Weeks In Dark Research Articles

Induction of somatic embryogenesis from young, fully expanded leaves of chilli pepper ( Capsicum annuum L.): effect of leaf position, illumination and explant pretreatment with high cytokinin concentrations

The effect of the explant position on the donor plant, illumination and explant pretreatment with high cytokinin concentrations on the induction, proliferation and development of somatic embryos from young, fully expanded leaves of chilli pepper ( Capsicum annuum L.) was investigated. Explants were cultured either directly on a solid Murashige and Skoog medium supplemented with 9 μM 2,4-dichlorophenoxyacetic acid + 12.9 μM 6-benzyladenine or incubated for 24 h in a liquid MS medium containing the cytokinin at a tenfold concentration (129 μM) and then transferred to the solid MS medium. Globular embryo proliferation depended on the leaf position on the donor plant: fewer embryos were derived from the third leaf (counting from the base of the shoot) than from the first two leaves. The initial pretreatment of pepper explants with increased 6-benzyladenine concentrations significantly reduced the overall proliferation of somatic embryos without affecting the percentage of globular embryos which further developed into the torpedo-shape stage and germinated. Depending on the leaf position, somatic embryo induction was significantly affected by the initial culture incubation under illumination or in darkness. Heart- and torpedo-shaped embryos could be observed only on callus pieces initially incubated for 3 weeks in darkness.

Effect of temperature and light on rooting in vitro and subsequent flowering of pot alstroemeria

SummaryA clone of dwarf alstroemeria cv. Rosalina propagated in vitro was used in experiments to develop a method for pot alstroemeria production. The aim of this study was to optimize the in vitro rooting conditions of the rhizomes so as to obtain early flowering after transplanting to a greenhouse. The effects of cooling in darkness or in light, the addition of paclobutrazol to the rooting medium and the effect of stage of rhizome development on subsequent flowering were studied. The in vitro rhizome cultures maintained on the rooting medium were cooled at 10°C for 2–5 weeks and at 5°C for five weeks in darkness or under a 16 h photoperiod. To allow root formation and growth, the rhizomes were kept for 10 d at 20°C and 16 h photoperiod before cooling at 5°C and 10°C. Pretreatment with 20°C for 10 d and 16 h photoperiod applied during cooling resulted in superior rooting in vitro and better plant survival in the greenhouse. The faster flowering and most plants flowering were obtained after five weeks of cooling at 10°C under a 16 h photoperiod using pre-rooted rhizomes. Paclobutrazol markedly decreased flower stem length.

Parvilucifera infectans norén et moestrup gen. et sp. nov. (perkinsozoa phylum nov.): a parasitic flagellate capable of killing toxic microalgae

Summary The toxic dinoflagellate Dinophysis, collected on the Swedish West Coast, was found to contain round bodies previously interpreted as the result of sexual reproduction. After two weeks of darkness in the refrigerator, all Dinophysis had died, however, and round bodies were present. These proved to be sporangia of a parasitic protist, here named Parvilucifera infectans gen. et sp. nov. Its identity was examined by LM, EM, and DNA sequencing. It is related to Perkinsus, an oyster-killing protist, and Colpodella, a phagocytic protist. Perkinsus has been indicated by 18S rRNA sequencing to be related to dinoflagellates, and the opportunity was taken to examine the ultrastructure of the flagellar apparatus of Parvilucifera in detail. Parvilucifera and its allies, known as perkinsids, share features with both dinoflagellates and apicomplexans. They do not fit readily into any of these groups but appear to form a missing link between them. They are described as a taxon on level with the other alvelolate phyla, as Perkinsozoa phylum nov. Infection studies showed that Parvilucifera infectans infects several other dinoflagellates, notably Alexandrium spp. which are responsible for PSP (paralytic shellfish poisoning). A discussion of the ecological role, in terms of biocontrol of harmful algal blooms, is included.

