Response of stem explants to screening and explant source as a basis for methodical advancing of regeneration protocols for chrysanthemum

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration, length of induction period and light quality on leaf regeneration of quince clone BA 29 were investigated. After 2, 4 or 6 days of induction with 2.5 mg l⁻¹ or 5.0 mg l⁻¹ 2,4-D, leaves were cultured under red, blue, red+blue, far-red+blue, white, far-red light or darkness conditions. Leaves thereby treated showed different responses, with respect to somatic embryogenesis, callus, red-nodular structures or roots. Callus production increased with increasing 2,4-D concentration and induction period, although it was not influenced by light quality; the only exception was far-red+blue light, which reduced callusing response. This result suggested involvement of the blue-absorbing photoreceptor system in the callus formation processes. A high regeneration of red-nodular structures with a meristematic appearance was also observed; from some histological characterizations, we presumed they were adventitious buds that were arrested at an early developmental stage. Red-nodular structures increased with decreasing 2,4-D concentration and induction period. In the regeneration of such structures, the blue-absorbing photoreceptor system appeared to have a negative effect but only at a low photoequilibrium value. In contrast, light quality which activated phytochrome induced an increment in regeneration, but the response did not vary for photoequilibrium values ranging from 0.43 to 0.86. For root regeneration, phytochrome seemed to be the only photoreceptor involved. This revised version was published online in June 2006 with corrections to the Cover Date.     

Endoreduplication in <Emphasis Type="Italic">Phalaenopsis</Emphasis> is affected by light quality from light-emitting diodes during somatic embryogenesis

Endoreduplication is a developmental process that is unique to plants and occurs in all plants. The present study aimed to assess endoreduplication in various explant tissues and regenerated somatic embryos of Doritaenopsis. We further investigated the effects of light quality on endoreduplication and somatic embryo proliferation. To this end, we studied endoreduplication in leaves and root tips from regenerated plantlets and somatic embryos and in developing somatic embryos under 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light. We found that the degree of endoreduplication varied in different explants, and that the choice of explants used also influenced the ploidy levels of the newly regenerated somatic embryos. The DNA content of the leaf (2C–8C) was less than that of the root tip (2C–16C) and somatic embryo (2C–64C). In terms of light quality, the combination of red and far-red light produced the highest number of somatic embryos, while maintaining a low degree of endoreduplication. The data obtained indicate that this light combination stimulates somatic embryogenesis in Doritaenopsis and may exert some control on endoreduplication during cell division. These findings can be applied to achieve a reduction in somaclonal variations for the purpose of mass proliferation and genetic improvement.     

The response of Cuscuta planiflora seedlings to red and far-red, blue light and end-of-day irradiations

Irradiation of excised stem segments from de-etiolated seedlings of Cuscuta planiflora for 24 h with mixtures of red and far-red light with red to far-red ratios between 0.02 to 1.0 enhanced coiling and formation of prehaustoria. Maximum number of prehaustoria were recorded when red:far-red was near 0.1. Coiling and prehaustoria were observed whenever estimated in vivo Pfr/Ptotal at photoequilibrium was between 0.06 and 0.67. Irradiation of excised stem segments from white light grown seedlings with 12 h blue light also promoted coiling and prehaustoria formation after another 38 h in darkness. Coiling and prehaustoria were not observed in segments pulsed with 10 min red light at the end of 12 h in blue light. Coiling and prehaustoria were observed after photoreversible end-of-day far-red/red/far-red pulses but not after red/far-red/red pulses. A far-red pulse may not reverse inhibition by end-of-day red pulse when far-red is given more than 12 h after the red pulse.     

