Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Role of other plant organs in gibberellic acid-induced delay of leaf senescence in alstroemeria cut flowers

Chlorophyll loss in leaves of cut flowers of alstroemeria (Alstroemeria pelegrina L. cv. Westland) was rapid in darkness and counteracted by irradiation and treatment of the flowers with gibberellic acid (GA₃). The mechanism of the effect of GA₃ under dark conditions was investigated. The content of various carbohydrates in the leaves under dark conditions rapidly decreased; this was not influenced by treatment with GA_3. indicating that the loss of carbohydrates in the leaves did not induce the loss of chlorophyll. Placing the cut flowers in various solutions of organic and inorganic nutrients exhibited no significant effect on the retention of chlorophyll in leaves of dark-senescing flowers. The total nitrogen content in leaves of dark-senescing cut flowers decreased with time. Leaves of GA₃-treated flowers retained more nitrogen. In contrast, the buds of GA₃-treated flowers retained less nitrogen during senescence in the dark than control buds. To investigate whether GA₃ affects export of assimilates from the leaf to various parts of control and GA₃-treated flowers, we labelled one leaf with radioactive carbon dioxide. ¹⁴C-assimilates accumulated preferentially in the flowers, in which the relative specific activity of the youngest floral buds was highest. No significant differences were observed in the distribution of ¹⁴C-labelled compounds between the buds of control and GA₃-treated flowers. To establish the importance of source-sink relations for the loss of leaf chlorophyll we removed the flower buds (i. e. the strongest sink) from the cut flowers. This removal only slightly delayed chlorophyll loss as compared to the large delay caused by GA₃-treatment. In addition, detached leaf tips exhibited chlorophyll loss in the dark, which was delayed by GA₃-treatment in a fashion comparable with that in flowers. Together these data demonstrate that interactions of the leaves with other plant organs are not essential for chlorophyll loss during senescence in the dark. Additionally, we have found no evidence that GA₃ delays the loss of chlorophyll by affecting the transport of nutrients within the cut flowers.     

Effect of Gibberellin and Auxin on Parthenocarpic Fruit Growth Induction in the cv Micro-Tom of Tomato

The effect of applied gibberellin (GA) and auxin on fruit-set and growth has been investigated in tomato (Solanum lycopersicum L.) cv Micro-Tom. It was found that to prevent competition between developing fruits only one fruit per truss should be left on the plant. Unpollinated ovaries responded to GA₃ and to different auxins [indol-3-acetic acid, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-D)], 2,4-D being the most efficient. GA₃- and 2,4-D-induced fruits had different internal morphology, with poor locular tissue development in the case of GA, and pseudoembryos development in the case of 2,4-D. Also, GA₃ produced larger cells in the internal region of the mesocarp (IM) associated with higher mean C values, whereas 2,4-D produced more cell layers in the pericarp than pollinated fruits. The smaller size of GA₃- compared with 2,4-D-induced fruits was due to them having fewer cells, only partially compensated by the larger size of IM cells. Simultaneous application of GA₃ and 2,4-D produced parthenocarpic fruits similar to pollinated fruits, but for the absence of seeds, suggesting that both kinds of hormones are involved in the induction of fruit development upon pollination. It is concluded that Micro-Tom constitutes a convenient model system, compared to tall cultivars, to investigate the hormonal regulation of fruit development in tomato.     

Phytohormones et transport orienté au niveau de la feuille isolée dePelargonium zonale: compétition phytohormonale

Hormone-directed transport is shown in a detached adult leaf ofPelargonium zonale, after a local application of solution (0.1 ml) of various groups of phytohormones (GA3 -BAP-AIA-ANA-2,4-D). Importance of isolated leaf as a simplified experimental system is underlined by the positive results obtained with³⁵S (or³²P). The idea of competition between two attractive foliar centers, induced by hormones is illustrated:

1. with regard to the concentration of the same hormone (higher effect at 25 mg than at 2.5 mg.l-1 with GA₃ for example);
2. when two different hormones are competitive on the same leaf. In this respect, GA3 appears to be the most effective hormone tested on this material.

Finally, the idea of a hormone-directed transport, independent of the antisenescence effect is supported by the fact that an orientated translocation remains induced after general pretreatment with an alternative hormone (BAP and GA₃, for instance).     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs

The interrelationship between ethylene and growth regulators in the senescence of romaine lettuce (Lactuca sativa L.) leaves was studied. Gibberellic acid (GA₃), kinetin, and 3-indoleacetic acid (IAA) retarded chlorophyll loss from leaf discs which were floated on hormone solutions. Abscisic acid (ABA) and ethephon enhanced chlorophyll loss and antagonized the senescence-retarding effect of GA₃ and kinetin. A high concentration of IAA (10^–4 M) caused accelerated chlorophyll loss, whereas a similar concentration of kinetin neither retarded nor promoted chlorophyll loss. The ineffectiveness of IAA and kinetin at their supraoptimal concentrations in retarding leaf senescence was related to increased production of ethylene induced in the treated leaf discs. GA₃ was the most effective in retarding chlorophyll loss and did not stimulate ethylene production at all. The senescence-enhancing effect of ABA was not mediated by ethylene. However, the moderately increased production of ethylene, induced by relatively high concentrations of ABA, could act synergistically with the latter to accelerate chlorophyll loss. It is proposed that the effectiveness of exogenously applied hormones, both in enhancing and retarding senescence, is greatly affected by the endogenous ethylene concentration of the treated plant tissue.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2571-E, 1988 series.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Endogenous gibberellin levels and senescence in Taraxacum officinale

