Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

EFFECT OF PICLORAM ON TRANSPORT IN YOUNG BEAN,MESQUITE, AND HUISACHE PLANTS

The effect of 4-amino,3,5,6-trichloropicolinic acid (picloram) on transport from leaves to the roots was studied using young bean (Phaseolus vulgaris L.), mesquite (Prosopis juliflora var. velutina (Woot.) Sarg.), and huisache (Acacia farnesiana (L) Willd.) plants. The only picloram treatments which were effective in enhancing transport of ¹⁴C-assimilate or ¹⁴C-picloram to the roots were those made to the shoots or roots one day or more before application of the label to the shoots. The enhancement of transport was not evident when un-labeled picloram or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or both, were applied at the same time as the ¹⁴C-label. Enhancement of transport was to the more mature stem or root tissues. Inorganic nitrogen applied to nitrogen-deficient bean plants also increased transport of ¹⁴C-assimilate to the roots, especially the rate.     

Phytotoxicity and transport of clopyralid from three formulations in honey mesquite

Foliar sprays of the monoethanolamine salt, oleylamine salt, and 1-decyl ester of clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) were about equally effective in killing greenhouse-grown honey mesquite (Prosopis glandulosa Torr.). Treated leaves absorbed more clopyralid within 15 min after pipet application of the oleylamine salt compared to the other formulations. After 24 h, treated leaves absorbed and transported more clopyralid into the plant after application of the salt formulations compared to that of the 1-decyl ester. There were no consistent differences among clopyralid formulations in transport of clopyralid from foliar sprays at 4 h or 1, 3, or 8 days after treatment. Only the acid form of clopyralid was transported from the site of application of either ester or the amine formulation.     

Phytotoxicity and transport of clopyralid from three formulations in honey mesquite

Foliar sprays of the monoethanolamine salt, oleylamine salt, and 1-decyl ester of clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) were about equally effective in killing greenhouse-grown honey mesquite (Prosopis glandulosa Torr.). Treated leaves absorbed more clopyralid within 15 min after pipet application of the oleylamine salt compared to the other formulations. After 24 h, treated leaves absorbed and transported more clopyralid into the plant after application of the salt formulations compared to that of the 1-decyl ester. There were no consistent differences among clopyralid formulations in transport of clopyralid from foliar sprays at 4 h or 1, 3, or 8 days after treatment. Only the acid form of clopyralid was transported from the site of application of either ester or the amine formulation.This paper reports results of research only. Mention of trademark name of proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply their approval to the exclusion of other products that may also be suitable.     

Comparative Effects and Concentration of Picloram, 2,4,5-T and Dicamba in Tissue Culture

In nutrient agar comparative concentrations (10^?3 to 10^?5M) of (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) were generally more inhibitory to the growth of tissue cultures of soybean (Glycine max (L.) Merrill cv. Acme) and cottonwood (Populus deltoides Marsh.) than were either 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloro-o-anisic acid (dicamba). Compared to untreated tissue dicamba or picloram at 10^?6M in the nutrient agar resulted in a 200 % increase in the growth of soybean tissue. At 10^?5 and 10^?6M dicamba also produced an increase in the growth of cottonwood tissue. Greatest absorption of picloram and dicamba by tissue cultures from agar occurred during the first 24 h after treatment. However, absorption remained nearly static thereafter for 14 days. More dicamba was absorbed by soybean and cottonwood tissue cultures than either picloram or 2,4,5-T.     

