ENVIRONMENTAL AND HORMONAL CONTROL OF TURION GERMINATION IN MYRIOPHYLLUM VERTICILLATUM

Germination, or outgrowth, of Myriophyllum verticillatum turions involves a series of visible changes starting with reflexing of leaves followed by extension and curving of the axis, and then by root formation. Before abscission, turions grow out in response to long days (16 hr) but not short days (8 hr). After abscission, turions show maximal dormancy which can be fully broken by a cold treatment (4 C). Turions are heterogeneous in degree of dormancy and ability to respond to less complete dormancy-breaking treatments, e.g., long days at 20 C. Cytokinins (10^-6 m) break dormancy of non-cold-treated turions, whereas gibberellic acid (GA₃) is ineffective except at high concentrations (10^-3 m). Continuous treatment with cytokinins causes abnormal development after germination. GA₃, on the other hand, induces apparently normal development even at high concentration. Indoleacetic acid (IAA) induces outgrowth only at high concentrations (10^-3 - 10^-4 m), but these concentrations also produce abnormal development. Abscisic acid (ABA, 10^-5 m) retards outgrowth of cold-treated turions and can completely suppress it in non-cold-treated turions. The activity of ABA-like substances in turions remains about the same before and during germination, whereas other (unidentified) acidic inhibitors decrease markedly. The cytokinin activity changes in a complex pattern.     

Ecophysiological Characterization of Dormancy States in Turions of the Aquatic Carnivorous Plant Aldrovanda vesiculosa

Two main dormancy states, innate and imposed dormancy, were characterized in turions (winter buds) of the aquatic carnivorous plant Aldrovanda vesiculosa L. (Droseraceae) kept at 3 ± 1 °C in a refrigerator over the winter. As a result of the breaking of imposed dormancy by a temperature increase (at 15 – 20 °C), some of the turions rose to the water surface within 1 – 3 d and germinated. Turion leaves contained large lacunae with a slimy reticulum and were filled by water over winter. As a result of breaking imposed dormancy, the proportion of gas volume in inner turion leaves rose from 10 – 20 % to 100 % of leaf lacunae volume. The aerobic dark respiration rate of the turions [0.74 – 1.5 μmol O₂) kg⁻¹(FM) s⁻¹] slightly increased during innate dormancy after 1 – 2 d at 20 °C, while it was almost constant during the breaking of imposed dormancy. The anaerobic fermentation rate of the turions was only 1.5 – 7 % of the oxygen respiration rate and also was constant during the breaking of imposed dormancy. In turions, the content of glucose, fructose, and sucrose was the same for the two states of dormancy, but starch content was greatly reduced for the imposed dormancy (10 – 11 vs. 32 % DM). It may be suggested that a temperature increase causes an increase of fermentation or respiration which is responsible for the evolution of gas in turion lacunae and, thus, for turion rising.     

Effects of gibberellin and auxin on the synthesis of abscisic acid and ethylene in buds of dormant and sprouting potato tubers

Gibberellic and β-indolylacetic acids at concentrations of 10⁻⁷-10⁻⁵ M were shown to change the hormonal status and duration of true dormancy in potato tubers. Gibberellic acid shortened the true dormancy and decreased the contents of abscisic acid and ethylene in the apical meristem. β-Indolylacetic acid elongated the true dormancy and decreased abscisic acid production, but caused a more than tenfold increase in the production of ethylene by apical tissues. The data suggest that β-indolylacetic acid and ethylene, as well as gibberellic and abscisic acids, are involved in the regulation of true dormancy in potato tubers.     

Effect of Methyl Jasmonate on Morphology and Dormancy Development in Lily Bulblets Regenerated In Vitro

Scales of lily bulbs are swollen petioles. Lily scale fragments cultured in vitro regenerate bulblets consisting of scales that may or may not carry a leaf blade. The bulblets are dormant and require a cold treatment to sprout. We added the gaseous plant growth regulator methyl jasmonic acid (MeJA) in the headspace of the tissue-culture container and studied the effect on plantlet morphology (scale/leaf-blade formation) and dormancy development in three lilies, Lilium speciosum “Rubrum No. 10,” L. longiflorum “Snow Queen,” and the Asiatic hybrid “Connecticut King.” Methyl jasmonic acid strongly reduced leaf-blade formation in Lilium longiflorum and Connecticut King. This was a specific effect as scale formation was affected much less. The specific inhibition of leaf-blade formation was not observed in Lilium speciosum. In this lily, high concentrations of methyl jasmonic acid (MeJA) inhibited leaf-blade and scale formation to similar extents. Methyl jasmonic acid reduced dormancy development in all three lilies, with the largest effect observed in Connecticut King. In this Asiatic hybrid, almost all bulblets that had regenerated at 300 or 1000 μl l⁻¹ MeJA in the headspace, did not require a dormancy-breaking treatment to achieve sprouting after planting in soil. Previously, it has been found in lily that treatments that reduce leaf-blade formation promote dormancy development. The present findings with MeJA do not agree with this. In the three lilies, the various parameters that were studied—regeneration, scale weight, leaf-blade weight, and dormancy development—were very differently affected by MeJA.     

