Development of a liquid flow-through system to inhibit browning in callus cultures of guayule (Parthenium argentatum Gray)

Calli of P. argentatum were grown on a newly designed liquid nutrient flow-through system which facilitated the subculturing of calli and delayed browning for 6 weeks. Friable calli were obtained on half-strength Gamborg B5-medium supplemented with 0.05 mgl⁻¹ 2,4-dichlorophenoxyacetic acid. Shoots developed on media supplemented with 0.2 mgl⁻¹ benzylaminopurine but lacking 2,4-dichlorophenocyacetic acid.     

Improved germination in seeds of guayule (Parthenium argentatum Gray) following polyethylene glycol and gibberellic acid pretreatments

Suitable conditions for the pretreatment of seeds of guayule were determined with a view to improving germination. These included the addition of thiram (500 mg litre^-1), and applications of GA₄₊₇ alone, or in combination with polyethylene glycol. Significant improvements in the seed germination were seen following applications of PEG at -0.10 MPa. Simultaneous application of GA₄₊₇ with priming improved soil emergence and germination over a wide range of temperatures. No beneficial effects of PEG priming were evident on seed storage.     

Responses of guayule (Parthenium argentatum) seedlings to plant growth promoting fluorescent pseudomonads

Summary The potential of a number of fluorescent pseudomonad strains to promote growth of guayule plants in the greenhouse and in the field was studied. A number of bacterial strains collected from guayule roots and rhizospheres promoted growth of greenhouse-grown plants but not field-grown plants. Percent increase in shoot dry weight of 12-week-old, greenhouseinoculated guayule plants ranged from 17 to 75 nine weeks after inoculation compared to non-inoculated plants. The increased growth of plants in the greenhouse could reduce production cost by shortening the time required to maintain plants in the nursery prior to transplanting to the field.Journal Series Article no 3816 of the Arizona Agricultural Experiment Station.     

A simplified protocol for micropropagation of guayule (Parthenium argentatum gray)

Summary A simple, efficient protocol for in vitro micropropagation of guayule is reported. Shoot cultures were maintained on MS (Murashige and Skoog, 1962) medium supplemented with 1.0 mg l⁻¹ (4.4 μM) 6-benzylaminopurine and 0.025 mg l⁻¹ (0.3 μM) α-naphthaleneacetic acid. Excised shoots were treated for 14–18 h with 100 mg l⁻¹ (492.1 μM) indole-3-butyric acid in 0.5 x MS salts to induce rooting. The shoots were subsequently inserted into cellulose plugs which were packed in sterile, ventilated plastic culture vessels and moistened with 0.5 x MS medium without growth regulators. Use of cellulose plugs, liquid medium and ventilated culture vessels facilitated acclimation. Rooted shoots were transplanted into potting medium and acclimated to greenhouse conditions by covering with a cloche for 2 d, followed by daily watering for the first week. Any mention of trade names or commercial products in this report is for informational purposes only and does not imply endorsement by the U.S. Department of Agriculture or the Agricultural Research Service.     

The effect of micronutrient supply on the growth and seed production of guayule (Parthenium argentatum Gray)

The effect of micronutrient supply on the growth and seed production of guayule (Parthenium argentatum Gray) was investigated. A deficiency of boron, but no other trace element, significantly reduced the growth, seed production and the percentage of seeds that germinated. The optimum concentration of B for seed production was between 20 and 100 μM. Results indicate that guayule may benefit from B fertilisation.     

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.     

A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.     

Comparative macro- and micro-morphological studies on varieties ofParthenium argentatum Gray (guayule)

We investigated the leaf shape, venalion pattern, trichomes, stomata, and branching of the inflorescence axes in 15 varieties of guayule(Parthenium argentaturo Gray) growing in India. Working from our study objective, we were unable to identify any specific correlation between these macro- and micro-morphological characteristics and rubber content.     

cis-Polyisoprene Synthesis in Guayule Plants (Parthenium argentatum Gray) Exposed to Low, Nonfreezing Temperatures

Exposure of guayule plants (Parthenium argentatum Gray) to 6 months of a night temperature of 7°C results in a 2-fold stimulation of cis-polyisoprene (rubber) formation over that of control plants exposed to 21 to 24°C night temperature. Control and cold-treated plants contained 2.18% and 5.69% rubber, respectively. Examination of the stem apices by transmission electron microscopy showed extensive formation of rubber particles in the cold-treated plants compared to the control plants. The rubber particles in guayule are formed in the cytoplasm and fuse to form large globular deposits. The surface area of the rubber particles and globules range from 4 × 10⁻⁶ to 2.9 × 10⁻³ square micrometers. The deposition of rubber in the cytoplasm of the cortical parenchyma cells differs from rubber deposition in the vacuoles of laticifers of Asclepias syriaca. Electron micrographs of stem cortical parenchyma in control plants show mature cells with large central vacuoles, thin layers of parietal cytoplasm, and smaller numbers of rubber particles. Radioactive acetate and mevalonate are incorporated into rubber at a faster rate in stem slices from cold-treated plants compared to slices from control plants. A faster rate of these reactions may account for the increase in rubber synthesis in the cold-treated plants.     