Application of the disk method: responses to growth regulators

The paper disk method of screening several plant growth regulators was evaluated. Leaf explants ofVigna unguiculata (L) Walp. were placed on solidified Murashige and Skoog's minimal organics medium containing 0.5 mg/l nicotinic acid. Hormones were tested, singly and in combinations, on paper disks in large Petri plates (150×20 mm). Hormones tested were 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), picloram (4-amino-3,5,6-trichloropicolinic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), BA (6-benzyladenine), 2iP (2-isopentenyl adenine), and kinetin [6-(furfurylamino)-purine]. Root formation was stimulated by IAA and IBA; dicamba, picloram, 2,4-D, and 2,4,5-T stimulated callus formation. All cytokinins tested suppressed root formation. Dicamba in combination with either 2iP or kinetin induced the greatest callus formation. Root formation was optimal with kinetin and either IAA or IBA. The disk method provided a rapid, nonquantitative evaluation of callus and root formation from leaf disks.     

Reductive Dechlorination of the Nitrogen Heterocyclic Herbicide Picloram

The anaerobic biodegradation of picloram (3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid) in freshwater sediment was favored under methanogenic conditions but not when sulfate or nitrate was available as a terminal electron acceptor. Under the former conditions, more than 85% of the parent substrate (340 μM) was removed from nonsterile incubations in 30 days, following a 50-day acclimation period. Concomitant with substrate decay, an intermediate transiently accumulated in the sediment slurries. By liquid chromatography-mass spectrometry, the intermediate was identified as an isomer of dichloro-4-amino-2-pyridinecarboxylic acid. Proton nuclear magnetic resonance evidence suggested that a chlorine was reductively removed from the parent substrate at the position meta to the nitrogen heteroatom. Upon continued incubation, the dechlorinated product was transformed into an unidentified compound which accumulated and resisted further decay. The addition of sulfate or bromoethanesulfonic acid to sediment slurries inhibited picloram dehalogenation, but molybdate reversed the inhibitory effect of sulfate on pesticide metabolism. These findings help clarify the fate of a halogenated nitrogen heterocyclic herbicide in anaerobic environments.     

An evaluation of the role of ethylene in herbicidal injury induced by picloram or clopyralid in rapeseed and sunflower plants

The role of ethylene in herbicidal injury induced by 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloropicolinic acid (clopyralid) was investigated in sunflower (Helianthus annuus L.) and rapeseed (Brassica napus L. cv Altex). Picloram induces herbicide injury in both species, whereas clopyralid induces injury only in sunflower. Picloram applied to the third leaf of a rapeseed plant increased ethylene evolution several-fold. Clopyralid had no effect on ethylene production in rapeseed. In sunflower, both picloram and clopyralid elevated ethylene production. Ethylene biosynthesis induced by the herbicide treatment was not restricted to treated areas. When clopyralid was applied only to the lower stem and cotyledons of sunflower, the herbicide treatment resulted in an increase in the rate of ethylene production from the true leaves. Increased ethylene production preceded or coincided with the onset of morphological responses induced by a herbicide application to a susceptible species. The contrast in ethylene production by these two plant species cannot be accounted for by differences in absorption and translocation of clopyralid and picloram.

Treatment with aminoethoxyvinylglycine (AVG) before picloram or clopyralid application prevented an increase in ethylene production. Pretreatment with AVG also delayed the development of morphological changes induced by picloram or clopyralid. It appears that enhanced ethylene biosynthesis after application of picloram or clopyralid to the susceptible plant species was a factor involved in resulting morphological changes.

     

