Physiology of gibberellin-induced elongation of epicotyl explants from Vigna sinensis

The physiological characteristics of the response of excised cowpea (Vigna sinensis cv Blackeye pea No. 5) epicotyls to gibberellins (GAs) were studied. Epicotyl explants, retaining the petioles and a 2-cm portion of hypocotyl, were placed upright in small vials containing water. Plant growth substances were injected into the subapical tissues as ethanol solutions.Epicotyl elongation resulting from treatment with 0.5 g of GA ranged between 5 and 13 times that of the control, depending on the GA applied. With GA₁, no differences were obtained with explants prepared from 5 to 9-day-old seedlings. The increase in elongation could be detected within 6 h of treatment, and the stimulus of a single application lasted at least 4 days. Final elongation was proportional to the logarithm of the amount of GA, applied, 0.01 to lug. The response to GA treatment was limited to the upper part, the most sensitive zone being located between 2 to 4 mm below the apex of the epicotyl; this effect was entirely due to cell elongation.The induction of epicotyl elongation by GAs seems to be specific and independent of the effect of auxin. IAA had no effect on elongation and 4-chloro-phenoxyisobutyric acid (PCIB) did not affect the response to GA₁ Abbreviations ABA abscisic acid - GA gibberellin - IAA Indole-3-acetic acid - TIBA 2,3,5-triiodobenzoic acid - PCIB 4-chloro-phenoxyisobutyric acid     

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l^–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l^–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l^–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.     

The role of acidification in gibberellic acid- and fusicoccin-induced elongation growth of lettuce hypocotyl sections

The roles of gibberellic acid (GA₃) and fusicoccin (FC) in the elongation growth and acidification of the medium by excised hypocotyl sections of lettuce (Lactuca sativa L.) were investigated. Hypocotyl sections incubated in buffer without GA₃ elongate optimally at pH 4.0–4.25 while sections incubated with GA₃ show the same growth between pH 4.25 and 6.0. Preincubation of sections at pH 6.0 for 6 h does not affect the subsequent elongation response to acidic medium (pH 4.25); however, the sections become refractory to further acid treatment after their initial burst of growth in response to pH 4.25. Sections made refractory to acid are responsive to GA₃ application, however, and the rate of growth in response to GA₃ of sections pretreated for 6 h at pH 4.25 is 85% of that of sections pretreated at pH 6.0. Although preincubation of sections for 48 h in medium at pH 6.0 abolishes the GA₃ response, it does not affect the response to buffer at pH 4.25. FC stimulates elongation growth in letuce hypocotyls at an optimal concentration of 1 M, and pretreatment of sections at pH 4.25 does not affect this elongation response. Although both GA₃ and FC increase elongation of the section, neither causes appreciable acidification of the medium. Addition of KCl or NaCl to FC-treated sections causes rapid medium acidification but addition of salts to GA₃-treated tissue does not cause acidification. Abrasion of the hypocotyl to remove the cuticle does not enhance acidification of the medium by the sections nor deos it affect elongation of the sections in response to GA₃ or FC. Medium acidification by the sections is not a passive process since it is abolished both by low temperature (2° C) and metabolic inhibitors (carbonyl cyanide-m-chlorophenyl-hydrazone, azide). The acidification of the medium by barley (Hordeum vulgare L.) roots in response to FC is also dependent on the presence of KCl. We conclude that the acid-growth hypothesis does not explain GA₃- or FC-induced elongation in lettuce hypocotyls.Abbreviations FC tusicoccin - GA₃ gibberellic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - MES 2-(N-morpholino)ethanesulphonic acid - Tris tris-(hydroxymethyl)aminomethane     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Regulation of cabbage (Brassica oleracea capitata) hypocotyl elongation, and infection by Peronospora parasitica, with B-indolylacetonitrile and chloramphenicol

Untreated excised segments of the hypocotyls of dark grown cabbage seedlings are always systemically infected when inoculated with the conidia of the obligate parasite Peronospora parasitica (Downy mildew). Cabbage hypocotyl elongation is promoted by 10^–4 M indolylacetonitrile (IAN) and this elongation is inhibited by 100 g mL^–1 chloramphenicol (CAM). The fungus remains localized in 5–8 day old hypocotyl segments exposed to CAM, but this inhibition is reversed by IAN. Indol-3-acetic acid (IAA) has the same effect as IAN. Both gibberellic acid and kinetin inhibit systemic infection. Conidial spore germination is not reduced by the CAM concentration used in these experiments. The success of the pathogen in the host is not correlated with host elongation, but is probably related to a common metabolic site in either host or pathogen affected by both CAM and IAN.Abbreviations IAN indolylacetonitrile - CAM chloramphenicol - IAA indol-3-acetic acid - G gibberellic acid - K kinetin     