Light-quality effects on Arabidopsis development. Red, blue and far-red regulation of flowering and morphology

Arabidopsis thaliana (L.) Heynh. race Columbia plants were grown in red. blue, red + far-red, blue + far-red and various light mixtures of red + blue + far-red light under 14 h light/10 h dark photoperiods. Each single light source and light mixture maintained a constant irradiance (50 μmol m⁻² s⁻¹) and the mixtures of red + blue + far-red maintained a constant ratio of red/far-red light, but varied in the ratio of blue to red + far-red light. Depending on the method used for calculation, values of the fraction of phytochrome in the far-red absorbing form (P_fr/P_tot) for these light mixtures were either constant or decreased slightly with increasing percentage of blue light in the mixtures. Arabidopsis flowered early (20 days) in blue, blue + far-red and red + far-red light and late (55 days) in red light. In mixtures of red + blue + far-red light, each of which established a nearly constant P_fr/P_tot flowering was in direct relation to time and irradiance level of blue light. Leaf area and petiole length were also correlated with blue light irradiance levels.     

Effect of long-term in vitro shoot culture on somatic embryogenesis of quince leaves treated with different light qualities

Summary The effect of long-term in vitro shoot culture on somatic embryogenesis in quince BA 29 was investigated. Three experiments were performed on leaves explanted at about 8-mo. intervals from the same culture stock and maintained under different light qualities. Embryo production was assessed either in terms of percentage of embryogenic leaves or number of embryos per leaf. By appropriate data processing both these responses were linearly related to photoequilibrium in each experiment. Statistical comparisons among the three experiments showed significant differences both in mean (computed over light qualities) and line slope values. In particular, with increasing shoot culture age, both percentage of embryogenic leaves and number of embryos per leaf progressively increased, while mean slope values decreased. The increase in mean values suggests a positive effect on somatic embryogenesis due to possible tissue rejuvenation when mother cultures were cultivated in vitro for longer periods. Slope decrease over time indicated the interactions between age of the in vitro culture and photoequilibrium. Thus, embryo production at different culture ages was consistently found to be highest at high photoequilibrium values; in contrast, if a low level of phytochrome was activated, embryogenesis in the youngest cultures was low or absent, but increased with the progressive tissue rejuvenation arising from long-term in vitro culture.     

Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somatic embryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot cells in blue light produced secondary embryoids and anthocyanin. Cultures in red light had multiple cotyledons and orange-pigmented radicles. Leafy cotyledons occurred in all light treatments. Abscisic acid production peaked at the heart stage of embryogenesis and synthesis was most pronounced in blue light. Red light enhanced development to the heart stage. Both the red and blue light spectra may be used to manipulate carrot cell cultures to optimize growth.     

Photocontrol of Growth, and of Anthocyanin and Chlorogenic Acid Production in Cultured Callus Tissues of Haplopappus gracilis

Cultures of dark-grown Haplopappus callus (strain AI) were exposedto continuous blue, green, red, far-red, and white light for33 days at energy levels of approximately 10 J m^-2s^-1. Growthwas suppressed in all but far-red. Blue had the greatest suppressiveeffect, green the least; red and white were about equally effective.Mean cell generation times were increased from 8–8 days(dark control) to 12.5 days in red light and 20.5 days in blue.There was a slight increase in mean wet weight per cell in bluelight but a slight decrease in red, whereas there was almosta twofold increase in mean dry weight per cell in blue and littlechange in red. In contrast, far-red stimulated growth; the meancell generation time was reduced to 6–5 days and therewas little change in wet or dry weight per cell. Anthocyanin synthesis was promoted by all wavebands except far-red.Blue had the greatest effect, then white, red, and green inthat order. In blue light the pigments accumulated rapidly,but only during the early stages of culture. The maximum amountper cell was attained after 7 days and thereafter the valuesdeclined. In red, however, the pigments accumulated relativelyslowly, and the maximum cell content was not attained until22 days; the amount attained was less than half that attainedin blue light. Initially, the ratio of cyanidine-3-glucosideto cyanidine-3-rutinoside exceeded 5.0 in blue light, but theratio fell to almost unity with time. This probably reflecteda rapid initial synthesis of the glucoside accompanied by asteady conversion to the rutinoside. Blue light was also more effective than red in acceleratingchlorogenic acid production. The response to blue light occurredafter the initial rise in anthocyanins and continued for therest of the culture period. The data are discussed in relation to similar high-energy photoresponsesreported for intact systems.     

