Role of Ethylene in Axillary Shoot Proliferation of Lavandin--Interaction with Benzyladenine and Polyamines

The role of ethylene in in vitro axillary proliferation of lavandin(Lavandula officinalis Chaix Lavandula latifolia Villars)was investigated. Basal ethylene production was modulated bythe addition of exogenous growth regulators (BA and polyamines),ethylene precursor (ACC) and inhibitors (AVG and SA). The resultsindicate that BA action is mediated by ethylene and, furthermore,that ethylene can substitute for BA in shoot proliferation.Among tested polyamines (Put, Spd, Spn) only Put was effectivein stimulating both the differentiation process and relatedethylene production. Moreover, Put appears to have a regulatoryfunction similar to BA. The data obtained in the present workoutline the complexity of axillary budding which is under multihormonalcontrol, ethylene playing an essential role. Key words: Axillary proliferation, benzyladenine, ethylene, lavandin, putrescine     

In vitro shoot development of Prunus GF 655–2: interaction between light and benzyladenine

Experiments designed to study the effect of light quality and quantity and their possible interaction with benzyladenine (BA) in the control of in vitro proliferation of Prunus insititia Schneider GF 655–2 microcuttings are reported. The action of BA as a promoting factor of shoot formation was expressed only in the presence of light. The concentration response curves for BA-induced proliferation were very similar under the different light sources, irrespective of proliferation rate values.
Shoot formation under blue, far-red and white light was enhanced by the highest photon fluence rates, while the efficiency of red seemed to be independent of this factor. The results suggest the action of a low-energy response in the red waveband and under the low photon fluence rates of blue and white. The inhibition of shoot elongation induced by BA in the dark as well as under all light treatments indicates that, while the BA-induced release from the apical dominance is light dependent. BA inhibition of shoot elongation is entirely light independent.     

Photoregulation of growth and branching of plum shoots: Physiological action of two photosystems

Summary Plum shoot proliferation was investigated in terms of two distinct processes: axillary bud differentiation and axillary shoot development. Results showed that light quality influenced bud differentiation and interacted with apical dominance in determining shoot outgrowth, resulting in a differentiated structure of shoot clusters and type of branching. Results suggested that blue light, acting through its photoreceptor, increased the number of axillary buds differentiated from apical meristem, but did not remove the apical dominance. Red light removed apical dominance, while reducing the formation of axillary buds; both events appeared to be dependent on the putative amount of phytochrome active form, and independent of light photon fluence rate. On the contrary, blue light action appeared to be dependent on photon fluence rate. In addition, apparent blue-red interactions related to photomorphogenic events fit an antagonistic model for branching regulated by light via cryptochrome and phytochrome photoreceptors. Our results show that the dynamics of shoot cluster development is the product of two events: the formation of new axillary buds and their release from apical dominance.     

Light quality growth promotion of Spiraea nipponica: the influence of a low photon fluence rate and transfer time to a higher fluence rate

Combinations of different light quality and fluence exposure times were investigated for their effects on in vitro growth of the woody plant Spiraea nipponica. An interaction was demonstrated between different levels of benzyladenine (BA) used for in vitro propagation and the specific light regimes investigated. This relationship was affected by the length of exposure to either white or red/FR light and the time of transfer from one fluence rate to another. In all instances exposure to red/FR light resulted in more extensive growth than under white light. Thus explants cultured under 0.25 and 0.4 mg l^-1 of BA exhibited high shoot proliferation rates when transferred, after 4 weeks of low photon fluence red/FR light, to higher fluence white light for a further week. The proliferation rates obtained were higher than any white light treatment including that with the highest BA level of 0.5 mg l^-1. In addition, the combination of red/FR light exposure with a white light stage of higher fluence improved proliferation at lower exogenous BA levels.     

Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro

Summary Continuous axillary shoot proliferation and in vitro flowering were achieved using single node explants from a mature (over 70-yr-old) field clump of Dendrocalamus giganteus (giant bamboo). The shoots proliferated in a basal Murashige and Skoog medium with 6 mgl⁻¹ (26.6 μM) N⁶-benzyladenine (BA) and 2% sucrose. The rate of shoot proliferation gradually increased to over three-fold before in vitro flowering took place. In vitro flowering was not the expression of a species-specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower. The rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation leading to build up, and release of stress in relation to flowering and its reversion are discussed.     

In vitro embryonic axis and seedling shoot tip culture of Juglans nigra L.

Embryonic axes and seedling shoot tips of Juglans nitra L., Black walnut, were cultured in vitro. Significant variation existed among progeny from individual trees for growth of radicles and epicotyls and production of callus and axillary shoots from embryonic axes. The concentration of 6-benzyladenine influenced the growth of the radicle and epicotyl and production of callus and axillary shoots of axes. Axes generally initiated growth quicker on solidified woody plant medium than on Driver and Kuniyuki's walnut medium, but axillary shoot proliferation and elongation were eventually better on liquid Driver and Kuniyuki's walnut medium than on woody plant medium which required an etiolation treatment for microshoot elongation. The concentration of BA also influenced both callus growth and axillary shoot proliferation from seedling shoot tips. Axillary shoots which formed in Driver and Kuniyuki's walnut medium rooted best in sterile vermiculite following a 15 s dip in 10 mM indole-3-butyric acid. Micropropagated plants are growing in the greenhouse.     

Interactions of green or red light with blue light on the dark closure of Albizzia pinnules

The interactions of green or red light with blue light on the dark closing of Albizzia julibrissin Durazz. pinnules have been investigated. Irradiations at 430, 450 and 470 nm progressively delay dark closing with increasing photon fluence rates. Red or green light alone has no effect. However, when the blue fluence rate is low, both red and green light interact with it and increase the delaying effect of the blue light. When the blue fluence rate is high, green light interacts with it to negate some of the effectiveness of the blue light, while red light has no effect. This is similar to results obtained previously with far-red light. It is suggested that the same unidentified photoreceptor is operating in both the far-red and blue regions. The results also indicate the presence of a blue-only absorbing photoreceptor whose action is increased by phytochrome.     

Light effects on in vitro rooting of pear cultivars of different rhizogenic ability

The effect of varying light regimes on in vitro rooting of microcuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy to-root Conference and the difficult-to-root Doyenne d'Hiver were incubated for 21 days with or without indole-3-butyric acid (IBA) in the medium in darkness or under continuous far-red (8 µmol m^–2 s^–1), blue, white or red (15 or 36 µmol m^–2 s^–1) light. Conference rooted without IBA when exposed to red, blue or white light while no rooting was observed under far-red light and in darkness. The high rooting efficiency under red and, by contrast, the inhibition under far-red light and darkness suggest the involvement of the phytochrome system in rhizogenesis. The addition of IBA to the culture medium enhanced root production under all light regimes in both cultivars. Red light, especially at the lower photon fluence rate, had a positive effect by increasing root extension (number × length of roots) and stimulating secondary root formation.Abbreviations IBA Indole-3-butyric acid - R red light - B blue light - FR far-red light - W white light - D darkness - P_fr active (far-red light absorbing) form of phytochrome - P_tot total phytochrome - BA benzyl-adenine     

Fluence dependence of the ultraviolet-light-induced accumulation of chalcone synthase mRNA and effects of blue and far-red light in cultured parsley cells

