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.     

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.     

Influence of Two Phenoxy Growth Regulators on the Uptake and Accumulation of Naptalam by Bean Plants

Bean plants (Phaseolus vulgaris L. cv. Black Valentine) treated with 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chlorophenoxyacetic acid (PCPA) absorb and accumulate considerably more N-1-naphthylphthalamic acid (naptalam) than untreated plants. All concentrations of 2,4-D from 5 x 10^-8 to 5 x 10^-4M were effective, peak stimulation occurring at 3 x 10^-5M. Plants treated with this concentration took up 186% more naptalam than control plants. It was shown that the leaf area (on a per g dry weight basis) was influenced most by growth regulator treatment. The leaf area of plants treated with 5 x 10^-5M 2,4-D contained 575% more naptalam than the leaf area of untreated plants. The influence of PCPA on naptalam uptake by bean plants was similar to that of 2,4-D but less effective.     

Effect of 4-Amino-3,5,6-trichloropicolinic acid on Protein Synthesis

The effects of 4-amino-3,5,6-trichloropicolinic acid (picloram) on protein synthesis in bean (Phaseolus vulgaris L. cv. ‘Astro’) hypocotyl and hook tissues were studied. Picloram (10^-4M) was shown to have a stimulatory effect on ¹⁴C-1-DL-leucine uptake in hook but not hypocotyl tissues. Maximum leucine incorporation and maximum total protein concentration occurred in hook tissues treated with 10^-4M picloram. Inhibition of protein synthesis with cycloheximide (CH) and erythromycin (ERY) indicates that endogenous and picloram-stimulated protein synthesis is a function of the 80S cytoplasmic ribosomes rather than 70S chloroplast or mitochondria ribosomes.     

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.     

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.     

Influence of adjuvants and plant growth regulators on herbicide performance in honey mesquite

Addition of surfactant I (trimethylnonylpolyethoxyethanol) or surfactant II (4-isopropenyl-1-methyl-cyclohexane) at 1.0 and 0.6% (v/v) of the spray solution enhanced the phytotoxicity of clopyralid (3,6-dichloro-2-pyridinecarboxylic acid), the triethylamine salt of triclopyr {[(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid}, picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid), and 2,4,5-T [(2,4,5-trichlorophenoxy)acetic acid)] applied at 0.14 kg ae/ha to greenhouse-grown honey mesquite (Prosopis glandulosa Torr.). Application of benazolin [4-chloro-2-oxo-3(2H)-benzothiazolacetic acid] increased the phytotoxicity of all herbicides, but ethephon [(2-chloroethyl)phosphonic acid] and mefluidide {N-[2,4-dimethyl-5-[[(trifluoromethyl)sulfonyl]amino]phenyl]acetamide} were usually ineffective. Clopyralid + picloram, triclopyr, or 2,4,5-T applied in 1:1 combinations at 0.07 + 0.07 kg/ha were usually equally or more effective than any one of the herbicides applied alone at 0.14 kg/ha. Adjuvants did not enhance the phytotoxicity of picloram, triclopyr, or 2,4,5-T on field-grown honey mesquite but sometimes did with clopyralid.     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

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.     

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.     

Calcium Ions Protect Beet Root Cell Membranes against Thermally Induced Changes

The initial heat induced increase in permeability of beet (Beta vulgaris L.) root cell membranes, as measured by betacyanin efflux, is reversible. The leakage of betacyanin stopped when tissue held for 90 min at 45°C was transferred to 27°C. Stronger heat treatments, 10 min at 60°C or 150 min at 45°C, resulted in a continuous release of betacyanin indicating an irreversible change in membrane structure. Calcium ions inhibited the heat induced betacyanin efflux only if this was due to a reversible increase in permeability.     

Translocation and distribution of picloram in bean plants associated with nastic movements

Nastic responses in bean plants (Phaseolus vulgaris L. var. Black Valentine) occur rapidly when very low concentrations of picloram (4-amino-3,5,6-trichloropicolinic acid) are applied to the root systems. The distribution of picloram associated with nastic responses was quantitatively determined in root-treated plants.     

Abscisic acid and water transport in sunflowers

The role of abscisic acid (ABA) in the transport of water and ions from the root to the shoot of sunflower plants (Helianthus annuus) was investigated by application of ABA either to the root medium or to the apical bud. The exudation at the hypocotyl stump of decapitated seedlings was measured with and without hydrostatic pressure (0–0.3 MPa) applied to the root. All ABA concentrations tested (10^-10–10^-4 mol·l^-1) promoted exudation. Maximal amounts of exudate (200% of control) were obtained with ABA at 10^-6·mol·l^-1 and an externally applied pressure of 0.1 MPa. The effect was rapid and long-lasting, and involved promotion of ion release to the xylem (during the first hours) as well as an increase in hydraulic conductivity. Abscisic acid applied to the apical bud had effects similar to those of the rootapplied hormone. Increased rates of exudation were also obtained after osmotic stress was applied to the root; this treatment increased the endogenous level of ABA in the root as well as in the shoot. Water potentials of the hypocotyls of intact plants increased when the roots were treated with ABA at 5°C, whereas stomatal resistances were lowered. The results are consistent with the view that ABA controls the water status of the plant not only by regulating stomatal transpiration, but also by regulating the hydraulic conductivity of the root.Abbreviations and symbols ABA abscisic acid - Tv volume flow - Lp hydraulic conductivity - PEG polyethyleneglycol - water potential - osmotic potential - osmotic value - P hydrostatic pressure     

