Adaptation to dim-red light leads to a nongradient pattern of stem elongation in cucumis seedlings

Relative growth rate determinations on 5-millimeter regions of cucumber (Cucumis sativus L.) hypocotyls show that dim-red light-grown seedlings have an even distribution of growth along the stem axis. This contrasts with the apical to basal graded decline in growth rate seen in dark-grown seedlings, including dark-grown cucumber seedlings used as controls in this study. Dark-grown seedlings convert to the nongradient pattern when transferred to dim-red light. The small amount of light required suggests that the change in developmental pattern may happen in the natural light environment.     

Slow wave potentials in cucumber differ in form and growth effect from those in pea seedlings

A positive hydraulic signal in the form of a xylem pressure step was applied to the roots of intact seedlings of Cucumis sativus L. and Pisum sativum L. Surface electrodes at three positions along the epicotyl/hypocotyl recorded a propagating depolarization which appeared first in the basal, then the central and sometimes the apical electrode positions and fitted the characteristics of a slow wave potential (SWP). This depolarization differed between pea and cucumber. It was transient in cells of pea epicotyls but sustained in cucumber hypocotyls. It was not associated with a change in cell input resistance in pea epicotyls but preceded an increase in the input resistance of cucumber hypocotyl cells. With the increased xylem pressure the growth rate (GR) of cucumber hypocotyls and pea epicotyls underwent a transient increase, peaking after 5 min. If the depolarization reached the growing upper region, it preceded a sustained decrease in the GR of cucumber hypocotyls but only a transient decrease in the GR of pea epicotyls. A temperature jump in the root medium (heat treatment) induced a steep pressure spike in the xylem of the cucumber hypocotyl which showed similar electric and growth effects as the previously applied, non-injurious pressure steps. We suggest that the observed differences in the electric and growth responses between the species were caused by the closure of ion channels in depolarized cells of cucumber but not pea seedlings.     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

The Influence of Cotyledons in Axillary and Adventitious Shoot Production from Cotyledonary Nodes of Cucumis sativus L. (Cucumber)

Cucumber explants including at least part of the cotyledon,a short section of hypocotyl, and the apical bud, are capableof producing multiple axillary buds from the seedling apex andadventitious shoots from the hypocotyl base in a medium whichcontains 2·0 mg dm^–3 of kinetin. Removal of theapical bud triples the number of shoots produced from the apexof explants with two intact cotyledons but does not affect shootproduction from explants with some or all of their cotyledonsremoved. The area of intact cotyledon also influences morphogenesis,as explants with both cotyledons removed, failed to produceadventitious shoots from the hypocotyl base. Culture in continuousdarkness entirely prevents shoot development from the explantbase, but has little influence on shoot production from theapex. The influence of endogenous growth regulators and apicaldominance on the morphogenesis of shoots in cucumber seedlingsare discussed. Key words: Cucumber, cotyledons, apical dominance, morphogenesis, adventitious shoots, Cucumis sativus     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration

The effect of addition of silver nitrate (AgNO₃) on organogenesis of proximal and distal cotyledon and hypocotyl explants of five cucumber (Cucumis sativus L.) cultivars was investigated. Distal cotyledon and hypocotyl were unresponsive while only poor shoot regeneration was observed in proximal cotyledon and hypocotyl explants of all cucumber cultivars. The addition of different concentrations of AgNO₃ (10, 30 and 50 μM) to the medium, however, induced shoot regeneration in distal cotyledon except Suyo Long cultivar and effectively increased shoot regeneration response as well as the number of shoots per explant in proximal cotyledon and hypocotyl of all cucumber cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation

A reliable plant regeneration system is described for the production of adventitious shoots from root explants of spinach. Explants from roots of axenic shoots and roots induced on cultured hypocotyl explants were used for adventitious shoot induction. Explants from apical, middle and basal root regions were incubated on Nitsch and Nitsch medium supplemented with α-naphthaleneacetic acid, gibberellic acid and kinetin. Optimum shoot regeneration was from explants of apical and middle root regions on medium with 20 μm α-naphthaleneacetic acid and 5.0 μm gibberellic acid. Shoots originated directly from root tissues without an intervening callus phase. Adventitious shoots were rooted and were grown to maturity in the glasshouse. This plant regeneration procedure has been exploited in preliminary studies of Agrobacterium-mediated transformation. Received: 27 February 1996 / Revision received: 22 August 1996 / Accepted: 30 September 1996     

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

The Role of Endogenous Auxins and Ethylene in the Formation of Adventitious Roots and Hypocotyl Hypertrophy in Flooded Sunflower Plants (Helianthus annuus)

