Dormancy and Impotency of Cocklebur Seeds: IV. Effects of Gibberellic Acid, Benzyladenine, Thiourea, and Potassium Nitrate on the Growth of Embryonic Axis and Cotyledon Segments

Germination of nondormant but impotent small cocklebur seeds (Xanthium pennsylvanicum Wallr.) was promoted profoundly with thiourea or benzyladenine, and slightly with gibberellic acid. Gibberellic acid was ineffective in causing the germination of dormant cocklebur seeds, although thiourea and benzyladenine were effective. Experiments with excised seed pieces showed that the promotive effects of thiourea, benzyladenine, and gibberellic acid on cocklebur seed germination were associated with the enhancement of growth of seed parts; thiourea stimulated predominantly the axial growth, whereas benzyladenine stimulated predominantly the cotyledonary growth.     

Growth and asexual reproduction of the sea anemone Metridium: comparative laboratory studies of three species

The rate of growth, individual size attained, and mode and frequency of asexual reproduction were investigated in three species of the sea anemone Metridium. The species differ in ecological distribution, characteristic habitat, and morphology. M. exilis Hand is characteristically intertidal and occurs in aggregations of numerous small (0.2 cm² pedal disc area, pda) individuals. Growth in laboratory culture ceased at 1.2 cm² pda despite copious feeding. Feeding accelerated the rate of asexual reproduction by binary fission but did not result in larger individuals. Individuals of M. senile (L.) are larger and reproduce both sexually and asexually by fragmentation. The species occurs from the mid-intertidal to the shallow subtidal in central California. Individuals from intertidal populations grew rapidly in laboratory culture when fed daily (mean pda after 5 months in culture was 45 cm²). The regenerative ability of M. senile is excellent: 100% of excised fragments (including column and pedal disc tissue) had completely regenerated, including tentacle formation, after 3 wk with no mortality. An undescribed species of Metridium is much larger (individuals grow >/ l m in height) and apparently is exclusively subtidal in central California. Asexual reproduction is rare or absent and no pedal lacerates were produced by individuals during the period of study. The regenerative ability of Metridium sp. is poor: 53% of the excised fragments had regenerated after 19 wk with 25% mortality. The three species thus show distinct patterns of growth and asexual reproduction, paralleling their morphological, ecological, and genetic distinctness.     

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- ¹) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization². These strains specifically colonize tumors while remaining essentially non-virulent to the mouse¹. Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.     

Growth response of excised ovaries of reseda odorata in sterile culture

In the basal growth medium only the ovaries excised 15 days after pollination reached maturity. In a medium containing IAA (5ppm) and kinetin (0.5ppm) ovaries excised even 10 days after polliration produced viable seeds. Young ovaries formed in the pedicel region a callus, which later differentiated into roots; older ovaries (excised 15 days after pollination) produced callus tissue and roots from the ovary wall.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

Growth, in vivo extensibility, and tissue tension in developing pea internodes

The relationship between growth, in vivo extensibility, and tissue tension in the first 3 internodes of 5, 6, and 7 day-old pea plants (Pisum sativum L. cv Alaska), grown under continuous red light was investigated. The upper 15 millimeters of each internode was marked with ink and its elongation growth measured over the next subsequent 8 hours. In vivo extensibility was measured by stretching living tissue at constant force (creep test) in a custom-built extensiometer. Tissue tension was determined by (a) measuring the rate of expansion of the isolated cortical cylinder after adding water and the amount of contraction of the epidermis after peeling, and (b) by use of the `split section test.' A good correlation between rate of elongation growth, in vivo extensibility, and tissue tension was established. The epidermis peeled from the growing third internode of 7 day-old plants and measured immediately showed a plastic extensibility (E<sub>pl</sub> twice that of peels from nongrowing excised sections. This high E<sub>pl</sub>-value was lost on incubation of the sections in distilled water, and was subsequently restored by incubating the sections in auxin (indole-3-acetic acid). We conclude that the in situ growth of the internodes is a function of tissue-tension, which provides the driving force of organ growth, and the extensibility (E<sub>pl</sub> of the outer epidermal wall, which is in the growing plant in a `loosened' state. We furthermore suggest that in the intact plant auxin is causally involved in the wall loosening process in the epidermis.     

