Influence of indole-butyric acid and electro-pulse on in vitro rooting and development of olive (<Emphasis Type="Italic">Olea europea</Emphasis> L.) microshoots

The effects of indole-butyric acid (IBA) and electro-pulses on rooting and shoot growth were studied in vitro, using olive shoot cultures. Tested shoots were obtained from seedlings belonging to three Spanish cultivars, ‘Arbequina’, ‘Manzanilla de Sevilla’ and ‘Gordal Sevillana’, which have easy-, medium- and difficult-to-root rooting abilities, respectively. The standard two-step rooting method (SRM), consisting of root induction in olive rooting medium supplemented with 0, 0.1 or 1 mg/l IBA followed by root elongation in the same rooting medium without IBA, was compared with a novel one-step method consisting of shoot electro-pulses of 250, 1,250 or 2,500 V in a solution of IBA (0, 0.1 or 1 mg/l) and direct transferral to root elongation medium. The rooting percentage of the seedling-derived shoots obtained with the SRM was 76% for ‘Arbequina’ and ‘Gordal Sevillana’ cultivars and 100% for ‘Manzanilla de Sevilla’ cultivar, whereas with the electro-pulse method, the rooting percentages were 68, 64 and 88%, respectively. IBA dipping without pulse produced 0% rooting in ‘Arbequina’ seedling-derived shoots. The electroporation in IBA not only had an effect on shoot rooting but also on shoot growth and development, with longer shoots and higher axillary shoot sprouting and growth after some of the treatments. These effects were cultivar-dependent. The electro-pulse per se could explain some of these effects on shoot development. I.M.G. Padilla and I. Vidoy contributed equally to this article.     

In vitro rooting of almond (Prunus dulcis Mill.)

Summary Shoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Effects of combinational treatment with ethephon and indole-3-butyric acid on adventitious rooting of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> cuttings

Adventitious rooting is essential for cutting propagation of pine wilt-resistant Pinus thunbergii. To examine a variety of adventitious rooting potentials among donor plants, cuttings were taken from 31 seedlings within a half-sib family. Rooting abilities of cuttings from each seedling ranged from 0 to 100%. When 11 ortets and 11 ramets (clonally propagated from each ortet) were used as donor plants, there was a positive correlation between rooting abilities of cuttings from ortets and ramets, suggesting that adventitious rooting is dependent on genetic factors in the donor plants. To promote adventitious rooting of cuttings by hormonal treatment, we examined the effect of soaking time in Oxyberon (19.7 mM indole-3-butyric acid (IBA) solution) on rooting. Ten minutes was the best soaking time for rooted cuttings to produce more adventitious roots without impairing normal growth. When cuttings were soaked in Ethrel diluent (69.2 μM ethephon) for 24 h before soaking in Oxyberon for 10 min, a significantly higher rooting ability was observed than those soaked in Oxyberon alone. Ethrel on its own barely affected rooting ability. The positive effect of the combinational treatment was confirmed in a similar experiment using authentic ethephon and IBA instead of Ethrel and Oxyberon. When cuttings were soaked in a mixture of ethephon and silver thiosulfate (STS), an ethylene action inhibitor, before IBA-soaking, the effect was partially diminished compared with combinational treatment without STS. These findings suggest that ethylene action caused by ethephon treatment promotes IBA-mediated adventitious rooting of P. thunbergii cuttings.     

Abscisic acid and Ethrel abolish the inhibition of adventitious root formation of paclobutrazol-treated bean primary leaf cuttings

Paclobutrazol (PB), a triazole growth retardant and an inhibitor of gibberellin biosynthesis, reduced at 17 μM concentration the adventitious root formation of bean primary leaf cuttings. Treatments with 5 μM ABA or 4 μM Ethrel, an ethylenereleasing compound, restored the rooting of PB-treated cuttings. Ethylene production and the content of the precursor 1-aminocyclopropane-l-carboxylic acid (ACC) were enhanced in root-forming tissues of PB-treated petioles 48 h after ABA application. The effect of ABA could be abolished by 10 μM CoCl₂, an inhibitor of ACC oxidase. Thus, ABA might stimulate rooting through its effect on ethylene release. 2 mM silver thiosulphate, an inhibitor of ethylene action, decreased the rooting of PB-treated cuttings similarly to Co²⁺, but failed to negate the ABA effect. These data indicate that the effect of PB on rhizogenesis is not associated directly with the inhibition of the biosynthesis of gibberellins Acknowledgements: We are grateful to Gabriella Biró. This work was supported by the Hungarian National Science Research Foundation (OTKA), Project No. 462.     