The role of ethylene and auxins in flood-induced adventitious root formation and hypocotyl hypertrophy in sunflower (Helianthus annuus L. cv. Russian) plants was studied. Flooding without aeration (F) resulted in a steady increase in ethylene in hypocotyls, and flooding with aeration (FA) caused a transient increase. Low light intensity increased ethylene levels but decreased adventitious root formation. Treatment of shoots with benzyladenine (BA) increased ethylene content in non-flooded (NF) but not in F or FA shoots. Twenty-four hours of flooding brought about a rise of endogenous indole-acetic acid (IAA) in hypocotyls. ¹⁴C-IAA applied to the shoot accumulated more in F and FA hypocotyls than in NF hypocotyls, and BA reduced this accumulation. There was less IAA metabolism in F and FA than in NF hypocotyls. Tri-iodo benzoic acid (TIBA) applied to the hypocotyls of F plants inhibited root production. Benzyladenine (BA) applied to the leaves had similar effect but was not effective when supplied to the shoot apex. BA did not inhibit flood-induced hypocotyl hypertrophy. Ethrel did not affect adventitious root formation in NF plants but did increase hypocotyl thickening. It is concluded that flood-induced adventitious root formation is stimulated primarily by an accumulation of auxins in the hypocotyls. Increases in ethylene might cause this auxin build up. Hypocotyl hypertrophy is presently thought to be the result of an interaction of auxin and ethylene with ethylene being the major factor.     

Protochlorophyll(ide) forms in hypocotyls of dark-grown bean (Phaseolus vulgaris)

The protochlorophyll(ide) forms and plastid ultrastructure were investigated in hypocotyls of dark-grown seedlings of kidney bean ( Phaseolus vulgaris L. cv. Brede zonder draad). By deconvolution of the fluorescence emission spectra into Gaussian components three protochlorophyll(ide) forms were found with maxima at 633, 642 and 657 nm, respectively. The ratio of protochlorophyll(ide) emitting at 657 nm to protochlorophyll(ide) emitting at 633 nm decreased downwards the hypocotyl. The gradient was established already after 4 days in dark-grown Phaseolus and was also seen in hypocotyls of 7-day-old dark-grown plants of 8 other species. Ultrastructural observations revealed a plastid developmental sequence along the hypocotyl. Plastids in the upper parts of the hypocotyl contained prolamellar bodies typical of etiolated leaves while those in the lower parts contained only stroma lamellae. Immunological detection of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) on nitrocellulose membranes after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated the occurrence of the enzyme in upper, middle and lower sections of hypocotyls and in the root tips.     

Differential Responses of Buds along the Shoot to Factors Involved in Apical Dominance

Intact and decapitated 6-node shoots of Hygrophila sp. weregrown aseptically immersed in liquid half-strength Knop's solutionwith microelements and 2% (w/v) sucrose (control medium), andin medium with 0.1 mg l^–1 benzyladenine (BA). In intactshoots grown in control medium apical dominance suppressed outgrowthof the lateral buds; in decapitated shoots buds grew out atseveral of the most apical nodes, increasing in size acropetally.There was a lag in outgrowth of the bud at the most apical node,attributable to its initially smaller size. Lateral shoots grewout first at basal nodes of intact shoots in BA medium, decreasingin size acropetally; in decapitated shoots in BA medium lateralshoots of approximately equal size grew out at all nodes. Differentialeffects of decapitation and cytokinin treatment on lateral shootoutgrowth along the shoot could be interpreted by postulatinga basipetally decreasing gradient of endogenous auxin concentrationin the intact shoot. Application of 20 mg l^–1 indoleaceticacid (IAA) in agar to decapitated shoots completely preventedbud outgrowth for at least 7 d in control medium, inhibitingit thereafter, and inhibited bud outgrowth in BA medium, thussupporting the hypothesis. Comparison of lateral shoot outgrowthin whole decapitated shoots and severed decapitated shoots (isolatednodes) lent no support to the alternative hypothesis that theremight be an acropetally decreasing concentration gradient ofa bud-promoting substance in the intact shoot, and demonstratedmuch greater lateral shoot growth in isolated nodes. The resultsemphasize important correlative relationships between the partsof a shoot with several nodes.     

Biological activities of indoleacetylamino acids and their use as auxins in tissue culture

THE AUXIN ACTIVITIES OF A NUMBER OF INDOLEACETYLAMINO ACID CONJUGATES HAVE BEEN DETERMINED IN THREE TEST SYSTEMS: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-l-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-l-alanine and indoleacetylglycine. The other conjugates inhibit shoot formation weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-l-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.     

Natural Inhibitors in Normal and Dwarf Peas

Extracts of light- and dark-grown, normal and dwarf pea seedlings(Pisum sativum L. cv. Alaska and Radio respectively) were purifiedby solvent partitioning, column, paper and thin layer chromatography.Conventional acid-base partitioning was modified because thelarge volumes of material processed caused considerable crosscontamination between neutral and acidic phases. At each stepof the purification, fractions were tested for inhibitory activitywith the wheat coleoptile and pea section tests. Recovery ofabscisic acid was monitored using ¹⁴C-abscisic acid. Estimatesof abscisic acid content were marie using gas-liquid chromatographyand the wheat coleoptile bioassay. Two main inhibitors were found; one of these was identifiedas (+)-abscisic acid, the other (inhibitor Y) has not been identifiedbut displays chromatographic properties which suggest that itis neutral in nature. Abscisic acid was found in both rootsand shoots of light- and dark-grown pea seedlings. InhibitorY was found in trace amounts in the roots of dark-grown plantsbut could not be detected in the shoots. Growth in light induceda manifold increase in inhibitor Y concentration compared withdark-grown plants. The level of Y was threefold greater in light-growndwarf shoots than in comparable light-grown tall shoots. Therewas, thus, a correlation between the concentration of inhibitorY and the light-induced inhibition of stem elongation.     

