Involvement of ethylene in growth induction of stationary tobacco pith tissue in vitro

The effects of ethylene on growth initiation of tobacco pith tissue in vitro were investigated. Pith explants were incubated on a double inorganic modified White’s media containing 0.2 mg l⁻¹ kinetin with and without indole-3-acetic acid (IAA) and the ethylene synthesis inhibitor aminooxyacetic acid (AOA). The burst of wound ethylene had no effect on growth initiation, was not affected by the AOA, and decreased to its minimum level during the initial 24 h in culture. Tissue growth was initiated after 72 h and continued on IAA-containing media only. A marked increase in ethylene evolution occurred only in tissues subjected to an IAA-containing medium prior to growth initiation. AOA inhibited this ethylene synthesis and the following growth of the tissues. The initial water uptake by the pith explants occurring even in the absence of IAA was also inhibited by AOA. The metabolic indicators for growth initiation such as enhanced respiration, increased activity of nitrate reductase, and initiation of cathodic isoperoxidases were all inhibited by AOA. It was concluded that the primer function of IAA in growth initiation is via inducing the biosynthesis of a marked ethylene signal, which in the absence of which active growth will not occur. The inhibiting effect of AOA is continuous and a transfer of the pith explants to fresh IAA-containing media did not result in a new ethylene burst nor tissue growth induction. The morphological changes in the tissues and cells during the initial stages of their development on the different media are demonstrated.     

Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth.

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco (Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis related proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of transgenic tobacco is discussed.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Callus formation and differentiation in tissue cultures of normal and cytoplasmic male sterile sorghum,pepper, sunflower,and tobacco

Summary The primary goal of this study was to establish callus cultures of selected plant taxa containing both normal (N) and cytoplasmic male-sterile (CMS) lines. A secondary goal was to attempt to differentiate the calli into whole flowering plants. Undifferentiated and organ differentiated calli were produced in specific lines of N and CMS sorghum, pepper, sunflower, and tobacco. Calli from both N and CMS lines of a given species developed similarly and grew well. Even though whole flowering plants have not yet been produced from any of the lines, this approach allows for good production of callus for studies dealing with CMS. Journal Paper No. J-7741 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1914.     

The influence of various carbon and nitrogen sources on oil production byFusarium oxysporum

The oil-synthesizing capacityof Fusarium oxysporum, cultivated on basal nutrient medium, was evaluated using different carbon and nitrogen sources. In one of the media, molasses was also used as a principal carbon source. Media containing glucose and ammonium nitrate were found to be most efficient for oil production. Fatty acid profile of the fungal oil indicated the presence of a wide range of fatty acids ranging from C₈ to C₂₄. Fatty acid composition largely depends on the type of carbon and nitrogen sources.     

Further studies on the growth and differentiation of single,isolated cells of tobacco in vitro

Summary Single cells of hybrid tobacco callus, isolated from germinating seeds, were grown individually in microchambers in the absence of any other cells, in fresh, liquid, coconut milk-medium. These produced a conlony of 50–75 cells in 10–15 days. In all instances the plane of the first cell division, and in most cases also that of the second division, were at right angles to the long axis of the cell. There was more variation in the size, shape and pattern of the second and subsequent divisions in single cells isolated from fresh stem pith and seed callus than those from old stem callus. Upon transfer from the microchambers to agar medium the colony of cells gave rise to a huge mass of callus tissue in about 3 months. In no instance did the cell colonies or any stages preceding them resemble or simulate any stages of normal embryogeny of tabacco. Single-cell clones obtained by this method from seed callus or fresh pith callus produced shoots with leaves and roots in synthetic medium with various indoleacetic acid-kinetin combinations. Normal plants established from the above cultures in the greenhouse flowered in due course of time. This method offers the possibility of producing a very large number of clones or identical plants in species where vegetative propagation is not otherwise possible, apart from its use in studies on the genetics and morphogenesis in higher plants.     

Effects of various concentrations of uranium tailings on certain growth and biochemical parameters in sunflower

Effects of different concentrations (25, 50, 75 and 100%) of uranium tailings conditioned with garden soil on growth and biochemical parameters in sunflower were studied. The shoot and root length, fresh and dry mass as well as leaf area and chlorophyll contents showed significant negative correlation with the applied uranium tailing concentrations. The influence on plant growth was also measured in terms of Tolerance Index (TI) and Grade of Growth Inhibition (GGI). Yellowing of leaves was recorded in all the tailing concentrations. Soluble proteins (leaf) showed significant enhancement as the concentration of uranium tailing increased indicating a breakdown of structural insoluble proteins. Survival of sunflower plants over 100 days on higher tailing concentrations (up to 75%) showed that sunflower may be helpful in revitalization of uranium mining waste.     

