Ethylene and ethane release during tobacco protoplast isolation and protoplast survival potential in vitro

Studies on stress ethylene and ethane during protoplast isolation from water-stressed and waterlogged donor plants Nicotiana tabacum L. xanthi-nc, show a correlation between ethane, but not ethylene, release and protoplast survival in vitro. Ethane release shows a high negative correlation with protoplast survival potential from donor plants subjected to both stresses. Ethylene showed a high negative correlation with protoplast survival potential in tissues from water-stressed but not from long-term waterlogged plants. The absence of correlation in the latter may be related to decreased ability to produce ethylene in hyperstressed plants.
The results are discussed in relation to the use of stress ethane release as a parameter of the physiological status of the plant.     

Flower formation in vitro in a quantitative short-day tobacco: interrelation between photoperiod and infructescence development

The flowering response of thin layers excised from branch internodes of Nicotiana tabacum cv. Maryland Catterton (quantitative short-day plant for induction) was studied under three photoperiodic treatments. The explants were excised from inflorescences bearing flowers only, flowers and green fruits, or from infructescences with green fruits only. The aim of the study was to investigate the post-inductive photoperiodic effects on in vitro flower bud formation in a quantitative short-day tobacco and the relation with infructescence development. Short days quantitatively enhanced the flower bud regeneration capacities of explants in all stages of development, both as number of explants induced to produce flowers and as mean number of flowers per explant. There was no significant difference in flower bud formation on explants of the first two stages, which produced much more flowers than those of the third stage. Observations in planta showed that, during the 20 days separating the second stage from the first stage, there was no significant difference in the number of floral buds and flowers present on the inflorescence; however, the branch internodes lengthened, as did the floral buds and flowers. During the 10 days leading to the third stage, the number of capsules did not change significantly, but a high rate of floral abscission occurred. The present results show that in Nicotiana tabacum cv. Maryland Catterton short day quantitatively controls not only the inductive step of the flowering process, but also affects the capacity to regenerate flower buds during the late post-inductive phases. The responsiveness to the photoperiodic signal decreases only when the plant exhibits only fruits.     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

The effect of photoperiod on flower formation in vitro in a quantitative short-day cultivar of Nicotiana tabacum

Superficial cell layers of a quantitative short-day tobacco plant ( Nicotiana tabacum L. cv. White Burley) were excised from different parts of the inflorescence (i.e. pedicels, branch internodes, rachises), and cultured in continuous darkness, continuous light or 8 h light/16 h dark daily. The flowering response in vitro of the different types of explants was investigated with respect to the effect of light on the post-evocation phases of the flowering process and explant commitment. Treatment effect was qualitatively and quantitatively influenced by explant origin. Three morphogenic features were observed: flower neoformation, caulogenesis and rhizogenesis (the latter on rachis explants only). Under all treatments, the highest flowering potential was shown by pedicels, while the highest vegetative potential was shown by rachises. Branch internodes showed an intermediate response, but with a tendency towards caulogenesis, which probably reflects their phylogenetic origin. Thus, opposite gradients of the neoformation of flowering and vegetative buds on explants were observed under all treatments. Pedicels formed new single flowers rather than inflorescences, while rachises regenerated mainly inflorescences. In darkness, flowering was limited mostly to pedicels. Vegetative bud formation was higher than floral bud regeneration in all types of explant. Continuous light enhanced the flowering response mostly in pedicel and branch internode explants. Short days enhanced flower bud formation in vitro on all types of explant. Results with respect to microsporogenesis, flower and inflorescence anomalies observed under darkness also seem to support the existence of a quantitative photoperiodic control on floral neoformation in vitro in this plant. These results suggest that in Nicotiana tabacum cv. White Burley in vivo floral induction, initiation and development are governed by the same photoperiodic requirements.     