替代物种与除草剂对紫茎泽兰的防效及其互作效应

[目的]研究替代物种与选择性除草剂,以实现对紫茎泽兰安全、高效和绿色的防控。[方法]采用植物间竞争试验法测定替代物种（高丹草、非洲狼尾草和黑麦草）和除草剂对紫茎泽兰的防效及其互作效应。[结果]高丹草、非洲狼尾草和黑麦草的竞争力依次强于紫茎泽兰。随着3种替代物种密度比例增加,紫茎泽兰的株高、分枝、根冠比、叶面积比均受到显著抑制,当替代物种与紫茎泽兰密度比为5∶1时,高丹草、非洲狼尾草和黑麦草对紫茎泽兰的替代控制效果分别为55.1%、46.9%和40.3%。除草剂氨氯吡啶酸、三氯吡氧乙酸、氯氟吡氧乙酸和二氯吡啶酸的毒力测定表明,二氯吡啶酸相对最高,LC₉₀为902.1 g·hm^-2,但三氯吡氧乙酸对替代物种的安全性相对最好,对高丹草、非洲狼尾草和黑麦草选择性指数分别为3.75、2.79和2.67。高丹草和非洲狼尾草与紫茎泽兰种植密度比例为3∶1时,结合二氯吡啶酸和三氯吡氧乙酸,非洲狼尾草和二氯吡啶酸结合表现拮抗作用,与三氯吡氧乙酸结合表现加成作用;高丹草和二氯吡啶酸结合表现加成作用,与三氯吡氧乙酸结合表现增效作用,在同等取得90%防效的情况下,可减少三氯吡氧乙酸用量20.0%~35.0%（即210.0~370.0 g·hm^-2）。[结论]合理的替代物种结合除草剂应用可实现互作增效,可作为紫茎泽兰综合防控的重要措施。     

Establishment and characterization of <Emphasis Type="Italic">Prosopis laevigata</Emphasis> (Humb. &; Bonpl. ex Willd) M.C. Johnst. cell suspension culture: a biotechnology approach for mesquite gum production

This study presents a protocol for the establishment of Prosopis laevigata cell suspension culture as a strategy to obtain an in vitro mesquite gum productive cell line. The callus used for this purpose was obtained with hypocotyls from 15-day-old plantlets, placed on Murashige–Skoog medium with two different plant growth regulators (PGRs), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T; 5.0 μM) and kinetin (KIN; 5.0 μM). With this PGRs treatment, after four subcultures (30 days each) an exuded gum-like substance was observed on the callus surface. The growth kinetics of the cell suspension culture showed a specific cell growth rate (μ) of 0.14 d⁻¹ and doubling time (t _d) of 6.6 days, respectively. The gum-like substance from callus culture and the broth from cell suspension culture were subjected to chemical analysis and compared with the mesquite gum exuded from wild trees. Both, gum-like substance from callus culture and the broth from cell suspension culture showed the presence of Arabinogalactan-proteins, and their polysaccharide fraction presented the same monosaccharides as those isolated from mesquite gum. In addition, the emulsifying properties of gum-like substance from callus culture and the broth from cell suspension culture were compared to those of mesquite gum and all three samples exhibited similar emulsifying capacity and emulsification stability.     

The discovery of Arylex™ active and Rinskor™ active: Two novel auxin herbicides

Multiple classes of commercially important auxin herbicides have been discovered since the 1940s including the aryloxyacetates (2,4-D, MCPA, dichlorprop, mecoprop, triclopyr, and fluroxypyr), the benzoates (dicamba), the quinoline-2-carboxylates (quinclorac and quinmerac), the pyrimidine-4-carboxylates (aminocyclopyrachlor), and the pyridine-2-carboxylates (picloram, clopyralid, and aminopyralid). In the last 10 years, two novel pyridine-2-carboxylate (or picolinate) herbicides were discovered at Dow AgroSciences. This paper will describe the structure activity relationship study that led to the discovery of the 6-aryl-picolinate herbicides Arylex™ active (2005) and Rinskor™ active (2010). While Arylex was developed primarily for use in cereal crops and Rinskor is still in development primarily for use in rice crops, both herbicides will also be utilized in additional crops.     