Releasing primary dormancy in <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses by smoke-derived butenolide

The effect of smoke and smoke-derived butenolide in releasing dormancy of caryopses (referred to as seeds) of the economically important weed Avena fatua L. was studied. Seeds of A. fatua are dormant after harvest. Both smoke-water and butenolide, applied continuously, removed dormancy in darkness at 15, 20 and 25°C and slightly at 30°C. Butenolide was very active at a concentration of 10⁻⁸ M. Butenolide at 10⁻⁸ M was also able to remove dormancy at 20°C when applied for 12 or 24 h at 4°C or for 3 to 24 h at 20°C. Sensitivity to butenolide decreased with longer preincubation times in water. This compound was less effective in releasing dormancy in the light than in darkness. Dormancy release by butenolide involves induction of cell-cycle activity just before coleorhiza protrusion. Stimulatory effects of smoke-water and butenolide were also observed in respect of seedling growth and vigor.     

Life cycle and growth ofPotamogeton crispus L. in a shallow pond,ojaga-ike

The life cycle and growth ofPotamogeton crispus L. were studied in a shallow pond, Ojaga-ike. With respect to the shoot elongation and seed and turion formations, the life cycle of this plant in the pond could be divided into following five stages: germination, inactive growth, active growth, reproductive and dormant stages. It was suggested that the plant showed these successive stages depending mainly upon water temperature. The turions germinated on the bottom in autumn when the water temperature fell below ca. 20 C. The plant showed hardly any growth during winter (December—early March) when the temperature was below 10 C. In the spring when the bottom water temperature rose to above 10 C (mid-March), the plant started to grow again and the shoot elongated rapidly at the rate of 4.2 cm day⁻¹ until the shoot apex reached the pond surface in late April. Both the increment of node number and the internodal elongation were associated with this rapid shoot growth. On 10 May (last sampling date), the mean values of shoot length, internodal length and the number of nodes estimated for 10 predominant plants were 238.2±5.6 cm, 7.1±0.8 cm and 34.9±4.0 cm, respectively. The turion formation and flowering occurred during the period from mid-April to mid-May when the surface water temperature ranged 19 and 22 C. The dry weight of a plant reached the maximum mean value of 1180 mg on 10 May. At its peak biomass, an individual plant produced 1–10 turions (5.5 on average) of which the mean individual turion dry weight was 53.2 mg. The turion dry weight accounted for ca. 42% of the total plant biomass m⁻² at that time.     

Effects of Cadmium on Root Growth, Cell Division and Nucleoli in Root Tip Cells of Garlic

The effects of different concentrations (10⁻⁷ to 10⁻² M) of cadmium chloride on root growth, cell division and nucleoli in root tip cells of Allium sativum L. were investigated. At lower concentrations of Cd²⁺ (10⁻⁷ to 10⁻⁶ M), Cd²⁺ did not influence the root growth, even had a stimulation effects during a short treatment. The results showed that the rate of root growth per day at the treatment groups (10⁻⁴ to 10⁻² M Cd²⁺) decreased with increasing duration of the treatment and increasing Cd²⁺ concentration. Cd²⁺ induced c-mitosis, anaphase bridges, chromosome stickiness and on nucleoli, causing some particles of similar silver-stained material scattered in the nuclei and making the silver staining reaction at the periphery of the nucleolus weaker. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cadmium-induced stress on the seed germination and seedling growth of <Emphasis Type="Italic">Brassica napus</Emphasis> L., and its alleviation through exogenous plant growth regulators