The nodulation and nitrogen fixation of isolated roots of Phaseolus vulgaris L.

Summary The exposure of isolated bean roots to white light immediately before inoculation with nodule bacteria stimulated nodulation during a subsequent 7-day dark period. Strong white light applied to inoculated roots for 3 days or longer suppressed nodulation except when the light was applied immediately after inoculation and the duration of the light period did not exceed 3 days. Alternating periods of 12 hours light and 12 hours darkness were almost as inhibitory to nodulation as a continuous exposure to light. A combination of blue and far-red light inhibited nodule formation more than a combination of red and far-red light. Light treatments that inhibited nodulation also suppressed the production of lateral roots, but to a lesser extent. The effect of light on nodulation and lateral root formation was not influenced by the presence or absence of an attached hypocotyl segment in the explant.     

Photocontrol of the cotyledonary blade epinasty of Sinapis alba seedlings. Effects of irradiance and quality of light

The effects of the fluence rate of continuous light (far-red, fluorescent white, blue and red light) on the increase of epinastic percentage of cotyledons were recorded for six days. The epinastic photoresponse was compared to the cotyledonary enlargement and to the inhibition of the hypocotyl extension of irradiated Sinapis alba L. seedlings. Fluence rate response curves and wavelength response curves showed that the photoepinasty is a typical high irradiance response (as is the inhibition of hypocotyl extension) with two maxima, one in the blue and one in the far-red. The epinastic photoresponse was not directly related to cotyledonary growth, which was greater in red and far-red light than in blue light.     

Photoperiod and light quality effects on rate of dark respiration and on photosynthetic capacity in developing seedlings of Chenopodium rubrum

Development and acclimation of energy transduction were studied in seedlings of Chenopodium rubrum L. ecotype selection 184 (50° 10' N; 105° 35' W) in response to photomorphogenic and photoperiodic treatments. Dark respiration and photosynthetic capacity [nmol O₂ (pair of cotyledons)⁻¹ h⁻¹] were measured with an oxygen electrode. Changes in chloroplast ultrastructure were analyzed concomitantly. After germination, seedlings were grown at constant temperature either in darkness or in continuous light (white, red, far-red and blue) or were subjected to diurnal cycles of light/dark or changes in light quality. Dark respiration was low in far-red light treated seedlings. In red light treated seedlings dark respiration was high and the mean value did not depend on fluence rate or photoperiod. Blue light stimulated transitorily and modulated dark respiration in photoperiodic cycles. Photosynthetic capacity was reduced by far-red light and increased by red light. In response to blue light photosynthetic capacity increased, with indications of a requirement for continuous energy input. Phytochrome and a separate blue light receptor seemed to be involved. In continuous red light a clear cut circadian rhythm of dark respiration was observed. Blue light had a specific effect on chloroplast structure.     

Control of chlorophyll a synthesis by phytochrome and cryptochrome in the red alga Corallina elongata Ellis et Soland

Chlorophyll a synthesis in the red alga Corallina elongata is controlled by phytochrome and by a specific blue light photoreceptor. Although the estimated photoequilibrium of phytochrome is similar in blue and red light, the amount of chlorophyll accumulated is greater in blue light, which implies the action of cryptochrome, according to the criteria for the specific blue light photoreceptor involvement. The amount of chlorophyll synthesized is greater when the level of photoequilibrium approaches 65% (in blue and red light) than with higher levels (72.7% in white light and 70.8% in green light). The action of phytochrome is demonstrated by the induction of chlorophyll synthesis after red pulses and the reversion after far red pulses. The reversion is not complete but the percentage of reversibility is high (85-90%). The amount of chlorophyll accumulated is greater in darkness after the application of red light pulses than in white light after the same light pulses. The induction of chlorophyll synthesis is greater after red pulses than after continuous red light. The existence of a fast destruction of chlorophyll in continuous light is observed. This destruction is greater in the high photoequilibrium of phytochrome (70-72%). The turnover times and the induction mechanism of chlorophyll synthesis must be very fast. This indicates the existence of a possible rapid adaptation to the change in light quality and intensity in the marine system.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

The Influence of Plant Growth Regulators and Light Quality on Somatic Embryogenesis in China Rose (Rosa chinensis Jacq.)