Fate of chloramphenicol in tobacco tissue culture under various light regimes

Abstract. Non-differentiated tissue cultures (calli) and differentiated tissues (shoots) of tobacco were found to differ in their sensitivity to chloramphenicol (CAP). This phenomenon is especially manifested in darkness and in an illumination regime lacking u.v. and blue light. When the latter are included, CAP's photodegradation products are shown to appear. It seems that one of the main photodegradation pathways is through the production of p -nitro-benzaldehyde ( p -NBA) which is further degraded. The possibility that either acetylation or physiological nitration of CAP is the cause for the differential tolerance was eliminated. The chromatographic and radiographic results indicate that in vivo degradation of CAP occurs both in calli and in shoot cultures. One of the in vivo degradation products is CAP-base.     

Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco

Benzyladenine (BA) was found to regulate the number of flower buds regenerated in vitro from pedicel tissue of tobacco. Flower bud induction was particularly sensitive to BA levels in the range of 0.45 to 1.0 μ M , where a two-fold increase in concentration caused a threefold rise in the number of buds. When tissues were fed radioactive BA for 24h, only 9–12% of the counts were recovered in the original compound. The rest was present in metabolites, tentatively identified as the mono-, di- and triribotides, 7- and 9-glucosides and 9-riboside of BA. The amount of growth regulator taken up and the quantities of BA and its metabolites in the explants were all linearly related to the concentration of the medium. The internal BA concentration was ca 60% of the level in the medium after 24 h. When the concentration in the medium was raised, relatively more BA remained in the non-conjugated form. However, this change in the equilibrium between BA and the conjugates is too small to account for the steep rise in the curve representing concentration vs effect between 0.45 and 1.0 μ M .     

Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

Carbohydrate Accumulation Affects the Redox State of Ascorbate in Detached Tobacco Leaves

A decreased utilization of NADPH for CO₂ fixation as a result of carbohydrate accumulation in chlorotic leaves is generally believed to be associated with an increase in oxidative stress. Molecular oxygen may serve as an alternative electron acceptor of photosynthesis under these conditions. In order to test this hypothesis mature leaves of tobacco plants (Nicotiana tabacum) were detached, fed with glucose (50 mM) via the petiole, and used to study the effect of carbohydrate accumulation on the pigment content and the components of the superoxide dismutase-ascorbate-glutathione cycle. During a period of five days the concentration of total soluble carbohydrates increased substantially in leaves supplied with glucose in comparison with control leaves supplied only with water. This increase was accompanied by a twofold decrease in the chlorophyll content. In detached water-fed leaves the levels of most of the antioxidative components increased, whereas glucose feeding had no or only little additional effect on the activities of the protective enzymes, but caused a 1.6- and 4-fold increase in the contents of glutathione and ascorbate, respectively. In relation to the total foliar ascorbate pool, the amount of reduced ascorbate increased from about 30 % to 60 % upon feeding with glucose. These results do not support the idea that the accumulation carbohydrates per se cause an increased rate of superoxide production which necessitates increased activities of antioxidative enzymes. It rather appears that glucose-fed leaves have an increased reductive capacity that can be released via glutathione into the ascorbate pool, thereby, regulating the redox state of ascorbate.     

茉莉酸甲酯对干旱及复水条件下烤烟幼苗叶绿素荧光特性的影响

以烤烟品种“龙江911”为试验材料,研究了干旱及复水过程中外源茉莉酸甲酯(MeJA)对移栽后烤烟幼苗叶绿素含量和叶绿素荧光特性的影响.结果表明： 干旱下烤烟幼苗叶绿素含量、PSⅡ反应中心完全关闭时荧光产量(F_m)、PSⅡ潜在活性(F_v/F_o)、最大光化学效率(F_v/F_m)、实际光化学效率(Ф_PSⅡ)、表观电子传递速率(ETR)和光化学猝灭系数(q_P)下降,而初始荧光(F_o)和非光化学猝灭系数(q_N)升高,0.2和0.5 mmol·L^-1的外源MeJA明显减缓了干旱下烤烟幼苗F_v/F_m、F_v/F_o、Ф_PSⅡ、ETR、q_P的下降和q_N的上升,而1.0 mmol·L^-1 MeJA效果不明显.复水后,烤烟幼苗各项叶绿素荧光指标均有明显恢复,并且MeJA处理后的幼苗恢复更明显.表明外源MeJA减轻了干旱胁迫下烤烟叶片叶绿素的分解,对PSII反应中心起到一定的保护作用,提高了电子传递速率,降低了干旱胁迫对烤烟幼苗的伤害,并且复水后叶绿素含量和叶绿素荧光参数能迅速恢复,从而保证了经干旱胁迫后烤烟幼苗能迅速缓苗.     

