Correlation of epiphyllous bud differentiation with foliar senescence in crassulacean succulent Kalanchoe pinnata as revealed by thidiazuron and ethrel application

Leaves of Kalanchoe pinnata have crenate margins with each notch bearing a dormant bud competent to develop into a healthy plantlet. Leaf detachment is a common signal for inducing two contrastingly different leaf-based processes, i.e. epiphyllous bud development into plantlet and foliar senescence. To investigate differentiation of bud and its correlation, if any, with foliar senescence, thidiazuron (TDZ), having cytokinin activity and ethrel (ETH), an ethylene releasing compound, were employed. The experimental system was comprised of marginal leaf discs, each harbouring an epiphyllous bud. Most of the growth characteristics of plantlet developing from the epiphyllous bud were significantly inhibited by TDZ but promoted by ETH. The two regulators modulated senescence in a manner different for leaf discs and plantlet leaves. Thus, TDZ caused a complete retention whereas ETH a complete loss of chlorophyll in the leaf discs. In contrast, the former resulted in a complete depletion of chlorophyll from the plantlet leaves producing an albino effect, while the latter reduced it by 50% only. In combined dispensation of the two regulators, the effect of TDZ was expressed in majority of responses studied. The results presented in this investigation clearly show that the foliar processes of epiphyllous bud differentiation and senescence are interlinked as TDZ that delayed senescence inhibited epiphyllous bud differentiation and ETH that hastened senescence promoted it. A working hypothesis to interpret responsiveness of the disc-bud composite on lines of a source-sink duo, has been proposed.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalanchoe) marnierianum: role of auxin and ethylene

Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.     

Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid

Summary The application of 2,3,5-triiodobenzoic acid (TIBA, 10 mg·g^-1 in lanolin) to the stem of intact pea seedlings (Pisum sativum L.) inhibited the basipetal transport of ¹⁴C from indoleacetic acid-1-¹⁴C (IAA-1-¹⁴C) applied to the apical bud, but not the transport of ¹⁴C in the phloem following the application of IAA-1-¹⁴C or sucrose-¹⁴C to mature foliage leaves. It was concluded that fundamentally different mechanisms of auxin transport operate in these two pathways.When TIBA was applied at the same time as, or 3.0 h after, the application of IAA-1-¹⁴C to the apical bud, ¹⁴C accumulated in the TIBA-treated and higher internodes; when TIBA was applied 24.0 h before the IAA-1-¹⁴C, transport in the stem above the TIBA-treated internode was considerably reduced. TIBA treatments did not consistently influence the total recovery of ¹⁴C, or the conversion of free IAA to indoleaspartic acid (IAAsp). These results are discussed in relation to the possible mechanism by which TIBA inhibits auxin transport,.Attention is drawn to the need for more detailed studies of the role of the phloem in the transport of endogenous auxin in the intact plant.Abbreviations TIBA 2,3,5-triiodobenzoic acid - IAAsp indoleaspartic acid     

Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism

The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H⁺ secretion, α-(p-chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA-induced inhibition. These results indicate a close relationship between the acid-growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism.     

江西铅山红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的建立

以江西铅山红芽芋脱毒苗为试材,研究不同因素对红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的影响,以期对红芽芋脱毒苗的再生体系进行优化。结果表明,红芽芋脱毒苗球茎愈伤组织诱导的最佳培养基是MS+TDZ 2 mg·L^-1+2,4-D 1 mg·L^-1。红芽芋脱毒苗球茎愈伤组织分化的最佳培养基是MS+TDZ 2 mg·L^-1+NAA 1 mg·L^-1。红芽芋脱毒苗不定芽生根的最佳培养基是1/2MS+NAA 0.5 mg·L^-1+PP₃₃₃ 0.5 mg·L^-1。红芽芋再生苗最好的移栽基质为发酵后的腐锯木屑。红芽芋脱毒苗球茎愈伤组织再生苗移栽时最佳的PP₃₃₃浓度为20～50 mg·L^-1。本试验成功建立了红芽芋脱毒苗球茎愈伤组织的再生体系,为红芽芋脱毒苗转基因的研究和种质创新奠定了基础。     

Thidiazuron-induced shoot-bud formation on root segments of Albizzia julibrissin is an apex-controlled, light-independent and calcium-mediated response

Root segments or entire roots of Albizziajulibrissin formed shoot-buds; the former were more responsive thanthe latter. The regeneration capacity of root segments increased with anincreasing distance from the meristem. Shoot regeneration on N₆mineral formulation required either a cytokinin (BAP) or thidiazuron (TDZ); thelatter was more effective than the former, inducing a higher number of shoots ata low concentration (0.1 M) in the light as well as in thedark. The frequency of shoot formation was reduced when the auxin inhibitorsmaleic hydrazide (MH) or triiodobenzoic acid (TIBA) were included, indicating anindirect role of auxin in shoot morphogenesis. Inhibitors of calcium uptake(lanthanum) and calmodulin, trifluoperazine (TFP) or chlorpromazine (CPZ) atvery low levels, resulted in inhibition to reduction in frequency of shootmorphogenesis. This indicates that TDZ-induced shoot formation may be acalcium-mediated response. Increasing the level of calcium in the medium did notpromote shoot formation in the presence of TDZ (0.1 M). At areduced level of calcium, which was ineffective in the presence of low TDZ (0.1M), shoot-buds appeared when the concentration of TDZ wasraised to 1.0 M. This provides indirect evidence that TDZmodulates the tissue level of calcium needed for shoot formation.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