Summary The level of endogenous gibberellins (GAs) in leaf tissue of Taraxacum officinale was high during leaf growth and expansion but declined progressively during leaf senescence. In the chromatographic system used, most of the GA from Taraxacum leaves moves with the R_f of GA₃. However, several other GAs were also effective in retarding senescence in Taraxacum leaves. It is concluded that ageing of dandelion leaves is associated with a deficiency of endogenous GA.     

Organ-specific effects of the auxin herbicide 2,4-D on the oxidative stress and senescence-related parameters of the stems of pea plants

Oxidative stress and senescence have been shown to participate in the toxicity mechanism of auxin herbicides in the leaves and roots of sensitive plants. However, their role in stem toxicity has not been studied yet. In this work, we report the effect of foliar or root applications of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the parameters of oxidative stress and senescence of stems of pea (Pisum sativum L.) plants. Contrary to their effect on the pea leaves, in the stems 2,4-D applications did not cause oxidative stress, as shown by the parameters of lipid peroxidation, protein carbonyls, and total and protein thiols. Moreover, they inhibited the superoxide radical (O₂^.−)-producing xanthine oxidase (XOD) activity and stimulated the antioxidant activities of catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST) and Krebs cycle NAD⁺-isocitrate dehydrogenase (IDH). Applications of 2,4-D also did not induce senescence in the pea stems, as shown by the increase of proteins, the lack of stimulation of proteolytic activity, and the inhibition of senescence-related isocitrate lyase (ICL) activity. However, they stimulated the H₂O₂-producing acyl-CoA oxidase (ACOX) activity of fatty acid beta oxidation. Results suggest that oxidative stress and senescence are not involved in the mechanism of toxicity of 2,4-D in the stems of pea plants, and that these phenomena are not whole-plant toxicity mechanisms for auxin herbicides in susceptible plants. Results also suggest that the effect of 2,4-D on the oxidative metabolism of pea plants might be organ-specific.     

Effect of gibberellic acid and 2,4-D on water hyacinths

Combinations of gibberellic acid (GA₃) and 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated to determine if GA₃ increased the effectiveness of 2,4-D on water hyacinths (Eichhornia crassipes (Mart.) Solms) under field conditions. Bulbous-leaved water hyacinths were grown outdoors in north Florida in 70-1 containers during the 1980–1982 growing seasons with combinations of GA₃ at 0, 23.5, 47.0, 94.0 and 188 g ha^?1 and 2,4-D at 0, 0.28, 0.56 1.12 and 2.24 kg ha^?1. Significant increases in the effectiveness of 2,4-D in combination with GA₃ were observed at lower rates of 2,4-D. However, these increases appeared to be additive since regression analysis revealed no significant interaction or synergism between GA₃ and 2,4-D at any level.     

The Effects of Gibberellin4+7 on the Vase Life and Flower Quality of Alstroemeria Cut Flowers

Leaf yellowing is a major problem in Alstroemeria and absence of leaf senescence symptoms is an important quality attribute. Two Alstroemeria cultivars ‘Yellow King’ and ‘Marina’ were sourced from a commercial farm and harvested when sepals began to reflex. Stems were re-cut under water and kept in vase solutions of gibberellin A₄₊₇ (0, 2.5, 5.0, 7.5, 10.0, 12.5 or 15.0 mg l⁻¹ [Provide^r]). Treatments and cultivars were combined in a factorial fashion and arranged in a completely randomised design. Application of GA₄₊₇ in the holding solution at 2.5–10.0 mg l⁻¹ significantly delayed the onset of leaf senescence by around 7 days and significantly increased days to 50% petal fall by ca. 2 days. Additionally, these GA₄₊₇ concentrations resulted in higher retention of leaf nitrogen, leaf chlorophyll and also increased leaf water content, while reducing leaf dry weight, all relative to untreated controls. Cultivar ‘Yellow King’ had significantly longer vase life and a better retention of leaf quality than ‘Marina’. Our results suggest that a concentration of 10 mg l⁻¹ GA₄₊₇ can be used to prolong vase life, delay leaf senescence and enhance post-harvest quality of Alstroemeria cut flowers during their transport to market.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997     

GA4 Does Not Require Conversion into GA1 to Delay Senescence of Alstroemeria hybrida Leaves

