Effects of plant growth regulators on the rapid proliferation of shoots and root induction in the Chinese traditional medicinal plant Atractylodes macrocephala

We developed an efficient plant regeneration protocol for rapidly propagating Atractylodes macrocephala Koidz,an important traditional Chinese medicinal plant,via shoot organogenesis.Shoot multiplication was induced on Murashige-Skoog (MS) medium supplemented with various concentrations of N-phenyl-N-1,2,3-thidia-zol-5-ylurea (TDZ),6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA).Rooting was induced on half-strength MS medium supplemented with NAA and indolebutyric acid (IBA).The maximum mean number of shoots (5.61) was obtained from a single explant by the combined effect of 1.08 μmol/L NAA and 2.25 μmol/L TDZ.The longest roots and a minimum number of roots were produced when they were cultured in a medium without plant growth regulators.The shortest roots and the largest number of roots were observed in the medium supplemented with 2.7μmol/L NAA.     

Stimulating effects on mouse splenocytes of glycoproteins from the herbal medicine Atractylodes macrocephala Koidz.

A traditional herbal medicine, Atractylodes macrocephala Koidz. (AMK), has long been used as a digestive and tonic. Recent investigations have suggested its potential ability in stimulating immune responses, although a scientific basis for this activity has not yet been elucidated. Based on previous results showing that the activity might be due to proteins, we purified protein samples from an original sample preparation of AMK and examined the stimulating ability of the protein samples on mouse splenocytes. The sample treatment markedly stimulated lymphocyte proliferation, antibody production, and cytokine secretion in mouse splenocytes. In particular, the samples showed the ability to induce the preferential stimulation of Th1 type, rather than Th2 type T lymphocytes. Stimulating activity of the samples was associated closely with glycoprotein(s) with molecular weights of around 30 kDa, especially with carbohydrate moiety rather than with protein residues of the glycoprotein(s). Our findings suggest that the glycoprotein(s) might play critical roles in modulating immune-response induction, and could potentially be used as medicinal and pharmacological agents.     

Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi

An efficient hairy root induction system for an important endangered medicinal plant, Dracocephalum kotschyi, was developed through Agrobacterium rhizogenes-mediated transformation by modifying the co-cultivation medium using five bacterial strains, A4, ATCC15834, LBA9402, MSU440, and A13 (MAFF-02-10266). A drastic increase in transformation frequency was observed when a Murashige and Skoog medium lacking NH₄NO₃ KH₂PO_4, KNO₃ and CaCl₂ was used, resulting in hairy root induction frequencies of 52.3 %, 69.6 %, 48.6 %, 89.0 %, and 80.0 % by A4, A13, LBA9402, MSU440, and ATCC15834 strains, respectively. For shoot induction, hairy roots and unorganized tumors induced by strain ATCC15834 were placed on an MS media supplemented with 0.1, 0.25, 0.5, and 1 mg/l BA plus 0.1 mg/l NAA. The high frequency of shoot regeneration and number of shoot were obtained in the medium containing 0.25 mg/l BA and 0.1 mg/l NAA. Root induction occurred from the base of regenerated shoots on the MS medium supplemented with 0.5 mg/l IBA after 10 days.     

Effects of plant growth regulators in soil on clover growth and nitrogen fixation

Effects of the growth regulators Barleyquat B, New 5c Cyocel, Cerone, Terpal and paclobutrazol on Rhizobium trifolii in vitro, on growth and root nodulation of clover grown in pots and on symbiotic nitrogen fixation were measured.Paclobutrazol in soil markedly reduced the weight of clover plants but had no effect on nodulation in relation to plant size. Paclobutrazol decreased the amount of nitrogen fixed per plant (measured by acetylene reduction) when present in soil at a concentration which would result from a single direct application at 0.25 kg a.i. ha^–1 remaining unchanged and evenly distributed in the top 5 cm of soil. A concentration in soil equal to that from an application at 0.125 kg a.i. ha^–1 had no significant effect on the rate of nitrogen fixation per plant and the rate relative to plants weight increased. It was evident that the inhibitory effect on plant growth was greater than on symbiotic nitrogen fixation. The other PGR's had virtually no effect on clover.None of the compounds seems likely to affect nitrogen fixation by a subsequent clover crop in the field if previously applied to a cereal crop once at typical rates of application.

