Regeneration Capacity of Calli Derived from Immature Embryos in Spring Barley Cultivars

Callogenesis, somatic embryogenesis and regeneration capacity in twenty-three agronomically important spring barley (Hordeum vulgare L.) cultivars on induction media with 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,6-dichloro-o-anisic acid (dicamba) and on modified regeneration media were studied. The frequency of zygotic embryos exhibiting callogenesis varied from 88 to 100 % according to genotype. Dicamba was more suitable for somatic embryogenesis induction and exhibited a higher frequency of regenerants than did 2,4-D. Green regenerants were obtained in all cultivars, and there were no albino plants. Except for cv. Victor all cultivars used in the experiment showed lower regeneration capacity as compared to the model cv. Golden Promise. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Somatic embryogenesis and plant regeneration from leaf, root and stem-derived callus cultures of Areca catechu

Plant regeneration through somatic embryogenesis of Areca catechu L. was established using leaf, root and stem segments as explants. Embryogenic callus was induced and maintained on medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,6-dichloro-2-methoxybenzoic acid (dicamba) at concentrations 2, 4, 6 and 8 mg dm⁻³ in darkness. Somatic embryos were found on primary callus in the presence of 2 and 4 mg dm⁻³ dicamba and during subculture on 2 – 8 mg dm⁻³ 2,4-D or 2 – 4 mg dm⁻³ dicamba-containing media. Plantlet conversion from embryos was successfully achieved on growth regulator-free medium. The plants grew well when transplanted to containers in shaded greenhouse.     

Relative toxicity of three wheat herbicides to two species of Coccinellidae

Abstract On the High Plains of the USA, herbicides specific for broad-leaf weeds are regularly applied to winter wheat in the early spring, sometimes late enough to coincide with the colonization of fields by cereal aphids and their natural enemies. We tested the toxicity of three such herbicides, Ally (Dupont), Rave (Syngenta) and 2,4-D ester (generic), to neonate larvae of two coccinellid species important in cereal aphid biocontrol, Coleomegilla maculata DeGeer and Hippodamia convergens Guérin-Méneville. Topical treatment of larvae with 2,4-D resulted in 25% and 60% mortality in the two species, respectively, with surviving C. maculata larvae experiencing a 5% increase in developmental time. No significant effects were noted for the other two materials, save for a 2.5% increase in developmental time for C. maculata larvae exposed to Rave. No material caused significant mortality in either species when larvae were fed on prey (Schizaphis graminum Rondani) treated with herbicide 24 h earlier, although 2,4-D reduced developmental time slightly in C. maculata. When herbicide applications are delayed enough in spring to coincide with aphid activity in wheat, farmers can reduce the risk of disrupting biological control by selecting an alternative to 2,4-D.     

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([¹⁴C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

Engineering dicamba selectivity in crops: a search for appropriate degradative enzyme(s)

The biotechnologial approaches to conferring crop selectivity to herbicides have been demonstrated for a number of compounds such as glyphosate, glufosinate, imidazolinones and cyclohexanediones. Imidazolinone-resistant and cyclohexanedione-resistant maize lines are already in the market. There are several other effective and environmentally benign herbicides such as dicamba, for which engineering crop selectivity is desirable, to broaden the product utility in different crops and provide new solutions for weed control. One of the most effective approaches to conferring dicamba selectivity in crops is to incorporate a gene for its rapid metabolism. It is advantageous to have different dicamba-metabolizing enzymes in order to maximize the chances of at least one functioning optimally in a plant environment. Three different metabolizing enzymes are currently available to engineer crop selectivity. The first one is the folate-dependent O-demethylase from Clostridium thermoaceticum, that converts dicamba to herbicidally inactive 3,6-dichlorosalicylate. The second enzyme is the NADH-dependent, multi-component monooxygenase from Pseudomonas maltophilia DI-6 that also converts dicamba to 3,6-dichlorosalicylate. The third enzyme is from corn endosperm cultures that catalyzes the 5-hydroxylation of dicamba. The merits of these three enzymes are discussed with respect to conferring crop selectivity to dicamba. In addition, a rapid microbial screen was conceived for discovery of new dicamba-degrading bacteria, which resulted in identification of Pseudomonas orvilla. This bacteria degraded dicamba by the same pathway, perhaps using a similar enzyme system as Pseudomonas maltophilia DI-6. However, the microbial screen has the potential to identify novel bacteria that degrade dicamba by a different pathway, providing more options for metabolizing enzymes to confer herbicide selectivity in crops. Received 13 February 1997/ Accepted in revised form 26 June 1997     

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O₂ into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (α₃) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co²⁺, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 Å, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.     

Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.     

Plant regeneration of mulberry (<Emphasis Type="Italic">Morus indica</Emphasis>) from mesophyll-derived protoplasts

A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.3 μM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 μM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 μM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 μM) and indole-3-acetic acid (IAA) (17.1 μM). The regenerated shoots were rooted on MS medium supplemented with 4.9 μM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.