Epidermis integrity and epicotyl growth in azuki bean

In order to verify if epidermis integrity played a determinant role in epicotyl elongation induced by fusicoccin (FC), buffers at different pH's, and indoleacetic acid (IAA), we studied the short-term kinetics of elongation growth, the increase of fresh weight in long-term treatment, and the H⁺ excretion in intact, abraded, and peeled azuki bean epicotyl sections. We demonstrated that the epidermis is more sensitive to IAA, whereas the cortex is highly responsive to protons. Our data are consistent with the acid growth theory. In addition, our studies support the idea that the epidermis may be the tissue target for auxin, but its integrity is necessary for IAA-induced elongation.     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Regeneration of peppermint and orange mint from leaf disks

Leaf disks from peppermint, spearmint, orange mint, lavender mint and Scotch spearmint were cultured on various Murashige-Skoog-based media in order to regenerate shoots. A significantly larger average number of orange mint leaf disks regenerated shoots on basal medium containing 44.4 M benzyladenine (BA) and 250 ml l^-1 coconut water (CW). Shoots regenerated from peppermint leaf disks cultured on basal medium containing 44.4 M BA and 250 ml or 450 ml l^-1 CW. The most shoots regenerated from orange mint leaf disks cultured on medium containing 10 g l^-1 washed Difco Bacto-agar. Disks excised from the bases of the first expanding pair of orange mint leaves cultured under dark conditions regenerated a significantly larger average number of shoots. Histological studies suggested that shoots regenerated from the palisade parenchyma cells associated with vascular tissue.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TIBA 2,3,5-triiodobenzoic acid - CW coconut water     

Initiation and propagation of Glycine max L. Merr.: Plants from tissue-cultured epicotyls

A successful technique for the initiation and proliferation of shoots from epicotyl tissue of soybean, Glycine max, has been developed and is described. Fertile plants were recovered. Seeds were germinated on Murashige and Skoog medium containing 5 M benzyladenine. Explanted epicotyl sections were induced to form callus and shoots on Schenk and Hildebrandt medium containing 5.2 mM monobasic ammonium phosphate, 74 M 3-aminopyridine, and 20 M kinetin for five weeks. Shoot proliferation was maintained on N6 medium containing 1.75 mM ammonium sulfate, 2.1 nM picloram, and 0.1 M benzyladenine. Shoots rooted on Gamborg's B5 medium without growth regulators. Shoot-forming cultures were maintained for 60 months. Although all varieties tested produced shoots, some variation in numbers of shoots obtained was observed.     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

Comparison of the effects of epibrassinolide and steroidal estrogens on adventitious root growth and early shoot development in mung bean cuttings

Concentrations of 24-epibrassinolide as low as 0.1 μ M consistently inhibited adventitious root formation and elongation in both hypocotyl and epicotyl cuttings from mung bean ( Phaseolus aureus L.). Similar, but less pronounced, inhibitory effects on root elongation were also observed with estrone sulphate and estradiol sulphate. With regards to root number, estrone sulphate enhanced this in both types of cutting, whereas estradiol sulphate was stimulatory in hypocotyl cuttings but inhibitory in epicotyl cuttings. Brassinolide caused a marked stimulation of epicotyl (but not hypocotyl) elongation and a swelling and splitting of the epicotyl in both types of cutting, whereas estrogens varied in their effect from inhibition of epicotyl growth to no effect. Root-applied brassinolide and estrogen sulphates brought about similar morphological abnormalities in shoots viz. epinasty and inrolling of primary leaves and delayed expansion of the first trifoliate leaf.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Regeneration of plants from leaf explants of Cucumis melo cv. Pusa Sharbati

Leaves of three different sizes excised from 14, 21, 28 and 35-day-old seedlings of Cucumis melo were cultured on a MS medium supplemented with a range and combination of growth regulators. Maximum shoot differentiation from the leaf explants occurred in the combined presence of BAP and 2iP at equimolar concentration of 1 M. Regeneration potential of leaves declined with increasing size of the leaves and the age of the donor seedlings. For elongation the shoots were transferred to MS+BAP [1 M]. Such shoots were rooted with 75% frequency on MS+IAA [0.5 M]. The plants have been established in pots.Abbreviations BM Basal Medium - MS Murashige and Skoog - BAP 6-Benzyl amino-purine - 2iP 6- V, V -dimethylallylamino purine - IAA Indole-3-acetic acid     

In vitro propagation ofMitragyna parvifolia Korth.

Multiple shoot formation and their elongation from excised apical vegetative shoots of a 40-year old-tree ofMitragyna parvifolia Korth. was achieved in Murashige and Skoog's medium supplemented with 4.44 M benzyl adenine. The in vitro regenerated shoots rooted when cultured on modified Murashige and Skoog's medium containing low inorganic salts and the three auxins. Regeneration by this method was suitable for mass propagation of the plant.     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 M N⁶-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 M indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 M IBA + 0.5% activated charcoal + 1 M BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.     