Thidiazuron: A potent regulator ofin vitro plant morphogenesis

Summary TDZ (N-phenyl-N’-1,2,3-thidiazol-5-ylurea) is a substituted phenylurea compound which was developed for mechanized harvesting of cotton bolls and has now emerged as a highly efficacious bioregulant of morphogenesis in the tissue culture of many plant species. Application of TDZ induces a diverse array of cultural responses ranging from induction of callus to formation of somatic embryos. TDZ exhibits the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured explants, although structurally it is different from either auxins or purine-based cytokinins. A number of physiological and biochemical events in cells are likely to be influenced by TDZ, but these may or may not be directly related to the induction of morphogenic responses, and hence, the mode of action of TDZ is unknown. However, the recent approaches applied to study the morphogenic events initiated by TDZ are clearly beginning to reveal the details of a variety of underlying mechanisms. Various reports indicate that TDZ may act through modulation of the endogenous plant growth regulators, either directly or as a result of induced stress. The other possibilities include the modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation. In this review, several of these possiblities are presented and discussed in light of recently published studies on characterization of TDZ-induced morphogenic effects.     

Thidiazuron-induced hypertrophic growth from foliar disks of <Emphasis Type="Italic">Kalanchoe pinnata</Emphasis> leads to visualization of a bioactive auxin gradient across the leaf plane

Thidiazuron induces a variety of effects in foliar explants of K. pinnata cultured in vitro. Of these, the induction of an organized hypertrophic growth at the vein ending on the proximal side of leaf disks is a significant morphogenetic effect. This polarized hypertrophy is considered an exclusive auxin-mediated response, as the effect is completely reversed by auxin efflux inhibitor 2,3,5-triiodobenzoic acid. With the magnitude of hypertrophic growth from individual disks taken as a manifestation of putative auxin levels, the occurrence of a gross, decreasing bioactive auxin gradient from median-basal to peripheral locations across the leaf plane has been observed.     

Hormonal control of the outgrowth of axillary buds in <Emphasis Type="Italic">Alstroemeria</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

We study apical dominance in Alstroemeria, a plant with an architecture very different from the model species used in research on apical dominance. The standard explant was a rhizome with a tip and two vertically growing shoots from which the larger part had been excised leaving ca. 1 cm stem. The axillary buds that resumed growth were located at this 1-cm stem just above the rhizome. They were released by removal of the rhizome tip and the shoot tips. Replacement of excised tips by lanolin with indole-3-butyric acid (IBA) restored apical dominance. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-napthylphthalamic acid (NPA) reduced apical dominance. 6-Benzylaminopurine (BAP) enhanced axillary bud outgrowth but the highest concentrations (> 9 μM) caused fasciation. Thidiazuron (TDZ) did not show improvement relative to BAP. Even though the architecture of Alstroemeria and the model species are very different, their hormonal mechanisms in apical dominance are for the greater part very similar.     

Inhibition of Auxin Polar Transport Affecting the Model of Leaf Growth and Development

Tobacco (Nicotiana tabacum L. cv. Gexin No. 1) leaf slices were cultured in MS medium with different concentrations of auxin polar transport inhibitors (2, 3, 5-triiodobenzoic acid (TIBA), trans-cinnamic acid (CA), and 9-hydooxyflurence-9-carboxylic acid (HFCA)) and their effects on bud formation were observed. Although the effective concentrations vary with different inhibitors, all of them induced the formation of trumpet-shaped leaves. The frequencies of trumpet-shaped leaves were increased with the concentrations of inhibitors in media, and it was up to 82.1% when cultured in the medium containing 7.5 mg/L TIBA. The trumpet-shaped leaves were formed in different sites of the adventitious buds. These results indicated that inhibition of auxin polar transport could affect the morphogenesis of leaves, so the polar transport of auxin is essential for the bilateral symmetry of leaf growth.     

Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.)

Summary Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N ⁶-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.     