Inheritance of resistance to clopyralid and picloram in yellow starthistle (Centaurea solstitialis L.) is controlled by a single nuclear recessive gene

The noxious weed yellow starthistle (Centaurea solstitialis L.) can be controlled effectively at the seedling stage with foliar application of the auxinic herbicides picloram or clopyralid. Although resistance to these herbicides is rare, a yellow starthistle biotype resistant to picloram and cross-resistant to clopyralid was observed in 1989 near Dayton, WA, in a pasture that had been subjected to intensive picloram selective pressure. Our objective was to determine the mode of inheritance for this resistance trait. Transmission of the resistant phenotype was monitored in reciprocal F(1) crosses between susceptible (SCI) and resistant (RDW) plants, their testcross and pseudo-F(2) progeny. Progeny from all crosses, as well as RDW and SCI seedlings of original populations, were sprayed with picloram or clopyralid to distinguish between susceptible and resistant individuals. All F(1) progeny were susceptible to both herbicides, indicating that the resistance trait was of nuclear origin and recessive in nature. Segregation of the resistant phenotype among pseudo-F(2) and testcross progeny of F(1) genotypes demonstrated monofactorial inheritance (P >.25) for resistance to both herbicides. The conclusion that resistance is conferred by a single recessive allele is consistent with the observation that no other picloram-resistant yellow starthistle populations have been identified in the area since picloram selection pressure was abated.     

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Summary Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - CA casamino acids - B5CA B5 with 0.2% casamino acids - IBA indolebutryric acid; Picloram (4-amino-3,5,6-tricnloro-2-pyridinecarboxylic acid) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - BA 6-benzylaminopurine     

Involvement of Ethylene in Picloram-induced Leaf Movement Response

The relationship of root-applied 4-amino-3,5,6-trichloropicolinic acid (picloram) to ethylene production and the leaf movement response in honey mesquite (Prosopis juliflora [Swartz] DC. var. glandulosa [Torr.] Cockerell) and huisache (Acacia farnesiana [L.] Willd.) was studied in detail. The threshold and saturation levels of exogenous ethylene and root-applied picloram necessary to inhibit leaf movement were determined. Internal levels of ethylene in excess of those necessary to saturate the leaf movement inhibition response occurred in tops of treated plants before and after symptom expression. These internal levels of ethylene, while averages for the entire plant tops, probably occur at the specific site of action and thus account for the action of picloram in inhibition of leaf movement and related responses. Quantitative differences in the leaf movement response of both species to picloram and ethylene were observed. In huisache alone, a very small change in ethylene levels was necessary to produce a major blockage of the leaf movement response, suggesting that the gas may have a natural function in that species.     

Chemical options for the control of Siam Weed (Chromolaena odorata) in natural ecosystems

The perennial shrub Siam Weed (Chromolaena odorata (L.) R. M. King & H. Rob.), a native of the rainforests of central and southern America, is predicted to invade most countries between the Tropics of Cancer and Capricorn. Subsequent to its discovery in northern Queensland, Australia in 1994, it was unsuccessfully targeted for eradication, with early control efforts restricted to manual removal and foliar spraying with triclopyr/picloram. A chemical trial involving five herbicides and three application methods (foliar, basal bark and cut stump) was undertaken in northern Queensland to identify effective chemicals to control this weed. Of the foliar herbicides tested, fluroxypyr (70 g/100 L) provided 99% mortality, triclopyr/picloram (105/35 g/100 L) 95%, while metsulfuron‐methyl (9 g/100 L) killed 85% of the treated plants. The herbicides fluroxypyr (300 g/100 L), picloram (43 g/1 kg) and triclopyr/picloram (400/200 g/100 L) killed greater than 98% of the plants when applied as basal bark or cut stump. The integration of fire into the management strategy either as a primary or secondary control option could have a major role, given that in this study, 99% of the Siam Weed soil seed bank was located on the soil surface.     

Effect of plant growth regulators on morphogenesis and forskolin production in Plectranthus barbatus Andrews