Microcutting leaf area,weight and position on the stock shoot influence root vigour,shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA₃ generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca⁺⁺ and Mg⁺⁺ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca⁺⁺ or higher Mg⁺⁺ contents. Both had comparable amounts of cytokinins in shoot and root tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. ‘Malagouzia’ and ‘Xinomavro’

The effects of six basal media on in vitro shoot proliferation of the greek grapevines Vitis vinifera L. cv. ‘Malagouzia’ and ‘Xinomavro’ were investigated. Galzy and Zlenco proved to be the most effective for ‘Malagouzia’ and ‘Xinomavro’, respectively. If only BA was present in the medium, shoot development was poor and the plantlets were chlorotic. When the medium was supplemented with BA and NAA, growth was enhanced. The best ratio (in μM) of growth regulators was 0.5/0.3 for ‘Malagouzia’, and 0.1/0.03 for ‘Xinomavro’, which resulted in the highest number of microshoots per explant and greatest proliferation rate. The development of ‘Malagouzia’ and ‘Xinomavro’ explants at 21±2 and 26±2°C was also investigated, revealing the higher temperature to be more effective. Regarding rooting, 0.5 μM IBA improved root formation at 26°C for ‘Malagouzia’ and 0.5 μM IBA at 21°C for ‘Xinomavro’. Moreover, 0.5 μM IBA resulted in a higher rooting percentage (>95%) and proved to be more beneficial for the overall morphological appearance of the plantlets of ‘Malagouzia’. After acclimatization, survival of microshoots cultivated in media with IBA was higher than those in NAA.     

Effects of ethylene and 1-MCP (1-methylcyclopropene) on bud and flower drop in mini <Emphasis Type="Italic">Phalaenopsis</Emphasis> cultivars

Phalaenopsis frequently exhibits bud drop during production and in response to adverse postharvest conditions. The effect of exogenous ethylene on bud drop of mini Phalaenopsis was studied and ethylene sensitivity of four cultivars was compared. Water content, membrane permeability and ABA (abscisic acid) content in floral buds and flowers were determined after ethylene treatment. Exogenous ethylene induced flower bud drop in all tested Phalaenopsis cultivars and the different cultivars showed distinct differences in ethylene sensitivity. The cultivar Sogo ‘Vivien’ exhibited the highest bud drop, water loss and change in membrane permeability in floral petals, while Sogo ‘Berry’ showed the lowest sensitivity. The ethylene inhibitor 1-MCP (1-methylcyclopropene) reduced ethylene-induced floral bud drop in the cultivar Sogo ‘Yenlin’. ABA content in floral buds was increased in response to ethylene and 1-MCP pretreatment inhibited the ethylene-induced increase in ABA levels efficiently. This finding suggests that the observed increase in ABA content during bud drop was mediated by ethylene. The interaction between ABA and ethylene is discussed.     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

Adventitious root formation in woody plant tissue: The influence of light and indole-3-butyric acid (IBA) on adventitious root induction in <Emphasis Type="Italic">Betula Pendula</Emphasis>

Summary The effects of auxin concentration and photoperiod on rooting were examined with a view to establishing a rooting regime for Betula pendula shoots cultured in vitro. Optimum concentrations of indole-3-butyric acid (IBA) were determined: the effects of a 16-h photoperiod and a pretreatment of 8d total darkness were examined. Maximum rooting rates and rooting densities (root number) were achieved using relatively low levels of IBA (0.39–0.74 μM). Both the dark and the light regimes produced roots, higher yields occurring with the latter. Maximum rooting percentage was reached after 30 d growth. in the light-treated cultures.     