We investigated the effect of red light and plant growth regulators on somatic embryogenesis in China Rose (Rosa chinensis Jacq.). Embryogenic calli that had been induced by combinations of 2,4-dichlorophenoxyacetic acid and thidiazuron in darkness were exposed to dark, red, and white light treatments. Cultures subjected to red light treatment generated the greatest number of embryos, with one (SE1 embryos) or two (SE2 embryos) expanded cotyledons. The largest numbers of shoot-like embryos without cotyledons (SE0 embryos) were produced in cultures subjected to dark treatment. The effects of different concentrations of abscisic acid (ABA) on the proliferation and germination of different types of somatic embryos were also evaluated. A concentration of 9.45 μM was found to be the most effective in promoting the proliferation and germination of SE2 embryos. The higher the concentration of ABA (from 0 to 18.90 μM), the higher the percentage of abnormal polycotyledonary embryos produced. The highest percentage of regenerated plants was obtained from SE2 embryos.     

Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida

Chlorophyll synthesis is stimulated by red light in the green alga Ulva rigida C. Ag. and in the red alga Porphyra umbilicalis (L.) Kützing. Because the effect of red light showed some far-red reversibility in successive red and far-red light treatments, the involvement of phytochrome or a phytochrome-like photoreceptor is suggested. The extent of the response is dependent on exposure and photon fluence rate of red-light pulses. In addition to the effect of red light, a strong stimulation of chlorophyll synthesis by blue light was only observed in Ulva rigida. The effect of blue light shows also some far-red reversibility. In the green alga the accumulated chlorophyll is higher after blue light pulses than after red light pulses. In Porphyra umbilicalis , however, the contrary is observed. In Ulva rigida the involvement of a blue light photoreceptor in addition to phytochrome or a phytochrome-like photoreceptor is proposed. The different responses to red and blue light in both algae are explained in terms of their adaptation to the natural light environment.     

Adventitious shoot regeneration from Begonia × erythrophylla petiole sections is developmentally sensitive to light quality

The influence of light quality on organogenesis in vitro was investigated using Begonia  ×  erythrophylla petiole explants. Pre-treatment of in vitro donor plants by growth in the dark or under far-red or blue light reduced their competence for shoot formation when compared with those grown under red or white light. Culture of competent petiole explants under far-red, blue light or in the dark reduced the number of shoots produced per explant compared to those cultured under red or white light. Explants were found to be developmentally sensitive to both far-red and blue light, because meristem, but not primordia development was inhibited. In addition, blue light inhibition of shoot formation is not mediated directly through phytochrome, as few shoots formed on explants cultured under a mixture of red and blue light which resulted in a high P _fr/ P _tot (0.82) and would allow shoot formation in the absence of blue light. Unlike the inhibitory influence of far-red light, which is reversible, exposure to blue light permanently reduces an explant's competence for shoot formation. Our results suggest that phytochrome and an independent blue light photoreceptor, possibly a cryptochrome, can regulate shoot production from B. erythrophylla petiole explants.     

Inhibitory Action of Blue and Far-Red Light in the Flowering of Lemna paucicostata

In a new strain of short-day duckweed (Lemna paucicostata T-101), blue and far-red light-induced inhibition of flowering was investigated. Flowering of this strain failed to be induced under a short-day photoperiod of blue and far-red light, although it responded as a typical short-day plant in red and white light. When the short-day photoperiod of blue or far-red light was terminated by a 15 min red light pulse, flowering recovered completely. This inducing effect of red light was reversed by subsequent exposure to far-red light. Furthermore, it could be demonstrated that 30 min of blue light completely reversed the flowering inductive effect of 5 min red light and vice versa. Evidence is presented suggesting that the inhibitory action of blue and far red light may be due to the lowering of phytochrome P_fr levels below those required to start the dark reactions which lead to flowering. These results are discussed in relation to the time measurement system of photoperiodism.     

