Induction of early flowering in Cymbidium niveo-marginatum Mak in vitro

 Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6-benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6-benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (α-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flower-promoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage. Received: 22 July 1998 / Revision revised: 23 November 1998 / Accepted: 2 December 1998     

Changes in peroxidase activity,auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloïdes) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA naphthaleneacetic acid - IAA indole-3-acetic acid - 2-iP 2-isopentenyladenine - IAAsp indole-3-acetylaspartic acid - IBA indole-3-butyric acid - GC gas-chromatography     

EXPERIMENTS ON THE PHOTOPERIODIC RESPONSE IN PINEAPPLE

Gowing , Donald P. (Pineapple Research Institute of Hawaii, Honolulu.) Experiments on the photoperiodic response in pineapple. Amer. Jour. Bot. 48(1): 16–21. 1961.—The initiation of flowering of ‘Smooth Cayenne’ pineapple plants is neither strictly a response to photoperiod (day lengths of 10 hr. 51 min.–13 hr. 24 min.) nor to a minimum temperature (minima from 50° to 72°F. in different areas) under natural Hawaiian conditions. Depending on the kind of planting material used and the time of planting, natural initiation of flowering may take place any month of the year. Slips planted in the fall generally initiate flowering in December of the following year. However, exposure of an 8-mo.-old slip-planting to a day length of 8 hours for 40 days starting Sept. 8 induced flowering irrespective of night temperatures from about 60 to 80°F. Interruption of the dark period by illumination at 30 ft.-c. from midnight to 1 a.m. suppressed the inductive effect. Lowering the night temperature to 60°F. was, of itself, non-inductive. Field-grown, 11-mo.-old plants treated in place responded similarly, in that 25 periods of 8-hr. day length starting Sept. 5 induced 60% of the plants to flower, and the night illumination suppressed the inductive effect as before. Daily application of 0.12 mg. of the major native pineapple auxin (indole-3-acetic acid) at the beginning of the dark period had no detectable effect on the short-day treatment, and similar application of an antiauxin (4-chlorophenoxyisobutyric acid) did not affect the suppression of flowering by the light-break. Supplemental illumination of field-grown 12-mo. plants to provide a photoperiod of more than 15 hr. daily from Nov. 4 to Jan. 30 did not suppress the natural initiation of flowering which occurred in early December (day length about 10 hr. 50 min.). ‘Smooth Cayenne’ pineapple is therefore a quantitative, but not an obligate, short-day plant.     

Apical Control of Flowering in an Orchid Hybrid, Aranda Deborah

Decapitation induced flowering in an orchid hybrid, Aranda Deborah.This flowering response was observed in both mature plants andcuttings. Flowering could also be induced by stem incisions. The flowering responses of the decapitated plants were shownto be inhibited by a continuous supply of 10^–4 M IAA solution.However, once the auxin supply was interrupted, floral initiationtook place. Renewed supply of exogenous auxin could not arrestfurther development of the inflorescence. Gibberellic acid had no significant effect on the floweringresponse of decapitated plants. CCC at 10^–2 M concentrationcaused slight decrease in the number of floral initials developedafter decapitation.     

Growth Hormone Changes in Chrysanthemum morifolium: Effects of Environmental Factors Controlling Flowering

Simultaneous quantitative analyses have been made of the endogenouslevels of auxin- and gibberellin like substances, growth inhibitors,and auxin-oxidizing enzyme activity in the cold-requiring Chrysanthemummorifolium cv. Sunbeam subjected to different daylength, lightintensity and temperature regimes known to affect flowering.While little hormone or enzyme activity was found in extractsfrom unvernalized plants, a striking rise in auxin-oxidizingenzyme activity occurred rapidly after the end of cold treatment.Increased auxin activity was also recorded shortly after vernalization.At 28 °C both enzyme and auxin activity declined over aperiod of 3–4 weeks; at 20 °C this response was delayed.Gibberellin activity at 28 °C rose steeply about 2 weeksfrom vernalization and declined several weeks later; at 20 °Ca similar response was less marked. Low light intensity treatment,which may have increased endogenous auxin levels, or exogenousauxin application reduced gibberellin-like substance levelsand cause d devernalization.Phosphon D treatment also loweredgibberellin levels and prevented flowering. An extract fromvernalized plants containing gibberellin-like substances intensifiedthe flowering of partially vernalized test plants. Persistenceof high auxin activity in vernalized plants on long days wasassociated with failure to form normal flower buds. Stem elongationrates correlated in general with levels of endogenous auxin-and gibberellin-like substances. Significant amounts of an abscisin-likeinhibitor were found in extracts of flower buds. The mechanismof natural devernalization is discussed in relation to theseobservations.     

Two different β-lactamase genes are present in Streptomycees cacaoi

Two beta-lactamase genes called blaL and blaU have been cloned independently in Liège and in Ume?, from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Liège and in Ume?.     

Kinetics of Determination in the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar α-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.     