Growth and betaine formation in Medicago sativa tissue cultures

Static and suspension tissue cultures of Medicago sativa were initiated from sterile seedlings, excised roots and aerial portions of the seedlings. Flask suspension cultures containing chlorophyll were also established. The various tissues were harvested after 30 days, growth rates were determined, and the tissue was later lyophilized. The lyophilized tissues from various batches were extracted separately with 70% EtOH. The betaine mixture was isolated from these extracts and from seeds of the plant as their tetraphenylborate salts. The betaines were separated from the latter complex via ion exchange and column chromatography and were identified by standard procedures. All of the tissues, regardless of origin, produced choline. Only chlorophyllous tissue biosynthesized stachydrine.     

Growth Capacity of Embryo Axes Excised from Dormant and Germinating Horse Chestnut Seeds and Their Response to Exogenous Abscisic Acid

In embryo axes excised from mature horse chestnut (Aesculus hippocastanum L.) seeds, both freshly-fallen and subjected to cold stratification, the ability for growth was studied. While excised axes were kept on water at 28°C for 3 days, their fresh weight and length increased, the polypeptide composition of soluble proteins changed, the content of some heat-stable polypeptides decreased, and the capacity for protein synthesis in vivo retained. All these processes were similar to those in the axes of intact seeds during stratification until radicle protrusion. Growth of excised axes accelerated with the increasing duration of stratification. Cycloheximide (50 mg/l) and -amanitin (7 mg/l) inhibited axis growth, but an inhibitor of ABA synthesis fluridone (5 mg/l) and a natural cytokinin dihydrozeatin (10^–5 M) did not influence the growth rate. The growth capacity of axes excised from dormant and germinating horse chestnut seeds indicates the absence of dormancy in the axes of mature seeds. ABA (10^–5 M) suppressed completely the growth of axes detached from seeds experiencing cold stratification but still not germinating, although protein synthesis was not inhibited. The axes excised from the seeds after radicle emergence were insensitive to ABA and grew actively in its presence. ABA-induced growth inhibition might be related to the suppressed synthesis of minor polypeptides required for growth or to the activated synthesis of some growth-retarding proteins. The conclusion was drawn that the excised axes could be used as a model for studying the processes preceding visible germination of recalcitrant seeds.     

The Effects of l-Naphthylacetic Acid on the Growth of Excised Tomato Roots

I-naphthylacetic acid (NAA) enhances main axis extension andlateral initiation of excised tomato roots cultured in 0.5 percent sucrose medium. NAA has a qualitatively similar effectupon the growth of root tips cultured in glucose medium or transferredafter 2 days' previous growth in standard (1.5 per cent sucrose)medium to a glucose medium containing NAA.NAA does not raisethe growth-rate of roots cultured in glucose medium to thatoccurring in presence of sucrose as carbon source. The enhancedgrowth resulting from addition of NAA to glucose medium is notdependent upon enhanced glucose uptake.The unique role of sucrosein the growth of excised tomato roots is discussed in the lightof the above observations and of studies on the ability of glucoseto maintain the growth of excised roots previously establishedby a period of culture in sucrose medium.     

pH-Dependence of Extension Growth in Avena Coleoptiles and Its Implications for the Mechanism of Auxin Action