351 Use of Promalin (GA4+7 and BA) Sprays to Prevent Cold Storage-induced Leaf Yellowing and Abscission in `Stargazer' Hybrid Lilies

Our previous research has demonstrated preventive effects of foliar sprays of growth regulators containing GA4+7 (ProVide or Promalin) on cold storage-induced leaf yellowing and abscission in `Stargazer' hybrid lilies. Further research was conducted to investigate the effective concentrations of Promalin and appropriate timing of promalin sprays. Lilies at “puffy bud” stage were sprayed with promalin at concentrations of 10, 25, 50 or 100 ppm (each GA4+7 and BA) just before placing them at 4 °C for 2 weeks in darkness. Promalin concentrations of 25 ppm or above completely prevented cold storage-induced leaf yellowing occurring during the poststorage evaluation phase in a simulated consumer environment, whereas 10 ppm sprays only partially prevented it. Foliar spray of Promalin (100 ppm each GA4+7 and BA) just before storage at 4 °C for 2 weeks was compared with spraying 2 or 4 weeks before cold storage. While spraying 2 weeks before storage prevented leaf yellowing to the same extent observed in plants sprayed just before cold storage, spraying 4 weeks before storage had very little preventive effect on leaf yellowing. To investigate the effectiveness of promalin sprays with different cold storage durations, puffy-bud stage plants were stored at 4 °C for 1, 2, 3, 4, or 5 weeks in darkness with or without promalin sprays (100 ppm each GA4+7 and BA) before storage. Longer cold storage durations increased the severity of leaf yellowing occurring during poststorage phase. Although promalin was able to prevent leaf yellowing completely up to 2 weeks of cold storage, beyond 3 weeks of cold-storage, effectiveness of promalin diminished with no apparent preventive effect on plants stored for 5 weeks.

<em>In vitro</em> neoformation of grape chimeras (<em>Vitis vinifera</em> L.)

<p style="text-align: justify;">Difference in grape sensitivity to <em>Botrytis cinerea</em> attacks between cultivars was explained by differences in the epidermic tissue of the fruit. Therefore, this work was conducted to create a grape periclinal chimera, whose fruits would combine the skin of a <em>Botrytis</em> tolerant cultivar with a pulp of an another cultivar admitted its good organoleptic quality and productivity. In a first time, graftings of two cultivars (Chardonnay and Pinot noir) were conducted <em>in vitro</em> on 5 different media supplemented with various plant growth regulators. Adventitious shoots were only observed on medium containing BAP and GA3 from a mixed callus structure after four weeks of darkness followed by a light/dark regime. In a second time, RAPD analysis, conducted on these plants, showed their chimerical characteristics.</p>

Somatic embryogenesis in Canary Island date palm

Zygotic embryo and shoot tip explants of Phoenix canariensis were cultured on MS (1962) basal medium supplemented with 100 μM Picloram and 9.5 μM kinetin or 10.8 μM or 45.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.8 μM N6-(2-isopentenyl) adenine (2iP). These explants after 12 weeks in darkness at 28 °C, produced embryogenic callus with very compact, pale yellow, nodular structures. Proliferation and maintenance of embryogenic callus was on MS basal medium with 2.26 μM 2,4-D, 0.83 μM kinetin and 2 μM abscisic acid (ABA), with a regular subculture every 3–4 weeks. Somatic embryo development was promoted by two months of culture on MS liquid medium enriched with 2 μM ABA, for torpedo stage development, then on liquid MS medium with 1 μM N6-benzyladenine (BA) and 0.46 μM kinetin, for shoot induction. Germinated embryos were transferred to basal media enriched with 0.45 μM BA and 0.06 μM naphthaleneacetic acid (NAA) for root and shoot induction and elongation. Viable plants were recovered on basal MS free of plant growth regulators (PGRs), but percentages of plant conversion have to be improved.

