Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999     

Synchronized shoot regeneration of rice (Oryza sativa L.) calli on solid medium by adjustment of intracellular 2,4-dichlorophenoxyacetic acid concentration

The present research aimed to establish conditions for synchronized plantlet regeneration from rice callus based on a quantitative analysis of the relationship between intracellular 2,4-dichlorophenoxyacetic acid (2,4-D) concentration and shoot regeneration rate. To prepare the rice calli with different intracellular 2,4-D concentrations prior to regeneration, callus precultures were carried out in medium containing 4 mg/l 2,4-D and in 2,4-D-free medium for predetermined periods. As the critical intracellular 2,4-D concentration of the calli precultured in 2,4-D-free medium was too low to analyze precisely by conventional analytical methods, it was estimated using a kinetic model which described the behavior of 2,4-D by taking its uptake, metabolism and/or inactivation rates during the callus preculture into consideration. An experimental relationship between intracellular 2,4-D concentration and regeneration rate of rice calli revealed that the intracellular 2,4-D concentration should be controlled as low as 2.6×10^–2μg/g fresh weight to reach the same synchronization in shoot regeneration as seen with rice seed germination. This condition was realized by feeding sugar into the 2,4-D-free medium after 4 days preculture when the carbon source was exhausted. Received: 29 June 1998 / Revision received: 24 September 1998 / Accepted: 27 October 1998     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

林生山黧豆幼苗2,4—二氨基丁酸的合成及其与胁迫条件的关系

林生山黧豆幼苗用［3H］－天门冬氨酸标记后,高丝氨酸在6h内迅速增加。高丝氨酸合成速率降低后,2,4－二氨基丁酸的合成量上升,于18h达到高峰。赖氨酸和苏氨酸与二氨基丁酸的合成表现有协同反馈机制。结果支持了天门冬半醛转氨生成二氨基丁酸的假说。盐胁迫、渗透胁迫和热激增加了二氨基丁酸的合成,可能是因为不同胁迫条件都造成了细胞脱水,从而促进了二氨基丁酸的合成。     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

A comparison of methods for callus culture and plant regeneration of RD25 rice (Oryza sativa L.) in two laboratories

While methodology is transferable from one laboratory to another, an exact transfer does not usually occur and even a nearly exact transfer of methods does not always result in repeatable data. Researchers should not expect that an effort to duplicate a published procedure will necessarily lead to identical results.In attempting to transfer rice tissue culture methods between laboratories in Fort Collins, Colorado, USA and Bangkok, Thailand, we discovered that a combination of the methods of each laboratory produced the best results in term of callus productions and plant regeneration. In the experiments reported here, the type of culture vessel used and the geographical location were also important variables.Supported by the USAID/Cooperative Agreement No DAN-4137-A-00-4053-00.     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.     

The influence of the jasmonates and abscisic acid on callus growth and somatic embryogenesis in Medicago sativa L. tissue culture

The jasmonates as well as abscisic acid were found to be inhibitors of callus growth and somatic embryogenesis in Medicago sativa L. tissue cultures. An exposure to these inhibitors during the induction as well as the differentiation stage reduced the number of somatic embryos obtained. The jasmonates showed to be less active in the inhibition of callus growth and somatic embryo production than abscisic acid.     

Influence of drought on the concentration and distribution of 2,4-diaminobutyric acid and other free amino acids in tissues of flatpea (Lathyrus sylvestris L.)

2,4-Diaminobutyric acid (A₂bu) may be responsible for the apparent toxicity of flatpea (Lathyrus sylvestris L.) forage to some livestock. To obtain information relative to environmental regulation of A₂bu, 3-month old flatpea plants, cv. “Lathco”, were subjected to water-deficit stress for 1, 2, and 4 weeks. A₂bu, the most abundant free amino acid in roots, stems, and leaves, increased nearly 100% in roots of stressed plants. Increases in the concentrations of asparagine (Asn), proline (Pro), and arginine (Arg) occurred in roots; Asn, Pro, and 4-aminobutyric acid (Abu) in stems; and Pro and homoserine (Hse) in leaves also occurred in response to drought stress. Proline was a minor constituent of the free amino acid pool, even under water-deficit stress. The distribution of A₂bu and Pro in the stressed plants (roots > stems > leaves) was the reverse of that in plants supplied with adequate water (roots < stems < leaves). As concentrations of Asn and Abu decreased from roots to leaves in control tissues, concentrations of Hse and A₂bu increased in roughly the same proportions. This observation suggests that Abu and Asn may be precursors of A₂bu and Hse, respectively. The increase in A₂bu levels in aerial parts of drought-stressed flatpea plants is probably not sufficient to lower the feed value of the forage.     