Because of its prolific growth, oilseed rape (Brassica napus L.) can be grown advantageously for phytoremediation of the lands contaminated by industrial wastes. Therefore, toxic effect of cadmium on the germination of oilseed rape, the capability of plants for cadmium phytoextraction, and the effect of exogenous application of plant growth regulators to mitigate phytotoxicity of cadmium were investigated. For the lab study of seedlings at early stage, seeds were grown on filter papers soaked in different solutions of Cd²⁺ (0, 10, 50, 100, 200 and 400 μM). In greenhouse study, seedlings were grown in soil for 8 weeks, transferred to hydroponic pots for another 6 weeks growth, and then treated with plant growth regulators and cadmium. Four plant growth regulators viz. jasmonic acid (12.5 μM), abscisic acid (10 μM), gibberellin (50 μM) and salicylic acid (50 μM); and three levels of Cd²⁺ (0, 50 and 100 μM) were applied. Data indicated that lower concentration of Cd²⁺ (10 μM) promoted the root growth, whereas the severe stresses (200 or 400 μM) had negative effect on the establishment of germinating seedlings. Plants treated with any of the tested plant growth regulators alleviated cadmium toxicity symptoms, which were reflected by more fresh weight, less malondialdehyde concentration in leaves and lower antioxidant enzyme activities. The application of abscisic acid to the plants cultivated in the medium containing 100 μM Cd²⁺ resulted in significantly lower plant internal cadmium accumulation. Huabing Meng and Shujin Hua contributed equally to this paper.     

Nitric oxide,hydrogen cyanide and ethylene are required in the control of germination and undisturbed development of young apple seedlings

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important regulators of plant development (germination, flowering, senescence), acting as secondary messengers in cooperation with classical phytohormones. Apple seeds are dormant, unless they undergo a 3 month long cold stratification. Deep dormancy of isolated apple embryos can also be broken by short pre-treatment with HCN or NO with the effect associated with enhanced ethylene synthesis. Non-dormant embryos germinate well and young seedlings grown from non-dormant embryos do not exhibit any morphological anomalies, such as asymmetric growth and greening of cotyledons. One of the aims of this work was to investigate the correlation between RNS- mediated (HCN- and NO-dependent) dormancy removal and ROS (H₂O₂ and O₂^−•) accumulation in the embryos. The beneficial effect of NO and HCN on germination of dormant apple embryos has been associated with marked increases in H₂O₂ and O₂^−• concentration in the embryos at early germination stages. We also analyzed growth of young seedlings developed from embryos pre-treatment with HCN or NO or exposed to ethylene (ethephone) and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC and ethephone removed all morphological anomalies of the seedlings (asymmetric growth and greening of cotyledons) but the radicle growth was rather slight. We propose that accumulation of ROS provoked by HCN and NO pre-treatment is required for embryo germination “sensu stricto”, while ethylene is required for post-germination seedling growth.     

Jasmonic acid affects changes in the growth and some components content in alga Chlorella vulgaris

The present study was undertaken to test the influence of exogenous applied jasmonic acid upon the growth and changes in some metabolites levels in the cells of green alga Chlorella vulgaris Beijerinck (Chlorophyceae). It was found, that JA in algal cells acted in a concentration-dependent manner. Treatment with JA at high concentrations range of 10⁻⁵–10⁻⁴ M, resulted in the decrease in cell number and reduction of major photosynthetic pigments, monosaccharides, soluble cellular and extracellular proteins levels as well as decrease in pH of the medium. In contrast to 10⁻⁵ and 10⁻⁴ M JA, this phytohormone applied at 10⁻⁸–10⁻⁶ M induced the increase in cell number, photosynthetic pigments and monosaccharides contents, significant accumulation and extracellular secretion of soluble proteins over control and neutralization of the medium. Quantitative changes in polypeptide pattern of total cellular proteins after treatment with the optimal concentration of 10⁻⁷ M JA on the 7^th day of cultivation as analyzed by SDS-PAGE, was also observed. JA induced synthesis de novo of 15 specific polypeptides with molecular weight 334-16 kDa which were’t detected in the control. The data suggest that JA plays a important role in algal growth and development.     

