Histochemical techniques to localize rubber in guayule (Parthenium argentatum Gray)

There are few histochemical techniques for staining rubber in the cells of guayule. With regard to specificity, clarity and retention of the stain we found staining with oil red O or iodine-potassium iodide for bright field, and a combination of oil red O and dansyl chloride for epifluorescence microscopy to be the best methods. The plausible mechanisms of staining are discussed.     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

In vivo biocompatibility of aliphatic segmented polyurethane in rabbit

An aliphatic segmented polyurethane with soft to hard segment ratio 3 was synthesised using hexamethylene diisocyanate, polypropylene glycol 400 and 1,4-butane diol.A stainless steel cage implant system has been used to study thein vivo biocompatibility of this polyurethane. United States Pharmacopoeia negative control polyethylene was used for the comparison. Three cages, one with polyurethane another with United States Pharmacopoeia polyethylene and the third control empty cage were implanted subcutaneously in the dorsal aspect of rabbits. The inflammatory exudate surrounding the material was aspirated from the cages on 4, 7, 14 and 21 days after implantation. The total protein content in the exudate aspirated from all the 3 cages was significantly higher at 7 days than in the reported normal rabbit serum of New Zealand white rabbit but equal to that of our rabbit colony. The albumin concentration was lower in the initial period but increased at 21 days post implantation period in all the cages. Concentration of α₁, α₂ and γ-globulin also decreased in all cages at 21 days. Neutrophils were predominant in all the exudates aspirated from polyurethane, polyethylene and empty control cages during whole implantation period. This is attributed to the profound effect of the cages on the surrounding vasculature. Macrophage was found to be seen during acute phase of inflammation due to the migration of macrophage along with neutrophil towards the inflammatory lesion. The percentage of neutrophils showed a faster decline in the cage containing polyethylene at 21 days. The extra cellular alkaline phosphatase activity, though higher in exudate from cages containing polyurethane at 14 days post implantation, was same in all 3 cages at 21 days. Leucine amino peptidase activity was found to be decreased at 21 days of post implantation time though the empty control cage exhibited an increase at 14 days post implantation. The inflammatory response at 21 days was similar in polyurethane and the control polyethylene     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

In vitro induction of mutation in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and isolation of mutants with improved yield and fiber characters

This paper describes a protocol to develop cotton mutant lines with improved yield and fiber characters. Immature ovules [(15-day post-anthesis ovules (dap)] were irradiated with 10–50 Gy gamma rays and treated with 1–5 mM EMS and SA to investigate the stimulatory effects of mutagenic treatments. During the subsequent field trials, the mutant lines showed significant variations from control lines. Lower dose/concentration of mutagenic treatments effectively stimulate the agronomical characters like early flowering, plant height, number of bolls, yield of seed cotton, ginning percent, seed index, harvest index and fiber characters while exposure at higher dose/concentration results in lowering the value of the parameters. Consequently, we use this approach to induce genetic variability for obtaining novel mutant cotton cultivars. Among the 19 different mutant lines isolated from the study, M7 showed higher morphological variations in terms of yield characters such as plant height, number of bolls, yield of seed cotton, ginning percent and fiber characters. Significant increase in cellulose content was also noted in mutant lines, whereas moderate increase was observed in total fiber units of the mutant lines. The selected cotton mutant lines for cultivars were investigated systematically; these lines significantly increased the potential for agronomical enhancement of cotton yield.     

Functional and Molecular Characterization of Hyposensitive Underactive Bladder Tissue and Urine in Streptozotocin-Induced Diabetic Rat

Background

The functional and molecular alterations of nerve growth factor (NGF) and Prostaglandin E2 (PGE2) and its receptors were studied in bladder and urine in streptozotocin (STZ)-induced diabetic rats.