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Zusammenfassung Die Einflüsse der Wachstumsregulatoren Barleyquat B, New 5c, Cyocel, Cerone, Terpal und Paclobutrazol auf das Wachstum von Rhizobium trifolii in vitro, auf das Wachstum und Knöllchenbildung des Klees und auf die symbiotische N₂-Bindung wurden bestimmt.Paclobutrazol im Boden führte zu einer bedeutenden Gewichtsverminderung der Kleepflanzen, blieb aber ohne Einfluß auf die Knöllchenbildung in bezug auf die Pflanzengröße. Paclobutrazol reduzierte die Menge Stickstoff gebunden pro Pflanze, (gemessen mittels Acetylenreduktion) wenn er im Boden in einer Konzentration enthalten war, die anwesend wäre, wenn eine einzige Applikation von 0,25 kg Wirkstoff.ha^–1 unverändert und gleichmäßig in den oberen 5 cm des Bodens verteilt bleiben würde. Eine Bodenkonzentration gleich jener von einer Applikation von 0,125 kg Wirkstoff.ha^–1 hatte keine signifikante Wirkung auf die N₂-Bindungsleistung pro Pflanze, und das Bindungstempo in bezug auf das Pflanzengewicht nahm zu. Offensichtlich war die Hemmwirkung auf das Pflanzenwachstum größer als auf die symbiotische N₂-Bindung.Wahrscheinlich wird keine der Verbindungen die N₂-Fixierung des Klees im Freiland beeinträchtigen, wenn sie einmal vorher auf eine Getreidearte in der üblichen Aufwandmenge appliziert werden.

     

Effects of plant growth regulators on three wood-rotting polypores

The results of the effects of plant growth regulators, kinetin, gibberellic acid, 3-indoleacetic acid and ethrel (2-chloroethylphosphonic acid) on three wood-rotting polypores,viz. Polyporus palustris, Daedalea flavida andTrarnetes badia, are given. 3-Indoleacetic acid, kinetin and gibberellic acid increased the dry mass and protein, and decreased free amino acids, while ethrel decreased dry mass and protein, and increased free amino acids inP. palustris. All the treatments decreased dry mass and protein, and increased free amino acids inD. flavida. Kinetin decreased dry mass and protein, and increased free amino acids whereas 3-indoleacetic acid, gibberellic acid and ethrel increased dry mass and protein, and decreased free amino acids inT. badia over the control. The effects were most pronounced in the three species upon treatment with 50 μm kinetin, 50 μm gibberellic acid, 50 μm 3-indoleacetic acid and 25 μm ethrel. Among the treatments, the effects of 3-indoleacetic acid were most marked in enhancing growth, measured by dry mass ofT. badia out of the three species studied.     

Effects of growth regulators on inflorescence proliferation of Bambusa edulis

The effects of various growth regulators in Bambusa edulis inflorescence proliferation were studied. Cytokinin is essential for inflorescence proliferation. Thidiazuron (TDZ) had been the most efficient cytokinin to induce inflorescence proliferation. The optimal TDZ concentration was 0.01–0.1 mg l^–1. Inflorescences did not proliferate in media containing auxin, gibberellic acid (GA₃), abscisic acid (ABA), or 1-amino- cyclopropane-1-carboxylic acid (ACC) alone. In TDZ-containing medium, the proliferation ratio decreased when the naphthaleneacetic acid (NAA) concentration higher than 5 mg l^–1.     

Establishment of the model of white blood cell membrane chromatography and screening of antagonizing TLR4 receptor component from Atractylodes macrocephala Koidz

A model of white blood cell membrane chromatography (WB-CMC) was established to screen active component from Atractylodes macrocephala Koidz. The component can antagonize Toll-like receptor 4 (TLR₄) and inhibit inflammatory reaction. In the model of WB-CMC, cell membrane stationary phase (CMSP) was prepared by immobilizing the rabbit white blood cell membrane (WBCM) onto the surface of silica carrier and taxinol was used as a model molecule. The active component which can act on WBCM and its receptor (such as TLR₄) as an effective target in A. macrocephala was determined by using a replacement experiment. The anti-inflammatory effects of the active component were tested by using pharmacological methods in vivo. The results indicated that the retention characteristics of atractylenolide I as active component was similar to that of taxinol in the model of WB-CMC. And so, atractylenolide I acted on the WBCM and TLR₄ and its anti-inflammatory activity was related with antagonizing TLR₄. Therefore, the interaction between the active component and WBCM and its receptor can be simulated by the model of WB-CMC in vitro. This model can be used to screen active components and to study effective characteristics for acting on definite targets.     