Gibberellin-induced ethylene production and its effect on cowpea epicotyl elongation

The effect of gibberellin A₁ (GA₁) on production of ethylene by cowpea (Vigna sinensis cv Blackeye pea no. 5) epicotyl explants and its relationship to epicotyl elongation was investigated. The explants were placed upright in water and incubated in sealed culture tubes or in large jars. GA, and IAA in ethanol solution were injected into the subapical tissues of the decapitated epicotyls. Cowpea epicotyl explants elongated after GA but not after IAA treatment, and they were very sensitive to exogenous ethylene. As little as 0.14 1/1 ethylene reduced significantly GA₁-induced epicotyl elongation.Treatment with GA₁ induced the production of ethylene which began 10 h after GA application, showed a peak at about 22 h and then declined. The yield of ethylene was proportional to the amount of GA, injected. The inhibition of epicotyl elongation in closed tubes was avoided by absorbing ethylene released with Hg(Cl0₄)₂ , or by adding AVG to the incubation solution to inhibit ethylene production. Treatment with IAA elicited a rapid production of ethylene which ceased about 10 h after application. The effects of IAA and GA₁ on ethylene production were additive.Abbreviations AVG aminoethoxyvinylglycine 2-amino-4-(2-aminoethoxy)-trans-3butenoic acid - ACC 1-aminocyclopropane-1-carboxylic acid - GA gibberellin - IAA indole-3-acetic acid     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Enhanced shoot regeneration from Brassica campestris by silver nitrate

Summary The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l^–1 NAA, 2.0 mg.l^–1 BAP, and 30 or 60 M AgNO₃ significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO₃ with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO₃. Cobalt chloride at 20 and 30 M increased cotyledon shoot regeneration but was inferior to AgNO₃. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 M prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO₃ after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl₂. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.Abbreviations NAA Naphthalene Acetic Acid - BAP 6-Benzylamino Purine - MS Murashige and Skoog Medium     

Changing proton concentrations at the surfaces of gravistimulated Phleum roots

The pH patterns at the surfaces of both vertically growing roots of Phleum pratense L. and roots tilted by 45° were recorded using H ⁺-sensitive microelectrodes. During vertical growth the root cap exhibited lower pH values than the meristematic zone. The highest pH values were found at the border between meristematic and elongation zones. In the apical part of the elongation zone the values strongly decreased basipetally. They reached a minimum value of pH 5.4–5.5 (medium pH of about 6.0) at a distance of 700 m from the root tip. This region of strongest acidification usually coincided with that of the highest relative rates of elongation. The region of the first visible curvature following gravistimulation was positioned at 100–200 m more apically. The pH values increased in the basal elongation zone towards the mature zone. The H⁺-flux pattern around a vertically growing Phleum root was characterized by high influxes in the meristematic zone and smaller effluxes in the elongation zone. Tilting the root by 45° induced changes in the pH values of the upper and lower sides of a Phleum root. At a distance of 300–500 m from the root tip, the upper side was strongly acidified while the pH of the lower side slightly increased compared with the values during vertical orientation. pH differences of up to 0.9 pH units between the two sides of a root were detected. These differences decreased basipetally and could not be measured more distant than 700–800 m from the tip. Compared with a vertically growing root, the H⁺-flux pattern of a Phleum root tilted by 45° exhibited effluxes on the entire upper organ flank while the pattern was scarcely altered on the lower side. The curvature-initiating zone in Phleum roots is positioned within that section of the root in which pH changes occur after tilting. The region of highest pH differences, however, is nearer to the apex of the root than the curvature-initiating zone. The pH changes began 8.2 min after a root had been tilted. The bending process started after 17.2 min, i.e. after double the time needed for differential acidification. After reorienting a root, which had just begun to bend, to its previous vertical position the inversion of the pH gradient could be measured within the same mean time of about 8 min. This is again significantly earlier than the beginning of the rebending process. The results indicate that, during the graviresponse, ionic movements occur much earlier than the changes in hormonal activities reported in the literature.Abbreviation CIZ curvature-initiating zone A preliminary report was presented at the 29th Plenary Meeting of the Committee on Space Research (COSPAR) in Washington D.C., USA, 28 Aug – 5 Sept 1992 (Zieschang and Sievers 1993)This work was supported by the Deutsche Forschungsgemeinschaft. We thank Professor H. Felle (Botanisches Institut, Universität Gießen, Gießen, FRG) for practical instructions concerning the method of H⁺-sensitive microelectrodes and Professor W. Simonis (Botanisches Institut, Universität Würzburg, Würzburg, FRG) for allowing to use the microelectrode amplifier.