Absence of Red Light-Induced Stimulation of Growth in the Hook of Etiolated Dwarf-Pea Seedlings

Red light inhibited the growth of the apical part of the hookin dark-grown seedlings of a dwarf variety (cv. Progress No.9) of pea (Pisum sativum L.), whereas it promoted such growthin a tall variety (cv. Alaska). In the elongation zone of theepicotyl of the dwarf variety the extent of inhibition of growthwas similar to or even smaller than that in the tall variety.Local irradiation of the apical part of the hook also causedinhibition of growth in the hook of the dwarf variety and promotionof growth in the tall variety. The inhibition of growth in theapical part of the hook of cv. Progress may be involved in thedwarfism induced by irradiation with red light of this cultivar. (Received May 15, 1989; Accepted April 27, 1990)     

Photomorphogenic effects of UV-B radiation on hypocotyl elongation in wild type and stable-phytochrome-deficient mutant seedlings of cucumber

Hypocotyl elongation responses to ultraviolet-B (UV-B) radiation were investigated in glasshouse studies of de-etiolated seedlings of a long-hypocotyl mutant ( lh ) of cucumber ( Cucumis sativus L.) deficient in stable phytochrome, its near isogenic wild type (WT), and a commercial cucumber hybrid (cv. Burpless). A single 6- or 8-h exposure to UV-B applied against a background of white light inhibited hypocotyl elongation rate by ca 50% in lh and WT seedlings. This effect was not accompanied by a reduction in cotyledon area expansion or dry matter accumulation. Plants recovered rapidly from inhibition and it was possible to stimulate hypocotyl elongation in plants exposed to UV-B by application of gibberellic acid. In all genotypes inhibition of elongation was mainly a consequence of UV-B perceived by the cotyledons; covering the apex and hypocotyl with a filter that excluded UV-B failed to prevent inhibition. These results indicate that reduced elongation does not result from assimilate limitation or direct damage to the apical meristem or elongating cells, and strongly suggest that it is a true photomorphogenic response to UV-B. The fact that UV-B fluences used were very low in relation to total visible light, and the similarity in the responses of lh and wild-type plants, are consistent with the hypothesis that UV-B acts through a specific photoreceptor. It is argued that, given the weak correlation between UV-B and visible-light levels in most natural conditions, the UV-B receptor may play an important sensory function providing information to the plant that cannot be derived from light signals perceived by phytochrome or blue/UV-A sensors.     

Phytochrome A and phytochrome B1 control the acquisition of competence for shoot regeneration in tomato hypocotyl

 We analysed the light-dependent acquisition of competence for adventitious shoot formation in hypocotyls of phytochrome A (fri) and phytochrome B1 (tri) mutants of tomato and their wild type by pre-growing the seedlings under different light quality. The regenerative response in vitro of explants from etiolated seedlings was reduced in comparison to that displayed by light-grown ones. Our results indicate that the light-dependent acquisition of competence for shoot regeneration in the tomato hypocotyl is regulated by phytochrome and antagonistically by a blue-light receptor. By using phytochrome mutants and narrow wave band light we showed that it is mediated at least by two distinct phytochrome species: phytochrome B1 and phytochrome A. The action of phytochrome B1 during seedling growth was sufficient to induce the full capacity of the subsequent regenerative response in vitro in explants from all positions along the hypocotyls. In contrast far-red light acting through phytochrome A did not induce the full capability of shoot regeneration from middle and basal segments of the hypocotyl when phytochrome B1 was absent (tri mutant). A few middle and basal hypocotyl explants pre-grown in blue light regenerated shoots. Received: 12 April 1999 / Revision received: 5 July 1999 · Accepted: 6 August 1999     

The Dormancy of Buds of Citrus sinensis (L.) Osbeck Inserted into Rootstock Stems: Factors Intrinsic to the Inserted Bud

Buds of sweet orange, harvested from shoots of different timeof flushing and from different positions along the shoot, wereused to examine whether lack of burst of inserted buds was acharacteristic of the bud. Bursting of inserted buds was significantlyslower in buds taken from (a) older branches (b) shoots producedunder winter conditions, and (c) basal rather than apical budson the same shoot. The slowness to burst when transferred matched a tendency todormancy in buds on shoot segments grown in vitro, suggestingthat the variation in budburst was intrinsic to the bud. Budburstwas correlated with the extent of secondary bud development;the majority of buds from apical regions of the shoot had developeda secondary bud by the time of implantation, but basal budshad not. Adequate vascular connections with the host tissueswere found in both burst and unburst buds. Citrus sinensis (L.) Osbeck, sweet orange, buds, endodormancy, budding