A comparative study of the seed germination biology of a narrow endemic and two geographically-widespread species ofSolidago(Asteraceae). 1. Germination phenology and effect of cold stratification on germination

AbstractSolidago shortiiis endemic to a small area in northcentral Kentucky (USA), whereas two of its sympatric congeners,S. altissimaandS. nemoralis, are geographically widespread. Seeds (achenes) ofS. shortii(0.370 mg) are significantly larger (PLSD,P=0.05) than those ofS. altissima(0.070 mg) andS. nemoralis(0.068 mg). Germination percentages of freshly-matured seeds of the threeSolidagospecies collected in November 1991, 1992 and 1994 were 0–2% in light at 15/6°C, 1–37% at 20/10°C, 9–56% at 25/15°C and 10–85% at 30/15 and 35/20°C. Stratification increased the percentage and rate of germination and decreased the time to the onset of germination (measured by Timson's index only at 20/10°C in light) in the three species. Following 12 weeks of cold stratification in light, seeds of the three species germinated to 72–100% in the light and to 22–100% in darkness over the range of thermoperiods; those cold-stratified in darkness germinated to 39–100% in light. Freshly-matured seeds ofS. altissimaand ofS. nemoralisgerminated to 0–4% in darkness, whereas those cold-stratified for 12 weeks in darkness germinated to 0–28% in darkness. On the other hand, freshly-matured and cold-stratified (in darkness) seeds ofS. shortiigerminated to 0–13 and 13–73%, respectively, in darkness. Under near-natural temperatures in a glasshouse without temperature control, germination of the three species peaked in March. Thus, the primary difference in dormancy-breaking and germination requirements of the three species is that the endemic germinates to a much higher percentage in darkness than its two congeners. Seeds ofS. shortiido not have any special dormancy-breaking or germination requirements that could not be fulfilled outside its present-day geographic range.

Direct Shoot Regeneration from Vaccinium pahalae (Ohelo) and V. myrtillus (Bilberry) Leaf Explants

Ohelo (V. pahalae Skottsb.) and bilberry (V. myrtillus L.) shoots were regenerated via direct organogenesis from whole leaves and leaf sections and also from hypocotyl explants of bilberry. Explants preincubated for 1 to 2 weeks in darkness yielded ≈75% regeneration frequencies and the highest number of regenerating shoots/explant on TDZ-supplemented media (0.9 to 2.7 μm). When 2iP or zeatin were substituted as the cytokinin source, frequencies of regeneration and shoot productivity were significantly lower. Explants held under constant illumination (no dark pretreatment) had significantly lower regeneration frequencies in all tested cytokinin-supplemented media. 2,4-D stimulated callus formation, but did not support regeneration from vegetative explants. Cells from callus and suspension cultures did not exhibit regeneration in any of the media that supported organogenesis from leaves. Regenerants were successfully micropropagated, although callus formation caused by zeatin and high 2iP levels interfered with shoot proliferation. Zeatin induced hyperhydricity in shoots from both species, but more severely in ohelo. Ex vitro rooting after treatment with 4.9 μm IBA or 5.4 μm NAA was 95% and 60% successful for bilberry and ohelo, respectively, and plants were readily acclimatized after an interval in a fog chamber. Bilberry microshoots also rooted in vitro in the absence of growth regulator treatment. Chemical names used: 1H-indole-3-butanoic acid (IBA); N-(3-methyl-2-butenyl)-1-H-purine-6-amine (2iP); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA); thidiazuron=1-phenyl-3-(1,2,3-thiadiazio-5-yl)urea (TDZ); 2,4-dichlorophenoxyacetic acid (2,4-D); 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

In vitro shoot apex micrografting of mature Acacia mangium

Prospects for in vitro micrografting shoot apices of mature Acacia mangium trees were investigated with the use of 432 micrografts. Overall success rates of 51.5%s% were obtained for shoot apices ranging from 200 to 400 μm in length, and a short basal wedge of underlying tissues top-grafted in aseptic conditions onto 2-to-3-month old in vitro grown Acacia mangium seedlings. The successfully established micrografts displayed, however, substantial variability in terms of further scion elongation as 41% of these micrografts, or 21.2% only of the total amount of the micrografts performed, had resumed growth two months after micrografting. The elongated scions exhibited different types of morphology, ranging from juvenile-like type composed leaves to the predominant mature-like phyllode morphology. Side-grafting, a more difficult procedure to perform than top-grafting, or placing the micrografts for 2 weeks in darkness after grafting, did not improve the scores. Moreover, attempts to micrograft meristems (150–200 μm) resulted in 5% success only.