Plant growth regulators and adventitious root development in relation to auxin

Adventitious root formation in stem cuttings of mung bean was enhanced by ethrel, which had an additive effect when employed simultaneously with indolebutyric acid (IBA). Abscisic acid (ABA) did not influence the number of roots per cutting whereas gibberellic acid (GA₃) and kinetin were without effect on rooting at lower concentrations but were inhibitory at higher concentrations. Nevertheless, all three of these chemicals showed synergistic interactions with IBA and/or indol-3-ylacetic acid (IAA) and thereby significantly promoted root formation. A localised application of morphactin to the epicotyl of cuttings totally inhibited root production irrespective of which of the foregoing growth regulators were suppliedvia the hypocotyl. Morphactin application also prevented root formation in cuttings treated with vitamin D₂. The various growth regulators employed had differing effects on growth of roots but there was no simple relationship between their effects on root formation and subsequent root growth.     

Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings

This study investigated the role of ethylene action in ethylene production and in poststorage performance of pelargonium cuttings. Cuttings of zonal pelargonium (Pelargonium x hortorum L.H. Bailey) of the cultivars Isabell and Mitzou were treated with ethylene and with the ethylene action inhibitors 1-methylcyclopropene (MCP), silver-thiosulfate (STS) and silver nitrate (SN) and were stored in the dark at different temperatures (5, 12, and 20 °C) for 48 h. Ethylene concentrations in the storage boxes were monitored and poststorage leaf senescence, survival and root formation of cuttings were determined. Applications of MCP resulted in a significant increase of ethylene evolution by cuttings of both cultivars which was more pronounced with increasing storage temperature. After 48 h of storage at 20 °C, ethylene concentrations were more than 20-fold higher for the MCP-treated cuttings as compared to those of the untreated controls. Also preharvest applications of STS and SN increased ethylene evolution by cuttings, even though these effects were less pronounced. However, application of these inhibitors caused severe poststorage leaf injury. Application of ethylene during storage had no effect on subsequent leaf damage. Leaf senescence during rooting and decay of cuttings, which raised with increasing storage temperature, could only partially been reduced by MCP. The results strongly support the conclusion, that in zonal pelargonium cuttings, ethylene production is controlled by autoinhibition, and clearly indicate, that temperature dependent processes other than ethylene action are substantially involved in storage-induced leaf senescence and decay.     

不同株龄日本落叶松插穗的内源激素含量与生根的关系

研究了日本落叶松母株年龄、插穗内源激素含量与生根之间的关系,以及外源IBA对插穗内源激素含量的影响及其对插穗生根的促进作用。结果表明：不同株龄插穗生根性状及插穗茎和叶中激素含量差异均达极显著水平,叶中激素含量对插穗生根力没有直接影响;插穗茎中生根抑制激素（ABA）含量随株龄增长而增加,生根促进激素与抑制激素的比值（IAA＋GA＋ZR）/ABA却随株龄的增长而递减,与生根力随株龄的变化趋势一致,且该比值与生根性状紧密相关,因此可作为评价母株（无性系）生根力的指标;插后13～32d是插穗愈伤组织形成和不定根诱导的关键期,此期生根促进激素消耗量大,茎中含量大幅度降低,进入根伸长生长阶段,含量上升;外源IBA促进插穗生根的机制在于通过外源激素的刺激,在不定根诱导期,插穗茎中ABA含量大幅度降低,从而有利于不定根的发生和发育。     

The effect of shading of stock plants on rhizogenesis in stem cuttings of Berberis thunbergii ‘Red Rocket’

In order to improve vegetative propagation of Berberis thunbergii ‘Red Rocket’, stock plants were subjected to shading and IBA treatment, as well as to the application of two commercially available rooting powders. Spraying stock plants with IBA 24 hours prior to cutting harvest gave the effect comparable to the use of rooting powders. Reduction of light intensity by 50% and 96% of ambient prior to harvest of cuttings improved rooting. Positive effect of shading results in changes in shoot anatomy, i.e. a weaker sclerenchyma development. Enhanced rooting in cuttings from shoots grown under reduced light intensity was accompanied by a decrease in the concentration of soluble proteins and by an increase in total chlorophyll content while the concentration of total soluble sugars remained unaffected.     

