MLA无性系(简称MLA)为难生根植物,尾叶桉U₆无性系(简称U₆)和刚果12号桉W₅无性系(简称W₅)为相对易生根植物。MLA插条内的吲哚乙酸氧化酶(IAAO)活性较U₆、W₅的高。用萘乙酸(NAA)处理桉树插条后,在扦插生根的不同阶段,插条内IAAO活性呈现规律性变化;可溶性蛋白质含量呈上升趋势。本文讨论了IAAO与桉树插条生根的关系。     

Plantlet multiplication from white pine (Pinus strobus L.) embryos in vitro: bud induction and rooting

White pine embryos were grown on 4 different media with 6 different benzyladenine (BA) concentrations. Maximum adventitious shoot initiation and growth were obtained on a modified Lepoivre medium with 20 M BA. Modified Schenk and Hildebrandt, Murashige and Skoog, and Gresshoff and Doy media were also tested. Shoot elongation was achieved on half-strength basal medium lacking growth regulators. Three rooting experiments involving indole-3-butyric acid (IBA), sucrose concentration, shoot orientation, IBA pulse length, and light or dark were carried out. Treatment of shoots in an upright position with 50 M IBA for eight days followed by culture in a medium with 3% sucrose in the light produced the most rooting (50% at 3 months). Rooted shoots were transplanted to the greenhouse for further growth.     

Maturation-related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity

A comparison of rooting ability of stem cuttings made from hypocotyls and epicotyls from 50-day-old seedlings of loblolly pine ( Pinus taeda L. ) reveals a dramatic decline by epicotyl cuttings, which do not root at all in 20–30 days in the presence or absence of auxin. In contrast, almost all the hypocotyls root during this time, but only in the presence of exogenously applied auxin. The failure of epicotyls to root does not appear to be due to differences in [¹⁴C]-labeled auxin uptake, transport, metabolism, or tissue distribution in the two types of cuttings. At the cellular level, initial responses to auxin, such as differentiation of the cambium into parenchyma, occur in both types of cuttings, but localized rapid cell division and root meristem organization are not observed in epicotyls. Autoradiography revealed that radioactivity from a -naphthalene acetic acid is bound in the cortex but not concentrated at sites of root meristem organization prior to the organization of the meristem in hypocotys. During the development of the epicotyl. cellular competence to form roots appears to be lost. Although this loss in competence is not associated with a concurrent loss in ability to transport auxin polarly, the latter process appears to play a key role in rooting other than to move auxin to the site of root formation. The phytotropin N-(1-naphthyl)phthalamic acid inhibits rooting if applied during the first 3 days after the cutting is made, but does not affect auxin concentration or metabolism at the rooting site.     

Synergistic effects of plant growth retardants and IBA on the formation of adventitious roots in hypocotyl cuttings of mung bean

The synergistic effect of plant growth retardants, such as daminozide, paclobutrazol and triadimefon, and of indole-3-butyric acid (IBA) on the formation of adventitious roots in hypocotyl cuttings of mung bean was studied. The three retardants and IBA all stimulated adventitious root growth, but IBA was the most effective. However, mixtures of the retardants with IBA have proven generally more effective than IBA alone in promoting adventitious root formation. When IBA was applied to the hypocotyls one day after cutting preparation followed by the growth retardant on the second day, there were even more adventitious roots produced than if applied in the reverse order. The effectiveness of the treatments were in the order, IBA followed by growth retardant, IBA + growth retardant together, and IBA alone.Abbreviations IBA indole-3-butyric acid - GA gibberellin     

Differences in endo/exogenous auxin profile in cuttings of different physiological ages

The process of physiological ageing in woody plants is a very important factor influencing adventitious rooting. However, there is a lack of knowledge of biochemical backgrounds triggering ageing and consequently, rhizogenesis. Experiments with Prunus subhirtella ‘Autumnalis’ leafy cuttings of three different physiological ages (adult (over 40-year-old stock plants), semi-adult (5-year-old cutting plants) and juvenile (5-year-old in vitro plants)) were conducted in 2009. Half of the cuttings were banded ca. 3 cm above the bottom of the cutting with aluminum wire prior to insertion into the substrate to block the polar auxin transport. IBA, which was exogenously applied to the cuttings, could only be detected in the base of the cuttings on the first day after severance. Juvenile cuttings tended to have the highest values, but the effect was age specific. Later, the detection was not possible, regardless of the age. The IAA profile in cutting bases was similar for all physiological ages, reaching the peak on the first day after severance. Juvenile cuttings, in which the stems had been banded before insertion, contained more IAA in their bases on day 1 compared to the stems, which were not banded. These cuttings presumably transported absorbed auxin mainly via phloem, and not via mass flow like semi-adult and adult cuttings, where IAA concentrations were similar or even greater in non-banded cuttings compared to banded ones. These cuttings also tended to exhibit the best rooting results. The IAA-Asp accumulation was especially strong in adult cuttings, which contained significantly more aspartate on the first and third days after severance when compared with semi-adult and juvenile cuttings.     