弱光下低温貯蔵によるナスセル成型苗の生長抑制および苗質維持

Eggplant (Solanum melongena L.) plug seedlings were stored for 3 weeks at 9°C air temperature and photosynthetic photon flux density (PPFD) of 0 (darkness), 2, 8, or 16μmol m-2s-1. The seedlings stored for 3 weeks in darkness or under 2μmol m-2s-1 PPFD reduced dry weight, while those under 8 or 16μmol m-2s-1 PPFD increased dry weight. The light compensation point of seedlings before storage was 8μmol m-2s-1 PPFD and it decreased to 4-5ymol m-2s-1 PPFD after storage regardless of PPFD in storage. All the plantlets stored under light survived at transplanting and grew successfully after storage. Dark storage lowered the photosynthetic capacity and, thus, lowered percent survival at transplanting and post-storage growth rate. For keeping seedlings at no-growth status and preventing quality degradation, it would be required to store the seedlings under conditions where they have null CO2 exchange rates throughout the storage period.

Regeneration of Peach [Prunus persica (L.) Batsch] Rootstock Cultivars from Cotyledons of Mature Stored Seed

Shoots were regenerated from cotyledons of mature stored seed of three peach rootstock cultivars (`Flordaguard', `Nemared', and `Medaguard'). Shoot regeneration rates were highest when cotyledons were cultured for 3 weeks in darkness on Murashige and Skoog (MS) medium with 2.5% sucrose and a combination of IBA (1.25 or 2.5 μm) and TDZ (6.25 or 12.5 μm). Regeneration rates for `Flordaguard', `Nemared', and `Nemaguard' were as high as 60%, 33%, and 6%, respectively. Length of seed storage (1 to 3 years) did not affect regeneration rates. Seventy percent of regenerated shoots produced rooted plants. This regeneration method is rapid and simple, and stored seed can be used year-round. It may be a useful regeneration system for gene transfer in seed-propagated peach rootstocks. Chemical names used: 5 indole-3-butyric acid (IBA); thidiazuron (TDZ).

Phalaenopsis Orchid Light Requirement during the Induction of Spiking

Potted mature Phalaenopsis `Joseph Hampton' orchid (clone Diane) plants were placed in each of four growth chambers with 0, 8, 60, or 160 μmol·m–2·s–1 photosynthetic photon flux (PPF) for 12 hours daily and at 20C day/15C night air. Plants under 160 or 60 μmol·m–2·s–1 PPF began spiking (an elongating reproductive bud protruding through the base of its subtending leaf) in an average of 28 or 34 days, respectively. None of the plants placed under 0 or 8 μmol·m–2·s–1 PPF started spiking within 6 weeks. These plants, following return to a greenhouse, spiked and flowered 8 weeks later than those receiving 160 μmol·m–2·s–1. In a second experiment, plants were placed in each of three growth chambers and kept in complete darkness at 20C day/15C night for 2, 4, or 6 weeks before exposure to 160 μmol·m–2·s–1 PPF. Air was maintained at 20C day/15C night for an additional 6 weeks and then raised to 25C day/20C night to accelerate flowering. Plants exposed to 2, 4, or 6 weeks of darkness required 45, 60, or 77 days, respectively, to reach spiking. However, all plants spiked at similar times (31 to 35 days) after lighting began. Anthesis occurred at progressively later dates for plants placed in darkness for increasing durations, but plants in all treatments required 123 days to reach anthesis following their exposure to light. Flower count and size were not affected in both experiments.

657 (PS 4) VARIETAL DIFFERENCES IN CALLUS AND SHOOT FORMATION OF PELARGONIUM SPP.

Before attempting transformation of Pelargonium spp., it is necessary to develop procedures for shoot regeneration under selective conditions (kanamycin resistance). Leaf disks of various cultivars of regal, zonal, ivy-leaved, and scented pelargoniums were compared for shoot regenerability and phenolic production. Disks were cultured on MS medium supplemented with 10.7 μM NAA and 8.9 μM BA. After three weeks in darkness, each disk was transferred to MS with 0.89 μM BA and evaluated for proportion of disks with callus, extent of disk development, and the rate and intensity of phenolic halo production. Phenolic production was not correlated with shoot regeneration ability. Regal pelargoniums had low phenolic production, while ivy-leaved pelargoniums had both the highest phenolic production and callus formation. Shoot regeneration from leaf disks was inhibited on media containing 100 μg/ml kanamycin.