Methodology/Principal Findings

Diabetes mellitus was induced with a single dose of 45 mg/kg STZ Intraperitoneally (i.p) in female Sprague-Dawley rats. Continuous cystometrogram were performed on control rats and STZ treated rats at week 4 or 12 under urethane anesthesia. Bladder was then harvested for histology, expression of EP receptors and NGF by western blotting, PGE2 levels by ELISA, and detection of apoptosis by TUNEL staining. In addition, 4-hr urine was collected from all groups for urine levels of PGE2, and NGF assay. DM induced progressive increase of bladder weight, urine production, intercontraction interval (ICI) and residual urine in a time dependent fashion. Upregulation of Prostaglandin E receptor (EP)1 and EP3 receptors and downregulation of NGF expression, increase in urine NGF and decrease levels of urine PGE2 at week 12 was observed. The decrease in ICI by intravesical instillation of PGE2 was by 51% in control rats and 31.4% in DM group at week 12.

Conclusions/Significance

DM induced hyposensitive underactive bladder which is characterized by increased inflammatory reaction, apoptosis, urine NGF levels, upregulation of EP1 and EP3 receptors and decreased bladder NGF and urine PGE2. The data suggest that EP3 receptor are potential targets in the treatment of diabetes induced underactive bladder.     

Callus induction and plantlet regeneration from mature cotyledonary segments of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

We evaluated the efficiency of callus induction and plantlet regeneration from mature cotyledonary segments of groundnut cultivars VRI-2 and VRI-3. Callus cultures were induced from mature tissues using NAA and IAA in combination with KIN or BAP. Maximum induction was recorded with 3.0 mg/L IAA and 1.0 mg/L BAP. However, green, compact, and nodular calli were obtained in 2.5 mg/L of IAA or NAA combined with 1.0 mg/L of either BAP or KIN. Fresh and dry weights were highly influenced by auxin concentration. Compact and nodular calli were then transferred to shoot induction media. The highest mean number of shoots was observed in 3.0 mg/L BAP plus 0.5 mg/L IAA. Finally, the resulting plantlets were rooted with IBA and NAA.     

Profilin isoforms in Dictyostelium discoideum

Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.     

Effect of growth regulators on rapid micropropagation and psoralen production in <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

A rapid and efficient micropropagation system was developed for Psoralea corylifolia, an endangered, valuable medicinal plant. Multiple shoot buds were obtained in half-strength liquid Phillips–Collins (L2) medium supplemented with 5 μM benzylaminopurine (BA) and 5 μM thidiazuron (TDZ) from apical bud explants of 1-week-old cultures. The shoot buds were subcultured on enriched solid L2 medium supplemented with different concentrations and combinations of BA, kinetin (KIN), 2-isopentenyladenine (2iP), TDZ, bavistin (BVN) and trimethoprim (TMP). Enriched solid L2 medium supplemented with 2 μM BA, 1 μM TDZ and 100 mg l⁻¹ BVN were more effective in producing greater number of shoots per explant (85.2 ± 0.9 shoots/explant) after 4 weeks of culture. The regenerated shoots (40–50 mm in length) rooted and accompanied by hardening upon transfer to 50 μM indole-3-butyric acid (IBA) for 15 min and followed by planting in sterile soil mixture and vermiculate (3:1 v/v), with 50 ml of one-eight strength L2 basal salt solution devoid of sucrose and inositol, supplemented with 5 μM IBA and 100 mg l⁻¹ BVN. The plants achieved 100% rooting with hardening. Subsequently the rooted plants were successfully established in the field. The survival percentage differed with seasonal variations. The concentration of psoralen was evaluated in different tissues of ex vitro and in vivo grown plants by high-performance liquid chromatography (HPLC). Psoralen content was increased in leaves (2.97%), roots (2.38%), stems (5.40%) and seeds (1.63%) of ex vitro plants than the in vivo plants. This system facilitates for commercial and rapid propagation of P. corylifolia for conservation strategies and phytomedicine production.     

Histochemical Techniques to Localize Rubber in Guayule (Parthenium Argentatum GRAY)

There are few histochemical techniques for staining rubber in the cells of guayule. With regard to specificity, clarity and retention of the stain we found staining with oil red O or iodine-potassium iodide for bright field, and a combination of oil red O and dansyl chloride for epifluoresence microscopy to be the best methods. The plausible mechanisms of staining are discussed.