The pH-dependence of acid-induced growth in excised segments of Avena sativa coleoptiles has been reinvestigated in the pH range 3 to 7. In contrast to previous reports (e.g. DL Rayle [1973] Planta 114: 63-73), only acidic buffers with a pH below 5.0 induce an extension response. A pH of 3.5 to 4.0 is required to mimic auxin-mediated growth. Very similar pH-response curves are obtained with both intact (abraded) and peeled coleoptiles. These results agree with the recent finding of a similarly low sensitivity to protons in maize coleoptiles. It is shown that the apparently much higher sensitivity to protons previously reported for peeled Avena coleoptiles is due to incubating the tissue in buffer of pH 6.8 between peeling and measuring the effect of acidic buffers. Neutral pH reversibly inhibits the spontaneous extension burst originating on release from tissue tension after removing the epidermis. Reversal of this inhibition can be achieved by buffers of pH 5.0 to 6.0 (or distilled water), thereby simulating an acid-induced growth response in this pH range. It is concluded that true acid-induced wall-loosening generally does not take place above pH 5.0 and that a pH considerably below 4.0 is required in order to stimulate growth to an extent comparable to that obtained in response to auxin. The “acid-growth theory,” which requires an acid-mediated loosening of the cell wall in the pH range 5 to 6, this pH being established by auxin-induced proton excretion, can therefore also not be substantiated in Avena.     

Short term phytochrome control of oat coleoptile and pea epicotyl growth

Continuous recordings of the effect of light on oat (Avena sativa L. cv. Victory) coleoptile and pea (Pisum sativum L. cv. Alaska) epicotyl growth were made. Using a single excised coleoptile 10 minutes of red light was found to promote growth after a latent period of 46 minutes. The stimulation was transient and was not far red-reversible. Blue and far red light also promoted growth with similar kinetics. The action of continuous red or far red light was similar to that of 10-minute light. The growth of the intact pea third internode (as well as excised segments) was strongly inhibited by red light, with a latent period of 80 minutes. This effect was far red-reversible, and far red and blue light caused only a slight inhibition of growth.     

Auxin-induced leaf blade expansion in Arabidopsis requires both wounding and detachment

Elevation of leaf auxin (indole-3-acetic acid; IAA) levels in intact plants has been consistently found to inhibit leaf expansion whereas excised leaf strips grow faster when treated with IAA. Here we test two hypothetical explanations for this difference in growth sensitivity to IAA by expanding leaf tissues in vivo versus in vitro. We asked if, in Arabidopsis, IAA-induced growth of excised leaf strips results from the wounding required to excise tissue and/or results from detachment from the plant and thus loss of some shoot or root derived growth controlling factors. We tested the effect of a range of exogenous IAA concentrations on the growth of intact attached, wounded attached, detached intact, detached wounded as well as excised leaf strips. After 24 h, the growth of intact attached, wounded attached, and detached intact leaves was inhibited by IAA concentrations as little as 1 µM in some experiments. Growth of detached wounded leaves and leaf strips was induced by IAA concentrations as low as 10 µM. Stress, in the form of high light, increased the growth response to IAA by leaf strips and reduced growth inhibition response by intact detached leaves. Endogenous free IAA content of intact attached leaves and excised leaf strips was found not to change over the course of 24 h. Together these results indicate growth induction of Arabidopsis leaf blade tissue by IAA requires both substantial wounding as well as detachment from the plant and suggests in vivo that IAA induces parallel pathways leading to growth inhibition.     

Compensatory abilities depending on seasonal timing of thallus excision of the kelp Undaria pinnatifida cultivated in Matsushima Bay, northern Japan

Improved cultivation technology for the kelp Undaria pinnatifida is greatly needed to increase production to meet increasing commercial demand. A previous cultivation trial indicated that the crop yield of U. pinnatifida sporophytes could be increased greatly by thallus excision in late February due to compensatory growth of the remaining tissues. To develop this potential new cultivation technology, it is essential to identify the time period during which this kelp can compensate and its physiological responses to thallus excision. In this study, U. pinnatifida sporophytes were excised at about 30 cm above the meristem at the beginning of January, February, March, and April, respectively. Morphological features, photosynthesis, nutrient uptake, and carbon and nitrogen contents of excised kelps were measured and compared with these parameters in control kelps grown without excision. Both experimental and control kelps were farmed together in Matsushima Bay, northern Japan. The kelps excised in January and February showed significant increases in the lengths and dry weights of the blade, photosynthetic rates, nutrient uptake rates, and carbon and nitrogen contents compared with the control kelps, and the growth phase was prolonged for at least 1 month. No significant increases were found in dry weights and carbon and nitrogen contents of sporophylls until early April, which indicated that the maturation period was delayed. At the end of this experiment, the nitrogen contents of sporophyll tissues formed after excisions were significantly lower than those of tissues formed before excisions. In contrast, the kelps excised in March and April showed no significant increases in morphological and physiological parameters compared with control kelps. These results suggest that U. pinnatifida sporophytes exhibited great compensation when excisions were conducted during the growing phase in January and February but not in March and April when the maturation phase had started. The regulation of resource allocation to growth and maturation after thallus excisions in January and February likely results in prolongation of the growth phase and maturation phase in excised kelps.     