Activity of salicylic acid on the growth and biochemism of Chlorella vulgaris Beijerinck

This study was conducted to investigate the influence of salicylic acid (SA) on the growth and changes of nucleic acids, protein, photosynthetic pigments, sugar content and photosynthesis levels in the green alga Chlorella vulgaris Beijerinck (Chlorophyceae). The most significant changes in the content of nucleic acids and proteins was observed at the concentration 10⁻⁴ M SA between 8 and 12 day of cultivation. This concentration of SA increased the number of cells (about 40 %) and content of proteins (about 60 %) and its secretion to the medium. The slight stimulation of protein secretion occurred on the 12th day of cultivation at concentration 10⁻⁴ M, while in the range of 10⁻⁵ M to 10⁻⁶ M the protein secretion was inhibited. SA also stimulated the content of nucleic acids, especially RNA by 20–60 %, compared with the control. The most stimulating influence upon the contents of chlorophylls a and b (50–70 %), total carotenoids (25–57 %), sugar (27–41 %) and intensity of net photosynthesis (18–33 %) was found at 10⁻⁴ M of SA. At the concentration of 10⁻⁶ M SA the slight inhibition of growth and biochemical activity of the algae was recorded at the first days of cultivation.     

IAA-induced hyperpolarization of the membrane potential in isolated cells fromMimosa pulvinus

Upon treatment with 10⁻⁴ M IAA the membrane potential of an isolated cell from the main pulvinus, ofMimosa pudica L. depolarized by about 6 mV in 2–5 min, but later it gradually hyperpolarized by about 30 mV. The membrane potential of a motor cell in the main pulvinar tissue hyperpolarized by about 80 mV 1 hr after application of 10⁻⁴ M IAA.     

Effect of jasmonic acid on in vitro explant growth and microtuberization in potato

The shoot fresh mass, root length and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and Helanwuhua were increased significantly by the application of 0.2 – 2 mg dm⁻³ jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of potato explants was inhibited by JA at high concentrations (20 – 50 mg dm⁻³). Chlorophyll content in explant leaves decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg dm⁻³ JA acid peroxidase activity increased, while in the leaves treated with more than 2 mg dm⁻³ JA peroxidase activity decreased. Under the dark, the microtuber numbers, fresh mass and percentage of big microtubers of two potato cultivars were not promoted by the application of 0.2 – 50 mg dm⁻³ JA.     

Ecological mechanisms involved in dormancy breakage in Ulex parviflorus seeds

Dormancy in the hard seed coats of Mediterranean species is considered a strategy that enables persistent seed banks to be formed in the soil. An important factor related to seed coat fracture and dormancy breakage in Mediterranean ecosystems is heat. Nevertheless, the effect of factors other than heat on dormancy breakage in these species has hardly been studied. To investigate the different ecological factors involved in germination, in the laboratory we applied several scarification treatments to seeds with chromatic polymorphism. We evaluated the effect of soil seed depth during experimental burns by sowing seeds at −1, −3 and −5 cm in the soil profile, and we also studied the effect of seed origin on the posterior germination of seeds from 4 and 10 year-old shrubs as well as from the soil seed bank. U. parviflorus shows clear chromatic polymorphism: its brown seeds present higher dormancy levels than its yellow seeds. The different techniques of dormancy breakage result in different degrees of germination; the highest degree of germination is generated by the mechanical treatment, followed by the acid and the heat treatments, in that order. The depth of the seeds in the soil determines the temperature thresholds and the residence times of these temperatures and whether they stimulate a massive germination at the −1 cm soil profile or only a slight germination at the −5 cm depth. Seeds recently produced by the plant show higher dormancy levels than seeds extracted from soil seed banks. Dormancy levels also depend on the shrubland age used for extracting the soil samples (3>9 years old). In effect, from the point of view of dormancy, the germination behaviour of U. parviflorus seeds seems to follow a multiresponse strategy based on different seed populations and involving both biological and abiotic processes to break dormancy.     

Photophysiology of turion formation and germination inSpirodela polyrhiza

Standardized laboratory techniques for the vegetative growth of the duckweedSpirodela polyrhiza (Lemnaceaé), and for formation as well as germination of their turions were described. Increasing photon fluence rates of blue or red light increased the yield of turions. A specific stimulating effect of blue light was demonstrated under autotrophic but not under mixotrophic conditions. Therefore the spectral composition of light is not important in mixotrophic formation of turions whereas in autotrophic formation light sources with a higher portion of blue light are recommended. Dark-grown (etiolated) turions showed accelerated germination and higher germination percentage in comparison with light-grown turions after induction by a single red light pulse. This difference was overcome in continuous red light by speeding up the germination response of light-grown turions. Use of Petri dishes (8 cm³ nutrient solution) instead of Erlenmeyer flasks (50 cm³ nutrient solution) retarded germination response. Especially for long term experiments the use of Erlenmeyer flasks is recommended. Storage of turions for 72 h at 25 ‡C following at 5 ‡C in darkness after-ripening resulted in a decreased lag phase of the light-induced germination both after induction by a single light pulse and in continuous light. We thank Dr. Halina Gabrys, University of Crakow, Poland for critical discussion.     