Obligatory short‐day plant,Perilla frutescens var. crispa can flower in response to low‐intensity light stress under long‐day conditions

An obligatory short‐day plant, Perilla frutescens var. crispa was induced to flower under long‐day conditions when grown under low‐intensity light (30 µmol m^?2 s^?1). Plant size was smaller under lower light intensity, indicating that the low‐intensity light acted as a stress factor. The phenomenon is categorized as stress‐induced flowering. Low‐intensity light treatment for 4 weeks induced 100% flowering. The plants responded to low‐intensity light immediately after the cotyledons expanded, and the flowering response decreased with increasing plant age. The induced plants produced fertile seeds, and the progeny developed normally. The plants that flowered under low‐intensity light had greener leaves. This greening was because of the decrease in anthocyanin content, and there was a negative correlation between the anthocyanin content and percent flowering. Treatment with L‐2‐aminooxy‐3‐phenylpropionic acid, an inhibitor of phenylalanine ammonia‐lyase (PAL), did not induce flowering under non‐inductive light conditions and inhibited flowering under inductive low‐intensity light conditions. The metabolic pathway regulated by PAL may be involved in the flowering induced by low‐intensity light.     

Changes in the content of endogenous auxins in apical buds ofchenopodium rubrum L. Induced with respect to the endogenous rhythm in capacity to flower

The content of endogenous auxins was examined in apical buds ofChenopodium rubrum plants induced by a photoperiodic cycle of 16h darkness and 8h light followed by a dark period of various duration so as to correspond with either maximal or minimal flowering response in the endogenous rhythm in capacity to flower initiated by the photoperiodic treatment. Apical buds of potentially generative plants contained less auxins than apical buds of plants which remained in the vegetative state. Apical buds from plants treated with kinetin (1. 10^-3 M) and therefore remaining in the vegetative state showed an auxin level comparable to that of untreated plants exhibiting minimal flowering response irrespective of the duration of the second dark period. Plants cultivated on a sucrose solution (0.6 M) during the second dark period became generative even at the normal minimum of flowering. The auxin content of the apical buds was low, similarly as in untreated plants induced for a period leading to maximal flowering response. On the other hand, apical buds from plants grown on sucrose solution during a dark period leading to the manifestation of maximal flowering response showed a relatively high auxin content comparable to that found in untreated plants which had obtained a more extended induction by three photoperiodic cycles. The results are discussed with respect to the possible role of endogenous auxins in the regulation of the changes in growth correlations occurring in the shoot apex during photoperiodic induction and in the expression of the competence to flower.     

Rhythmic changes in ribonuclease activity in relation to nucleic acid synthesis in cotyledons ofChenopodium rubrum L. and to floral induction of the plants

Ribonuclease (RNAse) activity was investigated in cotyledons ofChenopodium rubrum plants subjected to various conditions of illumination (photoperiodic induction, continuous light, induction cancelled by interrupting the dark period by a light-break). At the end of the dark period of the single inductive cycles RNAse activity of induced plants was inferior to that of plants grown in continuous light. At the end of the first two cycles the activity was lowest after the interruption of the dark period by light. The investigation of the enzyme in 6h intervals showed rhythmic changes in activity to occur in induced plants. Enzyme activity followed a pattern opposed to this of nucleic acid (NA) synthesis in the cotyledons. In plants from continuous light the enzyme activity did not show any rhythm and in plants having obtained a light-break during the inductive period the rhythm was less distinct than in the induced ones. The period length of the endogenous rhythm of NA synthesis in the cotyledons is about half as long as this of flowering and the peaks of flowering coincide with the throughs of NA synthesis.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.     

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.     

Sequential rooting media and rooting capacity of Sequoiadendron giganteum in vitro. Peroxidase activity as a marker

The rooting capacities of tips of seedling, juvenile and mature shoots of Sequoiadendron giganteum were compared on different rooting media (inductive and expressive media) after passage on an elongating medium. None of the cuttings rooted when continuously kept on medium containing the auxin NAA and vitamin D2. Peroxidase activity of all those cuttings on NAA+D2 first increased during the 7–9 first days and decreased in the days after. Rooting was obtained by transfer of the cuttings after periods longer than 7–9 days from the NAA+D2 inductive medium to a basal medium supplemented or not with rutin (expressive medium). The rooting capacity was emphasized by rutin treatment and was in correlation with the peroxidase peak reached on the NAA+D2 medium. Seedlings, characterised by the highest peroxidase activity, were most performing in rooting.Abbreviations BM basal medium - D2 ergocalciferol - NAA naphtaleneacetic acid     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Effects of exogenous cytokinins on flowering of the short-day plantChenopodium rubrum L.