Effects of Growth Retardants on the Growth of Excised Roots of Dolichos lablab L. in Culture

The effects of various growth-retarding chemicals on the growthof excised roots of Dolichos lablab in sterile culture are investigated.Application of a range of concentrations of CCC, phosfon, andB-995 inhibited growth in length of the roots by reducing thefrequency of cell division, with little or no effect on cellelongation. Treated roots had generally lower DNA, RNA, andprotein contents than controls, although CCC-treatment significantlyenhanced the soluble nitrogen content of the roots. The inhibitoryeffects of growth retardants were not reversed by IAA or GA,but substances like choline chloride and pyridoxine hydrochloridewere partially effective in reinstating normal growth in rootsgrowing in concentrations of CCC producing about 50 percentinhibition of growth in length.     

Growth of Alaskan tundra plants in relation to water potential

The relationship of growth to plant water potential was studied in several arctic tundra species by experimentally altering the water regimes on excised tussocks, by clipping leaf area, and by clipping roots. Most species, including evergreen shrubs, showed greater growth under waterlogged conditions. The reduction of leaf area did not affect plant water potentials or growth. Root pruning decreased both plant water potentials and growth. Growth was suppressed when plant water potentials were −12 to −15 bars.     

Growth and Morphogenesis in Tissue Cultures of Anagallis arvensis

Morphogenesis has been induced in excised organs and callus tissue cultures obtained from various parts of the seedling and mature plants of pimpernel (Anagallis arvensis). Vigorously growing cell cultures capable of being periodically subcultured have been established in liquid as well as on the agar-solidified Murashige and Skoog's medium supplemented with 2,4-D (0.1 mg/1) + kinetin (0.1 mg/l) + coconut milk (10%). The callus tissue obtained from excised hypocotyl segments is white, soft, friable and fast growing, and has been subcultured over a period of two years without showing any sign of decline in growth. The optimum conditions for growth are at pH 5.9, temperature 27°C, and with 4% sucrose as the carbon source. Under appropriate nutritional supply these cultures can be manipulated to induce rhizogenesis in the suspension cultures, and buds and “embryo-like” structures on agar-solidified media. The excised leaves, hypocotyl and stem segments regenerate buds. Of the cytokinins used, 6-(y,y-dimethylallylamino)-purine proved to be the best for the number of cultures producing buds, as well as for the number of buds per culture. Anatomical studies revealed that buds arise from the epidermal and subepidermal layers of leaves and hypocotyl; these buds form shoots which eventually develop into plantlets.     

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.     

Studies in the Vernalisation of Cereals. IV: The Effect of Preliminary Soaking of the Grain on the Growth and Tropic Responses of the Excised Embryo of Winter Rye

Rye grains were soaked for various periods and the effect onthe subsequent growth of the excised embryo (grown on sucroseagar) studied. Soaking the grain for as short a period as two hours producesincreased growth of both roots and shoots in the excised embryo.This increase takes place irrespective of whether the embryois grown in light or darkness and is still manifested twentydays after the excision of the embryo. Production of lateral roots, both in light and darkness, andof anthocyanins in etiolated embryos is also increased by preliminarysoaking. Preliminary soaking of the grain has a marked accelerating effecton the tropic responses of the excised embryo. Concentration of sugar and duration of prelutllnary soakingboth effect the growth of the excised embryo, but neither constitutesa simple controlling factor in this growth. It is concluded that during the preliminary soaking, both auxinand a ‘regulator’ controlling its production anddistribution, enter the embryo from the endosperm and aleuronelayer.     

Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.