Responses of Populus tremula to Picloram and Other Translocated Herbicides

Whole aspen plants and isolated aspen root segments were three to ten times more sensitive to the growth-inhibitory and toxic effects of picloram than to those of 2,4-D, 2,4,5-T and dicamba. The activity of picloram in the inhibition of root growth was about ten times higher than that of 2,4-D and dicamba when tbe substances were added to the nutrient solution. Epinastic responses indicated a very rapid translocation of picioram from the roots to the growing shoot parts. When the herbicides were applied to the mature leaves dicamba rapidly caused a complete inhibition of root growth indicating a rapid translocation of this compound from the leaves to the root tips. Leaf-applied picloram, 2,4-D and 2,4,5-T affected root growth considerably more slowly. Dicamba, 2,4-D and 2,4,5-T rapidly killed the directly treated leaf tissue due to high acute toxicity while picloram did not show this type of toxicity. It is concluded that the higher activity of picloram in killing the plant and in inhibition of root and shoot growth can only partly be explained as a result of greater uptake and translocation of this compound.     

Callus production,regeneration and evaluation of plants from cultured inflorescences of tall fescue (Festuca arundinacea Schreb.)

Twenty genotypes (individual plants) of Kentucky 31 tall fescue (Festuca arundinacea Schreb., 2n=6x=42) were evaluated to determine regeneration response, and meiotic and isozyme changes in the regenerants. Six genotypes (K8, K16, K25, K27, P3 and P13) were selected for study of efficiency in producing calli and regenerating plants. Panicle pieces (3569) were plated on Schenk & Hildebrandt medium with one of three auxins, 2,4-D (2.0 mg 1^-1), pCPA (3.8 mg 1^-1) or 2,4,5-T (2.5 mg 1^-1). Square-root transformed data were analysed as a completely randomized 6×3 factorial with genotype and auxin as the main effects. F-protected means were compared by the Waller-Duncan K-ratio T test. Genotype K8 produced significantly more calli per panicle piece (88/877), whereas genotype K25 regenerated significantly more plantlets per panicle piece (58/214) than the other genotypes. Callus production was significantly higher using 2,4-D (102/1244 calli per piece) than pCPA or 2,4,5-T. The type of auxin did not have a statistically significant effect on plant regeneration, which suggested that genotype was the most important variable. Twelve of the 210 regenerants were albino. Cytological evaluation of 95 green regenerants showed that all plants had 21 bivalents and pollen stainability ranged from 43.0–98.4%, which suggested all plants were male fertile. Zymograms of these 95 regenerants for ACPH, ADH, GOT, MDH, 6-PGD, PGI and SOD showed no differences from that of the parental genotype, which suggested that the green plantlets regenerated from somatic tissue.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pCPA parachlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - ACPH acid phosphatase - ADH alcohol dehydrogenase - GOT glutamate oxaloacetate transaminase - MDH malate dehydrogenase - 6-PGD 6-phosphogluconate dehydrogenase - PGI phosphoglucoisomerase - SOD sulfoxide dismutase     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.     

Potassium influx and efflux of 2,4-D- and MCPA-treated rice plants

Summary A study was made of the effects of the herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methyl-phenoxyacetic acid) on ion uptake, leakage and growth of rice seedlings. Using isotopically-labelled solutions containing different concentrations of 2,4-D or MCPA, it was established that 10^–4 M 2,4-D or MCPA effectively inhibited potassium ion uptake, while K-ion leakage from the roots occurred only at 10^–3 M. The growth of the rice seedlings was markedly retarded even at low (10^–6 M) concentrations, and the roots and shoots showed different tolerances to the herbicide. At 10^–8 M herbicide, the effects were not injurious, but rather favourable. Reduction in root length by herbicides was not in accordance with dry-matter production.     

The relationship between pathogenic fungal metabolities (fusaric and picolinic acid), endogenous ethylene evolution and the development of ethylene-like symptoms

Summary The effects of fusaric acid (5-n-butylpicolinic acid), picolinic acid (2-pyridine carboxylic acid), and picloram (4-amino-3, 5, 6-trichloropicolinic acid) on endogenous ethylene production by tomato cuttings and elongation growth of oat coleoptile sections were measured. Ethylene production by tomato cuttings was substantially stimulated by treatment with 1×10⁻³ and 1×10⁻⁵ M picoloram and to a lesser extent by 1×10⁻³ M fusaric acid; picolinic acid had little effect. The ethylene levels produced in response to fusaric acid are not high enough to account for the ethylene injury observed in Fusarium wilt. Fusaric acid inhibited oat coleoptile extension, picolinic acid had little effect, and picloram promoted growth.     

Effects of decenylsuccinic Acid on the permeability and growth of bean roots

Decenylsuccinic acid (DSA) at 10⁻³ m has been reported to increase the permeability of bean root systems to water without seriously injuring the plants. We have confirmed the increase in permeability at 10⁻³ m, but have found that 10⁻⁴ m DSA reduces the permeability. Both concentrations cause leakage of salts from the roots and cessation of root pressure exudation. The roots of intact bean plants are killed by 1 hour's immersion in 10⁻³ m DSA, but the plants may survive by producing new roots. Up to 4 hours in 10⁻⁴ m DSA causes only temporary cessation of growth. Comparisons are made between the effects of DSA and some metabolic inhibitors. It is suggested that DSA is acting as a metabolic inhibitor, and that increase in water permeability is the result of injury to the roots. Experiments with 3 other species indicated variations in response to 10⁻³ m DSA. These could be largely attributed to differences in susceptibility to injury.