Juvenile stockplant material enhances root development through higher endogenous auxin level

Stockplants with various physiological characteristics were compared in a propagation experiment with leafy cuttings of ornamental cherry Prunus subhirtella ‘Autumnalis’. At the time of severance, cuttings harvested from juvenile 3-year-old in vitro-derived plants contained in their bases nearly twice as much IAA (indole-3-acetic acid) as cuttings derived from mature 40-year-old trees. Juvenile cuttings showed better rooting success in the propagation season. They developed a significantly higher number of primary roots and grew better than physiologically older cuttings. IAA time-course levels in cutting bases in the days after severance were similar in both cuttings types. They decreased over the first day (rooting late initiation phase) after severance until the third day after severance when the levels increased again (rooting induction phase and beginning of the root developing phase). At the time of severance, juvenile cuttings also contained higher concentrations of IAAasp (indole-3-acetyl aspartic acid) in their bases than mature cuttings. IAAasp time-course levels were similar to those measured for IAA.     

不同溶液浸泡处理对鹅掌柴扦插繁殖的影响

以清水对照,用多菌灵 福美双溶液和NAA IBA溶液对鹅掌柴插穗浸泡处理后扦插,试验对插穗的形态生长指标和生根过程进行了统计,并对插穗内源性激素IAA、ABA的含量进行了分析。结果表明:NAA IBA和适宜的多菌灵 福美双溶液均可提高插穗的生根率,促进根生长。经浸泡处理的插穗的成活率显著地高于对照(p<0.01)。多菌灵 福美双溶液浸泡插穗3h的理论最佳浓度为519mg/L。519mg/L多菌灵 福美双和200mg/LNAA IBA处理都提高了插穗IAA的含量,ABA的含量却有所降低。用519mg/L多菌灵 福美双和200mg/LNAA IBA分别浸泡插穗1.5h,成活率达到95.6%,比单独用200mg/LNAA IBA处理的成活率提高了10%(p<0.05);实验结果还表明,鹅掌柴的生根属混合生根型。     

Assessing competence for adventitious shoot formation in hypocotyl explant cultures from<Emphasis Type="Italic">Catharanthus roseus</Emphasis> cultivars

Hypocotyl expiants from 22 cultivars ofCatharanthus roseus were cultured on various shoot-inducing media to assess their competence for adventitious shoot formation. The Murashige and Skoog (MS) media had been supplemented with 14 μM zeatin and 2.5 μM indole-3-butyric acid (IBA), 4.5 μM BA and 0.5 μM α-naphthaleneacetic acid (NAA), or 14 μM thidiazuron and 2.5 μM IBA. After eight weeks, the expiants from ‘Cooler Raspberry Red’ showed the greatest frequency of adventitious shoot formation, followed by ‘Cooler Orchid’ and ‘Cooler Treated’. The highest frequency (86.7%) for ‘Cooler Raspberry Red’ was attained on the medium enhanced with 14 μM zeatin and 2.5 μM NAA. Excised adventitious shoots were then readily rooted on a half-strength MS basal medium. Afterward, the regenerated plantlets were transferred to potting soil and grown to maturity in a greenhouse.     

Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings

The role of ethylene in adventitious root formation and its involvement in auxin-induced rooting were investigated in cuttings ofVigna radiata (L.). Treatment with 30 M indole-3-acetic acid (IAA) for 24 h slightly inhibited rooting, whereas the same concentration of indole-3-butyric acid (IBA) significantly stimulated it. Ethylene derived from 1-aminocyclopropane-1-carboxylic acid (ACC) increased the number of adventitious roots but inhibited their emergence and elongation. Endogenous levels of ethylene, ACC, and malonyl-ACC (MACC) were initially higher in cuttings treated with IAA. This trend was quickly reversed, and cuttings, particularly hypocotyls, treated with IBA produced higher levels of ethylene and had more ACC and MACC during most of the rooting process. Aminoethoxyvinylglycine significantly inhibited rooting, but its inhibitory effect could not be reversed by ACC. The data suggest that the stimulating effect of IBA on rooting is closely associated with its induction of ACC and ethylene biosynthesis.     

Plant regeneration from in vitro leaf tissues of <Emphasis Type="Italic">Viburnum dentatum</Emphasis> L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.