吲哚丁酸对桉树插条多酚氧化酶的影响及其与生根的关系

尾叶桉MLA无性系(简称MLA)为难生根植物,尾叶桉U6无性系(简称U6)和刚果12号桉W5无性系(简称W5)为易生根植物。MLA插条内的PPO活性比U6、W5的低。用吲哚丁酸(IBA)处理桉树的插条后,在扦插生根的不同阶段,插条内的PPO活性呈现规律性的变化。蛋白质含量呈上升趋势。PPO同工酶谱带也随生根的进程出现增多现象。讨论了多酚氧化酶与桉树插条生根的关系。     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

Initiation of roots on hypocotyl cuttings of Pinus contorta in vitro

The origin of roots and wound tissue after treatments for induction of roots on hypocotyl cuttings of three-week-old Pinus contorta Dougl. ex Loud, is discussed. The cuttings were cultured in vitro and treated with 1.2 μ M to 1.5 M IBA (indole-3-butyric acid) for 6 h to 10 days. The control, which was not treated with IBA developed a wound tissue from which roots formed. Cuttings treated with IBA developed roots directly from the hypocotyl. Direct rooting was faster than indirect rooting via a wound tissue. Rooting was considered to be optimal if more than 80% of the cuttings rooted within 19 days and half of the cuttings which possessed roots after one month had acquired them within 14 days. This type of rooting was obtained after treatment with either 80 μ M IBA for 4 to 6 days or 1.25 to 5.0 m M IBA for 6 h. Suboptimal treatments gave lower rooting percentages and superoptimal treatments resulted in delayed rooting. In IBA-treated cuttings, large increases in mitotic activity (number of mitoses per mm hypocotyl) were found in the pericycle and parenchyma inside endodermis. However, the control also had similar mitotic activities as the IBA-treated cuttings but closer to the cut surface. This led us to the conclusion that similar tissues may produce either wound tissue or roots. Almost all roots obtained through direct rooting originated outside resin ducts.     

Endogenous levels of abscisic acid and indole-3-acetic acid during in vitro rooting of Wild Cherry explants produced by micropropagation

Levels of endogenous ABA and IAA were quantified during the first week of in vitro rooting of Wild Cherry (Prunus avium L.) using IBA in the culture medium. Hormones were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme linked immunosorbent assay (ELISA), after a purification of the methanolic extracts by high-performance liquid chromatography (HPLC).Root primordia started to differentiate from day 5 at the basal part of the explants. ABA and IAA showed considerable changes and high levels were detected during the first week of culture. ABA levels increased transiently mainly in the apical part during root formation. Exogenous IBA was possibly transformed into IAA mainly in the basal part of the explants.     

Improved in vitro rooting of Prunus dulcis Mill. cultivars

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47–100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm⁻³ gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.     

RNA and protein metabolism during adventitious root formation in stem cuttings of Phaseolus aureus

Indole-3-butyric acid (IBA, 10⁻⁴ M ), spermine (7 × 10⁻⁵ M ) and vitamin D₂ (6.3 × 10⁻⁵ M ), all of which enhance rooting in mung bean cuttings ( Phaseolus aureus Roxb. cv. Berkin), influence RNA metabolism. Total and poly (A)⁺-RNA synthesis within the hypocotyl is inhibited by each of these chemicals within 24 h. These changes precede induced cell division and are therefore associated with the so-called inductive period of regeneration during which some cells in the hypocotyl undergo dedifferentiation. However, following subsequent transfer of cuttings to borate, which is an essential prerequisite for development of root primordia in these cuttings, RNA synthesis is enhanced by pretreatments with IBA, spermine or vitamin D₂. Furthermore, IBA inhibits synthesis and turnover of protein within the hypocotyl.     

Endogeneous levels of indole-3-acetic acid and abscisic acid during the rooting of Cotinus coggygria cuttings taken at different times of the year

Cuttings of Cotinus coggyria cv Royal Purple rooted well in the spring but not at all later in the season. Levels of free and conjugated IAA and ABA were measured in cuttings taken at different times of the year. Hormones were measured in the leaf, the upper stem and the lower stem (rooting zone). In cuttings taken in early June the level of IAA was much higher than that of conjugated IAA. In late July the opposite was found. No significant differences in ABA levels were found although the ABA/IAA ratio changed dramatically.