The effects of various growth factors on endothelial cell survival in vitro

The effects of various growth factors on endothelial cell survival in vitro were studied. Using rat heart endothelial cells, the cell survival curves were obtained; the cells were cultured until confluent, the medium was changed to serum-free medium with or without growth factors, and the cells were counted after 3, 6, 9, and 12 days. Transforming growth factor-beta, which is known as a potent growth inhibitor for vascular endothelial cells, shortened the rat heart endothelial cell's survival period, while epidermal growth factor or transforming growth factor-alpha prolonged survival. Insulin did not affect the rat heart endothelial cell's survival. Our data indicate that growth factors play a role not only in cell proliferation but also in cell survival in vitro. In addition, elevated levels of growth inhibitors such as transforming growth factor-beta may cause tissue damage in vivo by affecting cell survival.     

Eicosanoids influence in vitro elongation of plasmatocytes from the tobacco hornworm, Manduca sexta

Nodule formation is the predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from hemolymph circulation. Hemocyte spreading behavior is a critical step in the nodulation process. It has been suggested that eicosanoids mediate several steps in the process. However, the influence of eicosanoids on hemocyte spreading has not been investigated in detail. To test the hypothesis that eicosanoids mediate hemocyte spreading behavior, I treated larvae of the tobacco hornworm, Manduca sexta, with eicosanoid biosynthesis inhibitors and later assessed plasmatocyte elongation on glass slides. Plasmatocytes from larvae treated with dexamethasone did not elongate to the extent of plasmatocytes from untreated control larvae. The dexamethasone effect on plasmatocyte elongation was expressed in a dose-dependent manner and was reversed by injecting dexamethasone-treated larvae with the eicosanoid-precursor fatty acid, arachidonic acid. Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on plasmatocyte elongation. Finally, plasmatocytes from larvae treated with a range of eicosanoid biosynthesis inhibitors did not elongate to the extent of plasmatocytes from control larvae. Plasmatocyte width did not appear to be influenced in this study. These findings strongly support the idea that insect plasmatocyte elongation is influenced by eicosanoids.     

污泥蚯蚓粪对万寿菊生长发育的影响

以赤子爱胜蚓（Eisenia foetida）处理混合比例为1∶1、2∶1、3∶1、4∶1、5∶1和1∶0的污泥和牛粪得到污泥蚯蚓粪,并以10%、20%和30%（干物质量）与黑土混合,研究其对万寿菊株高、茎粗、叶片数、分枝数、地上部生物量、地下部生物量、根冠比、花蕾数、产花量、花直径和花生物量等生长发育指标的影响.结果表明：蚯蚓粪明显促进了万寿菊的生长发育;污泥和牛粪比值越小,对植物生长发育越有利;蚯蚓粪含量超过一定量,万寿菊生长发育状况有所下降,20%配比处理对万寿菊生长发育最为有利.     

The relation of DNA synthesis and mitosis in tobacco pith tissue cultured in vitro

Summary DNA synthesis and mitosis were initiated in cultured tobacco pith tissue by means of IAA and kinetin. DNA classes were determined by microspectrophotometric measurements (Feulgen); autoradiographs (tritiated thymidine) served to ascertain whether or not nuclei had undergone DNA synthesis during culture.All mitoses in new cells (resulting from divisions in culture) were diploid and had been preceded by DNA synthesis in culture.Whereas many of the old cells (which had not previously divided in culture) found in diploid or polyploid mitosis had undergone DNA synthesis during culture, others had not. Such non-radioactive mitoses still occurred after 16 days.In view of this, a 4 C nucleus in differentiated tissue should be considered as potentially both diploid and tetraploid, for it appears impossible to predict whether it would, upon restoration of conditions conducive to DNA synthesis and mitosis, enter a diploid mitosis or, after undergoing DNA synthesis, a tetraploid one.A high nuclear DNA content seems to have a much more inhibiting effect on the onset of DNA doubling than on that of mitosis.Somatic polyploidization is understood as the result of two DNA doublings between which mitosis was omitted, or aborted, or in effect undone by a failure of cytokinesis leading to fusion during a later mitosis.This work has been supported by research grants to K. Patau from the U.S. Public Health Service (grant No. C-3313) and the American Cancer Society.     

The effect of sugars on inverse relation between the growth and chlorophy11 synthesis in tobacco tissue

Cytokinin-dependent and cytokinin-autonomous strains of tobacco callus tissue (Nicotiana tabacum L. cv. ‘Wisconsin 38’) were grown on media containing sucrose, glucose and fructose, respectively. The tissues were kept 14 days in darkness and then transferred for 9 days to continuous light after which time the fresh weight and chlorophyll content were estimated. The highest chlorophyll concentration was recorded at sugar levels which were either suboptimal (sucrose in the case of cytokinin-dependent strain) or supraoptimal (all other sugars for both strains and sucrose for the cytokinin-autonomous strain) for tissue growth. The chlorophyll concentration was increased when the tissue was cultured on media containing glucose or fructose,i.e. sugars whioh did not support the growth as well as sucrose. Chlorophyll synthesis in the cytokinin-autonomous strain is significantly lower than in the cytokinin-dependent strain. This difference was independent of either sugar source or concentration. These results support the observed inverse relationship between tissue growth and plastid development and the limited metabolic activity of plastids in cytokinin-autonomous tissues.     

The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.