High-frequency Somatic Embryogenesis from Quiescent Seed Cotyledons of Cucumis melo Cultivars

A protocol for high-frequency somatic embryogenesis in Cucumis melo L. was developed using `Male Sterile A147 as a model cultivar. Basal halves of quiescent seed cotyledons were cultured on embryo induction (EI) medium containing concentration ranges of the auxin 2,4-D and the cytokinins BA, Bin, TDZ, or 2iP before transfer to embryo development (ED) medium. Medium with 2,4-D at 5 mg·liter-1 and TDZ at 0.1 mg·liter-1 was superior, with 49% of explants responding and an average of 3.3 somatic embryos per explant (6.8 somatic embryos per responding explant). More explants produced embryos when incubated on EI medium for 1 or 2 weeks (30% and 33%) than for 3 or 4 weeks or with no induction. However, 2 weeks was 2.9 times better than 1 week in terms of number of embryos per explant. One week of initial culture in darkness, followed by a 16 hour light/8 hour dark photoperiod, produced more responding explants (26%) than two or more weeks in darkness or no dark period at all; but 1 and 2 weeks of darkness resulted in a similar number of embryos per explant (2.1 and 2.8). Sucrose concentration in EI and ED media had a highly significant effect on embryo induction and development. EI medium with 3% sucrose resulted in more embryogenic explants than EI medium with 1.5% or 6% sucrose. However, treatments with 3% sucrose in EI medium and 3% or 6% sucrose in ED medium produced significantly more embryos per explant (8.5 and 11.9) than other treatments. Treatments did not affect embryo induction directly and regeneration per se but, instead, frequency and efficiency of somatic embryo development. The optimal treatments were tested with 51 other commercial varieties. All varieties underwent somatic embryogenesis, exhibiting a response of 5% to 100% explant response and 0.1-20.2 embryos per explant. Chemical names used: N-(phenylmethyl)-lH-purin-6-amine (benzyladenine or BA); N-(2-furanylmethyl)-lH-purin-6-amine (kinetin or BIN); N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ); N-(3-methyl-2-butenyl)-lH-purin-6-amine (2iP); (2,4-dichlorophenoxy) acetic acid (2,4-D).

Effects of nutrients on anthocyanin production in cultured cells of Aralia cordata

Good results for anthocyanin production in dark-grown calli of Aralia cordata Thunb. were obtained on the addition of sucrose as the carbon source and NH 4 + and NO 3 − at a molar ratio of 1:4 (15 mM total nitrogen). Also, by supplementing NAA as auxin and kinetin as cytokinin, good results were obtained. The anthocyanin yield of high-selected callus cultures was 13.0% maximum dry weight on being cultured for three weeks in darkness. The major anthocyanin, about 70% of the total pigment in the cells, was identified as cyanidin 3-xylosylgalactoside.

Changes in the levels of abscisic acid and its metabolites resulting from chilling of tomato fruits

The 6,6,6-[2H]-analogues of abscisic acid (ABA), phaseic (PA) and dihydrophaseic (DPA) acids were used in GC-MS-SIM determination of free and total alkali hydrolyzable ABA, PA and DPA in the pericarp of tomato (Lycopersicon esculentum L. cv. Pik Red) fruit. Determinations were made on breaker-stage fruit stored 1, 2, 3 or 4 weeks at 2.5°C or at 10°C, and after subsequent ripening for 1 week in darkness at 20°C. Two-fold increases in levels of ABA occurred after storage at low temperatures with a slightly but significantly greater increase in ABA level occurring with 2.5°C storage. These increases in ABA levels were not associated with the appearance of damage symptoms that occurred with storage at the chilling temperature (2.5°C). Differences in ABA metabolism were found resulting from storage at the two temperatures, 2.5 or 10°C. Significantly greater DPA levels were found after 10°C storage than after 2.5°C storage (2 weeks). Levels of ABA ester-conjugates increased with 20°C ripening only after 10°C storage while free ABA levels decreased after both storage temperature conditions. Levels of DPA conjugates also increased only after 20°C ripening following 10°C storage. A longer period of storage resulted in decreases of free DPA levels after 10°C storage but increased DPA levels were found after 2.5°C storage.