The biological activity and metabolism of applied GA₁ and GA₄ were studied in leaves of alstroemeria (Alstroemeria hybrida). It appeared that GA₄ was 2 orders of magnitude more active in delaying leaf senescence than GA₁. GA₃-13-OMe, a GA analog that cannot be hydroxylated on the 13-C position, also retarded chlorophyll loss, although less efficiently. Tritiated and deuterated GA₁, GA₄, and GA₉ were applied to leaves, and their metabolites were analyzed. According to high performance liquid chromatography and gas chromatography-mass spectrometry analyses, GA₉ was converted into GA₄ and GA₃₄, and GA₄ was converted into GA₃₄ and more polar components. No evidence was found for the conversion of both GA₉ and GA₄ into GA₁, even at the relatively high concentrations that were taken up by the leaf. The results strongly suggest that GA₄ is recognized directly by a receptor involved in regulation of leaf senescence in alstroemeria. Received November 24, 1997; accepted February 17, 1998     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

Effect of 2,4-dichlorophenoxyacetic acid on the dark- and light-induced pulvinar movements in Cassia fasciculata Michx

2,4-dichlorophenoxyacetic (2,4-D) applied to excised leaves of Cassia fasciculata modified the dark-induced (scotonasty) and light-induced (photonasty) leaflet movements, showing that this compound acts on rapid turgor variation and the concomitant ion migrations, in particular K⁺. 2,4-D inhibited the scotonastic closure in a dose-dependent manner from 10^–8 M to 10^–5 M and promoted the photonastic opening in the same range of concentrations. The compound acted rapidly since a treatment as short as 5 min gave an obvious effect on the motile reaction; however, a lag period of 45–60 min was needed to observe its effect. Although 2,4-D is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. The physiological results are discussed in relation to the chemical properties and the characteristics of transport of the molecule.Abbreviations ABA abscisic acid - 6-BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)-ethanesulphonic acid     

Morphogenesis from cultured leaf tissue ofSorghum bicolor-culture initiation

Summary In this paper we present further studies on the generation of tissue cultures from leaves of the cerealSorghum bicolor (L.) Moench. It could be shown that during differentiation the leaf tissue rapidly loses the ability to respond to conventional tissue culture techniques. This was probably related to a loss of sensitivity towards 2,4-D, an otherwise most potent growth regulator in tissue culture. The immature tissue which proved to be sensitive proliferated over a wide range of concentration with a broad optimum of about 0.6–6 mg 1^–1 2,4-D. This concentration range appears to be only slightly higher than that described for many dicotyledonous tissue cultures. The relevance of these findings is discussed with reference to the well known dual function of 2,4-D, namely as a selective herbicide and a potent artificial auxin. The implications of these attributes to the practical application of cereal tissue culture is stressed.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - 4-CPA 4-Chlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA 3-Indoleacetic acid - Kinetin 6-Furfurylaminopurine - 6-BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - ABA Abscisic acid - MS Murashige and Skoog     

Ammonium and proline accumulation in senescing cut leaves of <Emphasis Type="Italic">Zantedeschia</Emphasis>

Accumulation of ammonium and proline were reported as phenomena associated with plant response to stress and/or senescence. The effects of a preservative (8HQC + sucrose) and 24 hrs pulse conditioning with GA₃ on the ammonium and proline contents were studied in senescing cut leaves of Zantedeschia aethiopica Spr. and Z. elliottiana Engl., grown for the florists green. Generally, accumulation of both compounds was observed in senescing leaves, however, the final ammonium and proline levels depended upon the species and the treatment applied. Conditioning with GA₃, a treatment known to delay leaf senescence in Zantedeschia sp., prevented the increases in the ammonium and proline contents. Standard preservative solution used to prolong the longevity of cut flowers enhanced the ammonium accumulation in senescing leaves of both species, and the proline accumulation in the leaves of Z. aethiopica, but not in Z. elliottiana. These observations suggest that neither ammonium nor proline accumulation would be fully reliable predictors of cut leaf freshness during their entire market life. However, proline accumulation could serve as a quick test of freshness in the first half of the useful market life of cut leaves of Zantedeschia.     

Hormonal Aspects of Senescence in Detached Tobacco Leaves

The objective of the present work was to describe the simultaneous changes in endogenous levels of cytokinins, abscisic acid, indoleacetic acid and ethylene in detached, senescing tobacco (Nicotiana rustica L.) leaves. These measurements were related to changes in chlorophyll contents, ¹⁴CO₂ fixation and proline contents — three parameters which have been considered to reflect senescence. Effects of exogenous hormonal treatments on these parameters, as well as on endogenous hormonal levels, provided further evidence for the interrelationships between hormones and for their roles in senescence. Starting with actively growing attached leaves and ending with well-advanced senescence in detached leaves, our data indicate a chronological sequence of three hormonal states: (a) cytokinins — high activity, abscisic acid, auxin and ethylene — low contents (actively growing, attached leaves); (b) cytokinins — low activity, abscisic acid — high, auxin and ethylene — low contents (apparent induction of senescence in detached leaves); and (c) cytokinins and abscisic acid — low, auxin and ethylene — high contents (senescence proper in detached leaves).     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.