High Humidity and Heat Stress Causes Dissociation of Endoplasmic Reticulum in Tobacco Pollen

High humidity (95 % RH) and temperature (38/45 °C) stress for 4h applied to pollen grains of Nicotiana tabacum did not affect pollen viability, assessed on the basis of the fluorochromatic reaction test, but affected in vitro germination; pollen grains treated at 38 °C showed marked delay in germination, while those treated at 45 °C failed to germinate in vitro. The major ultrastructural effect of the stress was on RER. Stacks of RER, characteristically present in fresh pollen, were largely dissociated in the stressed pollen. The extent of dissociation of RER was greater in pollen samples stressed at 45 °C than at 38 °C. The generative cell did not show any obvious change in the stressed pollen. RER was restored in pollen grains which showed germination following culture; but not in those which failed to germinate. Apart from affecting other RER-related functions the dissociation of RER is likely to result in the destruction of long-lived mRNA and thus affect the ability of pollen grains to initiate protein synthesis needed for germination.     

盐胁迫对植物叶绿素荧光影响的研究进展

盐胁迫是制约植物生长发育的主要非生物胁迫之一, 研究植物的耐盐机理对开发和有效利用盐碱地有重要的意义。叶绿素荧光动力技术作为研究植物光合生理状况及植物与逆境胁迫关系的理想方法, 可表明外界胁迫环境对植物光合器官的伤害程度。通过总结性阐述盐胁迫对植物叶绿素荧光的影响, 分别从盐分类型、植物类型、光照强度以及盐旱交互作用等方面分析了植物叶绿素荧光对盐胁迫的响应, 进而反映盐胁迫对植物光合能力的影响程度, 并提出增强植物抗盐性的途径, 包括施加外源物质、利用转基因技术、真菌的协同效应和培育耐盐品种。最后对叶绿素荧光动力技术在抗盐胁迫的运用前景进行了展望, 提出了当前研究需要解决的问题, 旨在为提高植物耐盐能力提供一定的理论依据。     

低温胁迫对2个茶树品种叶片叶绿素荧光特性的影响

以茶树〔Camellia sinensis ( Linn.) O. Ktze.〕品种‘黄金芽’(‘Huangjinya’)和‘迎霜’(‘Yingshuang’)为实验材料,研究了4℃低温胁迫1、2、4和6d对茶树叶片叶绿素荧光特性的影响。结果表明：4℃低温胁迫条件下2个茶树品种叶片的PSⅡ最大光化学效率( Fv/Fm )、PSⅡ潜在活性( Fv/F0)和表观光合电子传递速率( ETR)均显著低于各自的对照(25℃),且总体上随胁迫时间延长逐渐下降;‘黄金芽’叶片的光化学淬灭系数(qP)随低温胁迫时间延长持续下降且低于其对照,而‘迎霜’叶片的qP较其对照的变幅较小,且2个品种的qP总体上与各自的对照无显著差异;随低温胁迫时间延长,2个品种叶片的非光化学淬灭系数( NPQ)均先升高后降低,并在胁迫2 d时达到最高,且总体上高于各自的对照;而2个品种叶片的光合功能相对限制值( LPFD )均随低温胁迫时间延长而增大,且大多高于各自的对照。与各自的对照相比,低温胁迫条件下‘迎霜’叶片的各项叶绿素荧光参数的变幅总体上低于‘黄金芽’。研究结果显示：低温胁迫可直接损伤茶树叶片的PSⅡ反应中心,致使过剩的激发能大量积累于PSⅡ反应中心,最终导致茶树光合作用能力减弱。根据叶绿素荧光参数的比较结果,可以初步判定品种‘迎霜’的耐寒性优于品种‘黄金芽’。     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.