Kinetin at a concentration from 3.10^-6 M to 1.10^-3 M was applied to the plumule ofChenopodium rubrum plants during photoperiodic induction. Different levels of induction were compared (one and three short days). The higher concentrations of kinetin applied to induced plants inhibited flower formation. The rate of leaf initiation was increased under these treatments. Lower concentrations of kinetin (from 3.10^-6 M to 1.10^-5 M) usually promoted lateral bud formation and flowering. The step-wise application of kinetin revealed that the inhibitory effect on flowering had been restricted to the inductive period. The effects of kinetin, benzyladenine and trans-zeatin were compared in plants partially induced by two short days. High concentrations always inhibited flowering. Benzyladenine was the most effective in this respect. Root removal diminished the inhibitory effects of cytokinins on flowering as was stated with benzyladenine. It is assumed that endogenous cytokinins play a role in the regulation of organogenetic activity of the stem apical meristem. Depending on the photoperiodic conditions, they presumably exert their activity by maintaining the vegetative functions of the apex.     

Gibberellin-Growth Retardant Interactions on the Growth and Flowering of Clerodendrum thomsoniae

Gibberellin-growth retardant interactions on the vegetative growth and flowering of the vine Clerodendrum thomsoniae Balf. were studied using both exogenous treatments and biologically testing the acid fraction attained from the plant extract. The growth retardant, ancymidol, greatly retarded stem elongation and markedly increased flowering under inductive environments. Gibberellin A₃ (GA₃) application to the shoot tip stimulated vine growth, prevented flowering under inductive environments, and completely overcame ancymidol-induced effects. In contrast to GA₃, treatment with GA₇ had little effect on vegetative growth but increased flowering under inductive environments. The elevated activity of gibberellin-like compounds, as determined by bioassay, were similar except for a marked increase in levels in ancymidol-treated plants grown under inductive environmental conditions. Microscopic examination of the stem tip indicated that the action of the growth regulators involved the induction of floral buds. Thus, in Clerodendrum, ancymidol appears to stimulate an unknown gibberellin(s) and simultaneously acts antagonistically with GA₃.     

Flowering Response of Pharbitis nil to Agents Affecting Cytoplasmic pH

Permeant weak acids and auxins have been shown to reduce the cytosplasmic pH in several systems. Lactic, citric, formic, butyric, salicylic, parahydroxybenzoic, propionic acid, and sodium propionate inhibited the flowering response of Pharbitis nil seedlings when applied immediately before an inductive dark period. The acidic auxins IAA, indolebutyric, and α-naphtaleneacetic acid, as well as the nonacidic auxin α-naphtaleneaceteamid, also inhibited the flowering response. Inhibition was generally more pronounced with a 12-hour than with a 16-hour dark period. Salicylic acid and sodium propionate shifted the response curve of the dark period by about 2 hours. Salicyclic acid, sodium propionate, and indolebutyric acid were inhibitory when applied during the first few hours of the dark period. The permeant weak bases NH₄Cl, procaine, and trisodium citrate enhanced the flowering response. NH₄Cl reduced the length of the critical dark period. The inhibition of flowering by acids and auxins as well as the promotion of flowering by bases was obtained even when only the cotyledons had been treated. The inhibition of floral induction by auxins may not be dependent on their effect on the cytoplasmic pH.     

REVERSION FROM FLOWERING TO THE VEGETATIVE STATE IN XANTHIUM

Lam , S. L. and A. C. Leopold . (Purdue U., Lafayette, Indiana.) Reversion from flowering to the vegetative state in Xanthium. Amer. Jour. Bot. 47(4): 256—259. Illus. 1960.–Plants of Xanthium pensylvanicum Wallr. which had been induced with short photoperiods were caused to revert to the vegetative state either by decapitation or by pinching and subsequent partial de-budding. The ability of induced plants to revert varies with the intensity of the inductive stimulus, and with the extent to which the plant is cut back. Four successive decapitation treatments caused complete reversion of almost all plants which had been given 7 inductive cycles. Reversion can be obtained after 3 inductive cycles even when all of the induced leaves remain on the plant. It is suggested that under the conditions of these experiments the synthesis of the flowering stimulus is stopped or the stimulus is immobilized in the leaves and made ineffective.     

Morphological and physiological analysis of cleistogamy in barley (Hordeum vulgare)

We previously reported that cleistogamy/chasmogamy (CL/CH) of barley ( Hordeum vulgare L.) is controlled by either two tightly linked genes or one gene with multiple alleles. To clarify the morphological and physiological mechanisms of barley CL, we analysed the lodicule size and auxin response of two cultivars whose CL/CH was controlled by two different genes, cly1 and Cly2 . In both cases, lodicules of the CL parent were smaller than those of the CH parent. Analyses of lodicule size and flowering phenotype of f ₁ plants and segregation analyses of the mapping population indicated that lodicule size co-segregated with the flowering phenotype. Indole-3-acetic acid (IAA) and other synthetic auxins, such as 2,4-dichlorophenoxyacetic acid, induced abnormal flowering in CH ears, in which florets remained open for a few days instead of the normal hour or so, but not in CL ears. This auxin effect also co-segregated with the flowering phenotype. Analyses of auxin-related compounds in the floret organs revealed that the anther contained high levels of IAA, whereas indole-3-carboxylic acid, a putative decarboxylated metabolite of IAA, accumulated only in lodicules of CH plants just at flowering. These results indicate that lodicule size and auxin response are pleiotropic effects of the CL gene, which may play a role in auxin response or metabolism.