Effect of auxin on mesocotyl elongation of dark-grown maize under different seeding depths

In order to elucidate the physiological mechanism of maize mesocotyl elongation induced by auxin under different seeding depths, seeds of five maize inbred lines, including 3681-4 line tolerant to deep seeding, were treated with IAA and triiodobenzoic acid (TIBA) under seeding depths of 20 or 2 cm. Under deep seeding conditions, maize mesocotyls grew by 1.5–2.0 times faster than under shallow seeding conditions. IAA (10⁻⁶ to 10⁻⁴ M) applied to roots stimulated mesocotyl elongation only of 3681-4 line and only under deep seeding conditions. TIBA (10⁻⁵ and 10⁻⁴ M) applied to roots inhibited mesocotyl elongation in all lines, but only 3681-4 was sensitive to 10⁻⁶ M TIBA. IAA promoted only cell elongation, and TIBA inhibited both cell elongation and cell division. After IAA and TIBA treatments, endogenous IAA content changed in parallel with the mesocotyl growth rate under different seeding depths. Furthermore, ABP1 gene expression changed in parallel with the mesocotyl growth rate under deep seeding conditions. Therefore, deep seeding tolerance of 3681-4 line was achieved due to auxin-regulated rapid mesocotyl elongation.     

Appearance of bulges on maize roots as affected by 6-benzylaminopurine and α-naphthylacetic acid

The thickening that appeared on maize roots under the influence of 6-benzylaminopurine and α-naphthylacetic acid (concentration 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M) were analysed. The changes in length and width of maize roots at the edge of elongation zone after 24,48 and 72 h of treatment were studied. The growth in length of cells at the edge of elongation zone stopped abruptly but the growth in width slowly continued. So, the growth of cells in length and width under the influence of growth regulators was not simultaneous. They had distinct time limits.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Improvement of echinacoside and acteoside production by two-stage elicitation in cell suspension culture of <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

At appropriate concentrations, polyamines promoted the callus growth and echinacoside content of Cistanche deserticola while Ag⁺ increased the content of echinacoside and acteoside. In a 20-day culture period, when putrescine (25 μM) and Ag⁺ (10 μM) were added on day 8 and day 16, respectively, the echinacoside production (1.7 g l⁻¹) and acteoside production (0.4 g l⁻¹) reached the maximum, which were 1.4-fold and 1.5-fold of those in single putrescine treatment, 1.6-fold and 1.4-fold of those in single Ag⁺ treatment, respectively. Exogenous putrescine enhanced cell viability and antioxidant enzyme activity markedly, so increased the final biomass. Ag⁺ addition increased H₂O₂ content and phenylalanine ammonia-lyase activity significantly which led to higher echinacoside and acteoside contents.     

Dihydroquercetin protects barley seeds against mold and increases seedling adaptive potential under soil flooding

Barley (Hordeum vulgare L.) seeds were soaked in aqueous 10⁻⁴ M dihydroquercetin (DHQ) to examine its influence on seed germination and further growth of seedlings under optimal soil watering and flooding conditions. The adaptive potential of the plants was estimated by the content of thiobarbituric acid reactive substances (TBARs) and the activity of ascorbate peroxidase (AsP). High-grade seeds were germinated evenly under (−DHQ)- and (+DHQ)-treatments. Low-grade seeds soaked in DHQ, showed no mold and twofold germination rate in comparison with the same seeds soaked in water. The seedlings grown from the similarly germinated seeds did not differ from each other in the shoot growth, independent of the DHQ-pretreatment. The root growth was higher in DHQ-pretreated plants. Soil flooding suppressed the shoot and root growth rates in non-pretreated and DHQ-pretreated plants, however TBARs content was lower in the roots and leaves of (+DHQ)-seedlings as compared to the (−DHQ)-ones. The activity of AsP increased more significantly in the (+DHQ)-plants. The ratio between TBARs content and the AsP activity was lower in the leaves of (+DHQ)-plants both under optimal soil conditions and flooding. Thus, the treatment of low-grade barley seeds with DHQ protects the seeds against mold and increases adaptive potential of the seedlings.