Pharmacological studies on the sedative and hypnotic effect of salidroside from the Chinese medicinal plant Rhodiola sachalinensis

This is the preliminary study of sedative and hypnotic activity of salidroside (a major component of Rhodiola sachalinensis) in mice by using synergism with pentobarbital as an index for the hypnotic effect. Loss of the righting reflex was used to determine the start of sleep. Sleep latency and sleeping time were evaluated in this experiment. The results showed that salidroside could obviously shorten the sleep latency and prolong the sleeping time of mice produced by pentobarbital sodium (55 mg/kg, i.p.). Salidroside produces significant sedative-hypnotic effect. The dose-effect relationship is remarkable.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic transformation and hairy root induction in Holostemma ada-kodien K. Schum--a vulnerable medicinal plant

Hairy roots were induced from shoot buds and seedling hypocotyls of Holostemma by infection with agropine type Agrobacterium rhizogenes strains. Type of explant, Agrobacterium rhizogenes strain used for infection, co-culture time and photoperiod influenced the transformation frequencies. Hairy roots were induced from seedling hypocotyls and shoot bud explants upon infection with agropine type Agrobacterium rhizogenes strains. The hairy roots were thin, whitish in colour and showed negatively geotropic growth. The transformed nature of hairy roots was confirmed by opine analysis.     

Effects of plant growth regulators on tiller bud outgrowth in uniculm cereals

Plant growth regulators were applied either to seeds or to leaves of uniculm selections of barley and wheat. None of the treatments stimulated the growth and development of tiller buds in the barley selection. In contrast, in wheat the development of tiller buds was readily stimulated and tillers were produced following treatment with Terpal, Cerone, TIBA or nitrate. In Terpal and Cerone treated plants, the stimulation of tiller bud outgrowth and tillering was accompanied by a marked retardation of growth in the main shoot. The growth of the tillers was however not sustained in any of the treatments, and so at maturity all plants were virtually uniculm. The results are discussed in relation to the different overall response of the two species, the death of tillers, and the hormonal regulation of tiller bud development.     

Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix

Although the effectiveness of topolins in plant tissue culture systems has recently been highlighted, there is a dearth of information on their interactions with auxins in relation to shoot organogenesis and secondary metabolite production. The current study evaluated the role of topolins singly or in combination with an auxin in comparison to 6-benzyladenine (BA) on shoot proliferation and secondary metabolite production of Huernia hystrix, a medicinal and ornamental stem-succulent of the endemic flora of southern Africa. Meta-topolin (mT) was more effective in improving shoot proliferation and phenolic production compared to BA. In general, the exogenous addition of α-naphthalene acetic acid (NAA) significantly increased shoot proliferation. The highest number of regenerated shoots (12.2 ± 0.98 shoots per explant) was recorded with medium containing 20 μM mT supplemented with 10 μM NAA and was three-times higher when compared to the treatments with cytokinin only. This suggests a synergistic interaction of auxin with cytokinin. On the other hand, supplementation with low NAA concentrations resulted in reduced in vitro flavonoid production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations (even when used in combination with NAA equimolar concentrations) significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on shoot proliferation and the production of secondary metabolites in H. hystrix.     