Plectranthus barbatus (syn. Coleus forskohlii) is the only known source of forskolin, a compound with a wide range of pharmacological activities. Here, an efficient protocol for adventitious root regeneration from leaf explants of P. barbatus was developed. Different concentrations of plant growth regulators individually and in combination were used to induce roots in vitro. Morphogenic responses and forskolin production varied depending on the concentrations of plant growth regulators added to the medium. Lower concentrations of auxins trigger callus proliferation while higher concentrations induced adventitious root regeneration. Of all the auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2 (2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP), and 4-amino-3,5,6-trichloropicolinic acid (picloram) induced callus, whereas α-naphthaleneacetic acid (NAA), indole-3-acetic acid, and indole-3-butyric acid induced rhizogenesis. Use of picloram at 1.0 and 0.5 mg l⁻¹ resulted in the formation of friable callus, and when combined with 0.5 mg l⁻¹ 6-benzylamino purine (BA), rhizogenic callus was produced. The cytokinins BA and kinetin produced a mixed response of multiple shoot regeneration, callus proliferation, and rhizogenesis. The maximum forskolin content of 1,178 mg kg⁻¹ dry weight was found in root cultures initiated on Gamborg’s B₅ medium supplemented with 0.5 mg l⁻¹ NAA. The biosynthesis of forskolin was differentiation dependent, and rhizogenic cultures exhibited the maximum biosynthetic potential for forskolin.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Screening and optimization of the derivatization of polar herbicides with trimethylanilinium hydroxide for GC-MS analysis

In the present study, a derivatization method for the determination of acidic herbicides has been investigated. This procedure involves the methylation with the quaternary ammonium salt trimethylanilinium hydroxide (TMAH) directly in the gas chromatographic auto-sampler vial for analysis by gas chromatography combined with mass spectrometry. The derivatization reaction has been screened for influential factors and statistically significant parameters. The identified factors, reaction time, temperature and hold-up time were optimized by a complete factorial response surface design and optimal reaction conditions were generated. Finally, the optimized methylation procedure was compared to different alkylation methods and obtained results demonstrated the applicability of derivatization with trimethylanilinium hydroxide. Acidic herbicides used in the study consist of several families of compounds like derivatives of acetic acid (2,4-D and 2,4,5-T), butanoic acid (MCPB), benzoic acid (chloramben, dicamba), phenol (dinoseb and dinoterb), propanoic acid (mecoprop) and other miscellaneous acids such as pyridinecarboxlyic acid (picloram). A reliably working, rapid method for the preparation of methyl compounds is generated with respect to automation for routine analysis.     

Effects of picloram and ethylene on leaf movement in huisache and mesquite seedlings

Application of 4-amino-3,5,6-trichloropicolinic acid (picloram) to roots stimulated the production of ethylene in both mesquite [Prosopis juliflora (Swartz) DC. var. glandulosa (Torr.) Cockerell] and huisache [Acacia farnesiana (L.) Willd.] seedlings. Herbicide levels rose in tissues before we detected increased ethylene production. Rates of ethylene production by various parts of the plant paralleled herbicide concentrations. In both species, picloram caused loss of leaf movement and epinastic curvature of leaves and stems. Only huisache was defoliated by picloram. Rates of ethylene production increased before we observed any leaf movement or defoliation responses. Fumigation of plants with levels of ethylene, calculated to approximate those in herbicide-treated plants at the initial loss of leaf movement, caused the same symptoms as picloram treatment. The time sequence of ethylene fumigation and loss of the ability for leaf movement is compatible with the hypothesis that there is a causal relationship between picloram and ethylene production and loss of leaf movement.     

Translocation and Metabolism of Picloram and 2,4-D in Populus tremula

Translocation and metabolism of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in small plants of aspen (Populus tremula L.) were studied. In most experiments ¹⁴C-carboxyl-labelled herbicides were used. Considerable quantities of both herbicides were retained in the treated leaf. Translocation was mainly upwards into the growing shoot tip. Only minute quantities were found in the roots. Injection of the herbicides through a cut stem surface increased the translocation into the roots very little. One of the reasons for the limited downward translocation is considered to be a ready transfer of the herbicides between the phloem and the xylem. Both herbicides were incorporated into complexes from which the active herbicides could be released. However, this complex formation can only partly account for the retention of the herbicides in the treated leaves. The differences in metabolism found between 2,4-D and picloram cannot explain the considerable difference in toxicity between the compounds.