HORMONAL REGULATION OF EPIPHYLLOUS BUD RELEASE AND DEVELOPMENT IN BRYOPHYLLUM CALYCINUM

The effects of various concentrations of several auxins and cytokinins separately and in combination on epiphyllous budding of Bryophyllum calycinum were investigated, using a marginal leaf strip method. The number of buds released per leaf in 10⁻⁶ m benzyladenine (BA) was 2.8 times greater than those in water, and 1.6 times greater in 10⁻⁵ m isopentyladenine (IPA). BA, IPA and kinetin were antagonistic to indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) when used in combination treatments. The IAA oxidase co-factor p-coumaric acid (PCA) increased the number of epiphyllous buds to twice that of the water controls. This stimulatory effect of PCA on bud release suggests that an increase in IAA oxidase activity may be one of the significant changes that precedes the release of epiphyllous buds.     

Response to strigolactone treatment in chrysanthemum axillary buds is influenced by auxin transport inhibition and sucrose availability

Axillary bud outgrowth is regulated by both environmental cues and internal plant hormone signaling. Central to this regulation is the balance between auxins, cytokinins, and strigolactones. Auxins are transported basipetally and inhibit the axillary bud outgrowth indirectly by either restricting auxin export from the axillary buds to the stem (canalization model) or inducing strigolactone biosynthesis and limiting cytokinin levels (second messenger model). Both models have supporting evidence and are not mutually exclusive. In this study, we used a modified split-plate bioassay to apply different plant growth regulators to isolated stem segments of chrysanthemum and measure their effect on axillary bud growth. Results showed axillary bud outgrowth in the bioassay within 5 days after nodal stem excision. Treatments with apical auxin (IAA) inhibited bud outgrowth which was counteracted by treatments with basal cytokinins (TDZ, zeatin, 2-ip). Treatments with basal strigolactone (GR24) could inhibit axillary bud growth without an apical auxin treatment. GR24 inhibition of axillary buds could be counteracted with auxin transport inhibitors (TIBA and NPA). Treatments with sucrose in the medium resulted in stronger axillary bud growth, which could be inhibited with apical auxin treatment but not with basal strigolactone treatment. These observations provide support for both the canalization model and the second messenger model with, on the one hand, the influence of auxin transport on strigolactone inhibition of axillary buds and, on the other hand, the inhibition of axillary bud growth by strigolactone without an apical auxin source. The inability of GR24 to inhibit bud growth in a sucrose treatment raises an interesting question about the role of strigolactone and sucrose in axillary bud outgrowth and calls for further investigation.     

生长素极性运输的抑制对叶生长发育模式的影响

以烟草 ( Nicotiana tabacum L.品种“革新一号”)无菌幼苗叶片为材料 ,在 MS培养基中分别加不同浓度的生长素极性运输抑制剂 (三碘苯甲酸、反式肉桂酸、9-羟基芴 - 9-羧酸 )和 2 mg/ L BA ,探讨不定芽分化的情况 ,在这些培养基上都观察到不同形态的喇叭状叶片形成。结果表明 ,再生喇叭叶的发生频率与培养基中抑制剂一定范围内的浓度密切有关。当三碘苯甲酸浓度为 7.5 mg/ L 时 ,喇叭叶的发生频率最高可达到 82 .1 % ,在再生不定芽的不同位置均观察到有喇叭叶的发生。实验证明 ,抑制生长素的极性运输可导致叶形态发生改变 ,说明生长素的极性运输在叶片两侧对称性生长中有重要作用     

Alteration in the Geotropic Response of Allium Seedlings by Growth Regulators

Indoleacetic acid (1AA) brings about a striking alteration in the normal geotropic response in the seedlings of onion and several other species of Allium. The seedlings which are normally positively geotropic, become ageotropic. Besides IAA several other auxins like IBA, NAA and 2,4-D also showed a similar response. An anti-auxin like triiodobenzoic acid (TIBA) enhanced the auxin-effect instead of reversing it. A marked enhancement of the auxin-effect was also brought about when sucrose (in the culture medium) was replaced by L-arabinose which appeared to act synergistically with the auxin.     

Morphogenetic responses obtained from a variety of somatic explant tissues of data palm

Shoot tip, bud, leaf, stem and root explants from bearing trees, offshoots, seedlings, and asexual plantlets ofPhoenix dactylifera L. were cultured on modified Murashige and Skoog nutrient media containing 3 g/l activated charcoal, 100 mg/l 2,4-dichlorophenoxyacetic acid, 3 mg/l N ⁶-(Δ²-isopentyl)adenine to obtain callus. Differential morphogenetic responses were obtained from calli dependent on the explant type and parent source. Subcultured shoot tips and leafy lateral buds callus on nutrient media devoid of charcoal and supplemented with 0.1 mg/l α-naphthaleneacetic acid (NAA) produced adventitious plantlets. Subcultured leaf calli produced roots only. Root callus failed to exhibit any morphogenetic response upon subculturing. Undifferentiated non-leafy buds and stem tissues did not give rise to callus, regardless of the parent source. Generally, the best callus and embryogenetic responses from explants were obtained from seedling and plantlet parent sources. Similarly, organogenetic responses such as root formation and shoot development from shoot tips cultured on media containing 10 mg/l NAA were also related to the parent explant source. Mention of a trademark or proprietary product in the paper does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be available.