Light and temperature action in germination of seeds of the empress tree (Paulownia tomentosa)

Seeds of the empress tree (Paulownia tomentosa Steud.) were imbibed for two weeks in darkness at constant temperatures (18, 23 or 28°C), and then irradiated with red light for 5 min. Germination was poor if it took place at the same temperature as imbibition, but a high percentage was achieved if the seeds were exposed to higher or lower temperatures before they were irradiated. Maximum germination was obtained when the difference between pretreatment and imbibition was about 10°C. The effect increased with the duration of the pretreatment and was optimal at 24 h. The effect decreased as the time lapse between temperature pretreatment and red light irradiation increased, and it was lost after two days. If pretreatment was shorter than 24 h (12 h). a high percent of germination was obtained by alternating pretreatment and imbibition temperatures. The germination of seeds imbibed in 40% heavy water was also stimulated by temperature pretreatments. Light and temperature also exhibited an interactive effect in the germination of seeds that were imbibed in darkness for only 3 days. For each of the germination phases there was a temperature at which the time needed for 50% germination was the shortest, namely 35°C during imbibition, 37.5°C in the period of Pfr activity. and 32.5°C during radicle protrusion. The data obtained are shortly discussed in relation to the domestication of empress tree in Southern Europe.

Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz. in vitro

Seeds of the difficult-to-germinate Paphiopedilum ciliolare Pfitz. were sown asymbiotically on a modified Thomale medium. Germination was evaluated by determining the germination percentage and the germination index, which were found to have a strong correlation. Germination of seeds was studied on different culture media and under various environmental conditions. Germination was strongly promoted by 12 weeks of darkness in comparison to light, and by lowering the macro- and micro-salt concentration. Optimal germination occurred at a pH of 6.0 and a low sugar concentration. Germination was not affected by adding the vitamins nicotinic acid, biotin or pyridoxin. Auxin had a slight promotive effect, but neither cytokinin nor gibberellin affected germination. The presence of tryptone appeared to be essential, whereas banana homogenate was slightly inhibitory. Optimal conditions for seed germination and seedling growth were different. Although the effect of active charcoal and banana homogenate were inhibiting during germination, they were promoting during further development of the seedlings.

The role of pattern vision in the increase of visual cortex acetylcholinesterase after first exposure of dark reared rats to light

Acetylcholinesterase (AChE) activity in homogenates from motor and visual cortex of rats for 7 weeks in darkness was compared to levels in normally reared and dark reared littermates exposed to 3-h periods of controlled illumination. AChE levels in the visual cortex of animals fitted with light-diffusing acleral contact occluders, and therefore lacking normally organized visual information, were elevated 17% over dark-reared animals, but were not significantly different from levels in animals exposed to lighting conditions permitting detailed pattern discrimination. There were no differences between conditions in motor cortex AChE. We conclude that higher order perceptual processing is not necessary for the light-stimulated increase in visual cortex AChE in the rat.

Regeneration of leaf explants of Anthurium andraeanum Lind. in vitro.

Leaf explants were cultured on a basic nutrient agar medium to which were added growth substances (adenine, benzyladenine, kinetin, 2-iP [isopentenyladenine], zeatin or 2,4-D) and NH4NO3, (NH4)2SO4 or NaNO3 in various concentrations. The effects of using the surfactant Tween 20 during leaf sterilization and of growing explants in light and/or darkness for 20 weeks were also investigated. There was a strong positive correlation between callus formation and shoot regeneration. Regeneration was optimal under the following conditions: addition of 0.25-1.00 ml/litre Tween 20 during leaf sterilization, adding a growth substance (adenine 0.1 mg/litre, zeatin 1 mg/litre, or 2,4-D 0.08 mg/litre), culture during 16 weeks in darkness followed by 4 weeks of light, and including 206 mg/litre NH4NO3 in the medium. The promoting effect of low levels of NH4NO3 on shoot regeneration in callus was caused by the NH4+ ion and not by the NO3- ion. (Abstract retrieved from CAB Abstracts by CABI’s permission)