The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg²⁺ isopentenyl pyrophosphate, and an allylic pyrophosphate. The K_m values for Mg²⁺, isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 × 10⁻⁴ molar, 8.3 × 10⁻⁵ molar, and 9.6 × 10⁻⁵ molar, respectively. The molecular characteristics of the rubber polymer synthesized from [¹⁴C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 × 10⁶ to 500 Ångstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 × 10⁶ compared to 1.49 × 10⁶ for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 × 10⁵ after 3 hours of incubation with Mg²⁺ and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer.     

Distribution, Development and Structure of Resin Ducts in Guayule (Parthenium argentatum Gray)

The distribution, development and structure of resin ducts inguayule (Parthenium argentatum Gray), the second best sourceof natural rubber, have been studied. Resin ducts are widelydistributed in stem, root, leaf, petiole and peduncle. The ductsin the primary tissues are initiated schizogenously and theirfurther development is schizolysigenous. The ducts in the cortexof the root do not have a well-defined epithelium. Ducts developedfrom the vascular cambium are initiated and develop schizogenously.Both resin and rubber are produced in the epithelial cells ofresin ducts. While resin is secreted into the duct lumen, rubberis stored within these cells. Epithelial cells store more thanneighbouring parenchyma cells. Guayule, rubber, resin, ducts, epithelial cells     

Partial purification and characterization of 3-hydroxy-3-methylglutaryl coenzyme a reductase from the leaves of guayule (Parthenium argentatum)

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been isolated and was partially purified from the leaves of Parthenium argentatum. The enzyme was found to be associated both with the cytosol and the chloroplasts. Ten mM dithiothreitol was essential to prevent loss of activity. Optimum activities of cytosolic and chloroplastic fractions were observed at pH 7.0 and 7.5 respectively. Preincubation of the reaction mixtures with CoA, acetyl-CoA, σ-phenanthroline and iodoacetamide resulted in the progressive loss of enzyme activity. 3-Hydroxybutyrate and mevalonate also inhibited the enzyme. The Michaelis constants of the enzyme for HMG-CoA and NADPH were 0.25 and 0.31 mM respectively for the cytosolic enzyme, while those for the chloroplastic enzyme were 0.018 and 0.42 mM respectively. Inhibition studies indicated that hydroxybutyrate was a competitive inhibitor with respect to HMG-CoA. The inhibition of mevalonate was competitive with HMG-CoA and non-competitive with NADPH.     

Differential Responses of Guayule (Parthenium argentatum Gray) Genotypes to Culture Filtrate and Toxin from Macrophomina phaseolina (Tassi) Goidanich

The potential for using cell-free culture filtrate (CFCF) and toxin (phaseolinone) from Macrophomina phaseolina for rapid and effective screening procedures for charcoalrot resistance in guayule (Parthenium argentatum) germplasm was assessed. The CFCF and partially purified phaseolinone were incorporated into modified Murashige and Skoog solid medium aat the rates of 0-100% (v/v) and 0-1000 μg ml^-1 respectively. The medium pH was adjusted to 5.8 before solidifying with 0.8% agar. Fourweek-old seedlings of 10 guayule genotypes were plantanted in the medium, incubated and rated for phytotoxic symptoms and tissue damage over a 15-day period. In a green-house study, seedling growth, phytotoxicity and damage severity were compared in 12-week-old guayule seedlings root-inoculated with M. phaseolina microsclerotia. There were significant differences (P = 0.05) in genotypic responses to the fungus, the filtrate and the toxin inoculations. Time until phytotoxic symptoms developed was inversely related to the concentrations of CFCF and the toxin. Phytotoxic symptoms were produced 6 days after exposure to 50% CFCF and 48 h after exposure to 1000μg ml^-1 of partially purified phaseolinone. A comparison of photomicrographs of the control and toxintreated root tissues revealed no damage to the control roots and extensive damage to epidermal layers of the treated roots, which was evident 48 h after exposure to 100μg ml^-1 level of phaseolinone. Significant correlations were found between tolerance to the fungus and insensitivity to the culture filtrate (r = 0.89, P = 0.05) and the toxin (r = 0.95, P = 0.001) suggesting the possibility of screening for resistance to M. phaseolina using CFCF or phaseolinone. The genotypic reactions to the CFCF were also correlated with reactions to the toxin (r = 0.90, P = 0.05). Guayule breeding lines‘UC101 and‘P3-1 exhibited the greatest tolerance to the pathogen and insensitivity to the CFCF or the toxin whereas‘Ca16′,‘Cal7′,‘N576′,‘N9-5′, 11605’and‘N6-5’were very susceptible to the pathogen and sensitive to the CFCF or the toxin.