The fluence dependence of the time course of accumulation of chalcone synthase mRNA in ultraviolet (UV)-light-irradiated cell suspension cultures of parsley (Petroselinum crispum) and the additional effects of blue and far-red light have been investigated. Variations of the UV fluence had no detectable influence on the initial rate of increase in mRNA amount or translational activity, nor on the preceding lag period of approximately 3 h, but strongly influenced the duration of the transient increase. The effects were the same whether the fluence rate or the time of irradiation was varied to obtain a given fluence. Blue-light pretreatment of the cells resulted in increased amounts of mRNA and abolished the apparent lag period. This effect remained cryptic without the subsequent UV-light treatment. Irradiation with long-wavelength far-red light following UV-light pulses shortened the duration of the mRNA accumulation period. This effect was not altered by a preceding blue-light treatment. Thus, three photoreceptors, a UV-B receptor, a blue-light receptor and phytochrome, participate in the regulation of chalcone synthase mRNA accumulation in this system.Abbreviations cDNA complementary DNA - UV ultraviolet - Pfr fai-red-absorbing form of phytochrome     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

Factors affectingin vitro establishment and shoot proliferation ofRosa hybrida L. andRosa Chinensis minima

Summary The effects of nodal explants collected at different plastochrones, use of various benzyladenine (BA) concentrations, sources of carbohydrates, and phases of the culture medium on shoot establishment and proliferation ofRosa hybrida L. andR. chinensis minima were evaluated. Higher numbers of shoots per explant were obtained fromR. hybrida cv. Carefree Beauty explants proximal to the apical meristem than those from distal nodes. However, proliferating shoots derived from plastochrones proximal to the apical meristem had a lower number of leaves/explant and were shorter than those derived from other distal plastochrones. Although shoot proliferation increased with higher BA concentration in the medium, a concentration of 4.4 μM BA was found optimum for axillary bud-break and shoot development forR. hybrida cvs. Adelaide Hoodless and Cuthert Grant. A higher shoot proliferation rate was observed forR. hybrida cv. Carefree Beauty explants grown on a medium containing 55.5 mM fructose than 58.4 mM sucrose. However, no differences were observed forR. hybrida cv. Cuthert Grant grown on a medium containing either fructose or sucrose. The mean number of shoots/explant was higher forR. chinensis minima cv. Red Sunblaze explants grown on a liquid (4.5) than on a solid medium (1.7) for the first reculture; while no significant differences between the two phases of the medium were observed for the second reculture. However, a higher mean number of shoots/explant was observed on solid-phase (4.0) than liquid-phase medium (3.4) for the third reculture. A higher mean number of leaves/shoot was obtained on a solidified medium rather than liquid medium in the first two consecutive recultures, while no differences were observed for the third reculture. Although a significant effect of BA concentration on mean number of shoots/explant was observed for Red Sunblaze nodal explants, the influence of BA concentration decreased in the two consecutive cultures for both phases of the medium. Hyperhydricity was observed on Red Sunblaze shoots grown on the liquid-phase medium.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.     

Micropropagation of calla lily (<Emphasis Type="Italic">Zantedeschia albomaculata</Emphasis>) via <Emphasis Type="Italic">in vitro</Emphasis> shoot tip proliferation

Summary A method for the micropropagation of Zantedeschia albomaculata is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. Of the four cytokinins tested, 6-benzyladenine (BA) and thidiazuron (TDZ) were found to be more effective. An optimal concentration of BA (8.87 μM) or TDZ (4.54 μM) developed an average of 3.8 and 3.2 shoots per explant, respectively, but increasing concentrations of cytokinins often led to lower proliferation rate and stunted growth. Addition of auxins to the MS medium supplemented with 8.87 μM BA slightly enhanced multiple shoot formation in the explants. Multiplication of six cultivars of Zantedeschia genus comprising different flower types and colors were tested and achieved using only one regeneration medium (MS+8.87 μMBA+2.46 μM IBA). Different MS medium strength, air temperature (15, 20, 25, and 30°C) and light quality [fluorescent, red + blue, red and blue light provided by a LED (light-emitting diode) system] were used (without phytohormone) with the aim of stimulating in vitro shoot and root development. Half-strength MS or MS and cultures maintained at 25°C were found to be equally suitable for shoot tip culture of Z. albomaculata. Shoot elongation as well as fresh and dry weight were significantly increased when cultures were kept under red or blue light.     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Summary Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N⁶-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.