Influence of plant growth regulators on volatiles produced by in vitro grown shoots of Agastache rugosa (Fischer & C.A.Meyer) O. Kuntze

The composition of volatile organic compounds emitted by in vitro shoots of Agastache rugosa (Fischer & C.A. Meyer) O. Kuntze (Lamiaceae) was studied using headspace solid-phase microextraction–gas chromatography–mass spectrometry and compared to the those emitted by adult plants and in vitro-germinated seedlings. Shoot-tip explants were cultured on a solid MS medium supplemented with either 4.4 μM 6-benzyladenine (BA), 9.3 μM kinetin, or 0.45 μM thidiazuron and with either 0.57 μM indole-3-acetic acid (IAA) or 0.41 μM picloram. Shoot proliferation was observed in all these treatments. The presence of these plant growth regulators in the culture medium significantly influenced the composition of volatiles as well as morphogenetic responses observed. The number and quality of regenerating shoots and frequency of axillary bud break were highest in medium containing the BA + IAA combination. Sixty-five compounds were identified in the headspace of the in vitro-produced material and plants cultivated in the field. The in vitro shoots emitted both hydrocarbon (limonene, α-pinene) and oxidized (menthone, isomenthone, pulegone) monoterpenes. The composition of monoterpenes differed depending on the type of auxin—rather than cytokinin—in the medium. The emission of phenylallyl compounds, such as estragole, a major compound in field-grown plants, was markedly lower in shoot cultures.     

Effects of plant growth regulators ambiol and phonk: biolphysical aspects

The antioxidative activity of two plant growth promoters, ambiol and phonk, in a model system of photo-induced glycyltriptophane oxidation was measured. It was shown that ambiol has a significant antioxidative activity, whereas phonk is a weak antioxidant. The effects of these compounds on DNA conformation were studied in vitro and in vivo (on wheat seed shoots). In vitro ambiol had a stronger effect as compared with phonk, whereas in vivo the latter produced a more essential effect than ambiol. The assumption was done that both compounds affect indirectly the genome expression activity. Possible mechanisms of biological activity of each compound are discussed.     

Effects of plant growth regulators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, nirgundi (Vitex negundo L.)

Summary A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.     

Effect of different plant growth regulators on micro-tuber induction and plant regeneration of Pinellia ternate (Thunb) Briet

An efficient micropropagation system for Pinellia ternate (Thunb) Briet, a traditional Chinese medicinal plant, has been developed. Petiole and lamina of P. ternate were used as explants and cultured on Murashige and Skoog (MS) medium containing different concentrations of different plant growth regulators. The results indicated that low concentration of 2,4-dicholorophenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) were suitable for micro-tuber induction, but callus induction rate increased with increasing concentrations of growth regulators. Tubers induction rates of petiole and leaf were (81.8 %–100 %) and (89.4 %–96.0 %) respectively, when 0.2 mg l⁻¹ 2, 4-dicholorophenoxy acetic acid, indole-3-acetic acid or α-naphthalene acetic acid were present in the medium. Tubers induction rates of petiole and leaf cultured on MS medium supplemented with 0.2–0.5 mg l⁻¹ 6-benzyl amino purine (6-BAP) were (94.1 %–100 %) and (96.0 %–100 %) respectively. When the concentration of 2,4-dicholorophenoxy acetic acid, α-naphthalene acetic acid and 6-benzyl amino purine was increased to 2.0 mg l⁻¹, callus induction rates of petiole and leaf were 100 % and 98.2 %, 91.0 % and 36.0 %, 62.3 % and 70.0 %, respectively. Different concentration of kinetin (KT) and zeatin (ZT) had no significant effect on micro-tuber induction of petiole. Most petioles showed polarity during the cultivation of explants, when supplemented with different concentrations of auxin or cytokinin in the MS medium.     

Effects of plant growth regulators on secondary wall thickening of cotton fibres

The effect of plant growth regulators on the secondary wall thickeningof cotton fibre was studied. The results indicated that the GA_S andiP+iPA levels in the fibre of field-grown cotton plantsremained almost constant but the IAA and ABA levels changed considerably duringfibre development. Although the change in both IAA and ABA levels seemed not tobe closely related with the rate of cellulose accumulation, there was still arelationship between the ratio of ABA to IAA and secondary wall thickening. Inin vitro studies, ABA (50mol·L^–1) markedly enhanced theaccumulation of dry matter and cellulose in the fibre cell wall duringsecondarywall thickening, but no similar effect was observed with NAA, GA₃ orkinetin treatments. The role of ABA in secondary wall thickening of cottonfibreis discussed.     

Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.