In vitro shoot development of Prunus GF 655–2: interaction between light and benzyladenine

Experiments designed to study the effect of light quality and quantity and their possible interaction with benzyladenine (BA) in the control of in vitro proliferation of Prunus insititia Schneider GF 655–2 microcuttings are reported. The action of BA as a promoting factor of shoot formation was expressed only in the presence of light. The concentration response curves for BA-induced proliferation were very similar under the different light sources, irrespective of proliferation rate values.
Shoot formation under blue, far-red and white light was enhanced by the highest photon fluence rates, while the efficiency of red seemed to be independent of this factor. The results suggest the action of a low-energy response in the red waveband and under the low photon fluence rates of blue and white. The inhibition of shoot elongation induced by BA in the dark as well as under all light treatments indicates that, while the BA-induced release from the apical dominance is light dependent. BA inhibition of shoot elongation is entirely light independent.     

Rapid photomodulation of stem extension in light-grownSinapis alba L.

Treatment of the whole of aSinapis alba plant with supplementary far-red light (FR), in back-ground white light (WL), induces a rapid increase in stem extension rate. This rapid increase is regulated by the light environment of the stem itself. Supplementary FR to the stem increases extension rate after a lag period of 10–15 min. A lag period of 3–4 h follows FR irradiation of the leaf, before an increase in extension rate is detectable. When the stem is given supplementary FR, the change in extension rate which is induced increases with increasing FR fluence rate, and with decreasing phytochrome photoequilibrium. There is no difference between the effects of supplementary FR _max 719 nm and supplementary FR _max 739 nm for these relationships. The increase in extension rate induced by supplementary FR is reversed by an increase in the fluence rate of red light (R). These data indicate that the response is controlled by phytochrome photoequilibrium.Abbreviations B blue light - FR far-red light - R red light - WL white light - Pfr far-red absorbing form of phytochrome - Pr red absorbing form of phytochrome - P_tot total phytochrome level (=Pr+Pfr); -Pfr/Ptot, measured - ER difference in stem extension rate, before and after treatment     

Oak Seedlings Grown in Different Light Qualities

Seedlings of oak (Quercus robur) were germinated in darkness for 3 weeks and then given continuous light or short pulses of light (5–8 min every day). The morphological development was followed during 25 days. In continuous white, blue, and red light the stem growth terminated after about 10 days by formation of a resting bud. At that time the seedlings were about 100 mm high. In con tinuous long wavelength farred light (wavelength longer than 700 nm) the stem growth including leaf formation was continuous without the formation of resting buds, and the stem length was about 270 mm after 25 days. The number of nodes developed became twice that of the seedlings grown in while light. The leaves became well developed in all light colours, but leaf areas were largest in plants cultivated in white light. Compared to dark grown seedlings the mean area per leaf was increased about five times in continuous long wavelength far red light. A supplement with short (5 min) pulses of red light each day increased the leaf area up to 20 times. The stem elongation showed a high energy reaction response, i.e. the stem length increased only in continuous long wavelength far-red light but was not influenced by short pulses of red light or far-red light. The leaf expansion, however, was increased by short pulses of red light with a partial reversion of the effect by a subsequent pulse of far-red light. The fraction of the plant covered with periderm was higher in plants given continuous light. In respect to periderm inhibition continuous long wavelength far red light was the most effective. The transfer of seedlings from darkness to continuous white light gave anthocyanin formation in the stem 10–20 mm below the apex. This formation took place in the cortex and was evident in plants grown in darkness or under short pulses of light. Plants grown in continuous red, blue or long wavelength Far red light showed only traces of anthocyanin.