Plant regeneration from in vitro culture of anthers of Solanum chacoense Bitt. and interspecific diploid hybrids S. tuberosum L. x S. chacoense Bitt.

Summary Production of plants from cultured anthers of Solanum chacoense clone IP 33, of its interspecific diploid hybrids with S. tuberosum clones IP 354 and IP 372, and of a complex Solanum hybrid containing in its genome S. ajanhuiri is reported. Genotypic differences were found to influence both the induction phase and the regeneration process. Hybrids derived from clone IP 354 of S. tuberosum were much more responsive in culture than hybrids from clone IP 372. Altogether, 507 plants were regenerated and 309 were cytologically analyzed. Of these, 52% were haploid, 47% diploid and 1% mixoploid or tetraploid. A number of diploid plants probably originated from unreduced microspores and some genetic consequences of this event are discussed.     

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l^–1 of 2,4-D and 30 g l^–1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l^–1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (6777%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.     

Endogenous plant hormones of the broad bean,Vicia faba L. (-)-jasmonic acid,a plant growth inhibitor in pericarp

(-)-Jasmonic acid was identified as a plant growth inhibitor of the pericarp of Vicia faba by means of gas-liquid chromatography, high resolution mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and ¹³C-NMR. Additionally, the pericarp contains very small amounts of abscisic acid (ABA) and 4-dihydrophaseic acid. The highest level of jasmonic acid was reached prior to full pericarp length. This amount (3 g g^-1 fresh weight) is similar to the maximal ABA content in the developing seed. Jasmonic acid is a plant growth inhibitor possessing a relative activity in the wheat seedling bioassay of 1–2.5%, compared to ABA. Contrary to ABA, jasmonic acid does not cause retardation of leaf emergence. The possible physiological role of jasmonic acid in the pericarp is discussed and compared with the assumed function of ABA in developing seeds.Abbreviations ABA abscisic acid - DPA 4-dihydrophaseic acid - DPAMeTMS methyl ester trimethylsilyl ether of DPA - EtOAc ethyl acetate - Et₂O ether - MS mass spectrometry - NMR nuclear magnetic resonance - GLC gas-liquid chromatography - TLC thin-layer chromatography - UV ultraviolet light     

Isolation of quinic acid derivatives and flavonoids from the aerial parts of Lactuca indica L. and their hepatoprotective activity in vitro

In our continuing study of biologically active compounds from Korean medicinal plants, we investigated the hepatoprotective constituents of the aerial parts of Lactuca indica L. (Compositae), since the methanolic extract of L. indica has hepatoprotective activity against hepatitis B virus (HBV) production. The bioactivity-guided separation of the methanolic extract of the aerial parts of L. indica resulted in the isolation of seven quinic acid derivatives (1, 3–4, 6, and 10–12), along with five flavonoids (2, 5, and 7–9). All the isolated compounds were evaluated for hepatoprotective activity by the HBV assay in vitro. In the human HBV-transfected liver cell line HepG2.2.15, all the compounds except 2 and 5 effectively reduced HBV DNA level in the release of mature HBV particles from HepG2.2.15 cultivation. Of the ten active compounds, treatment with 1, 3, and 12 led to significant reduction in the extracellular HBV DNA level, suggesting that they could be potent phytochemical agents against hepatitis B virus.     

2′-hydroxymethyl abscisic acid: A major catabolite of (±)-abscisic acid in leaves of Hordeum vulgare L.

Radioactive (±)-abscisic acid (ABA), supplied via the transpiration stream to light-grown leaves of Hordeum vulgare was catabolized to 2′-hydroxymethyl ABA. Identification was made by capillary gas chromatography-mass spectrometry (GC-MS).     

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.     

An efficient in vitro shoot regeneration from leaf petiolar explants and ex vitro rooting of Bixa orellana L.- A dye yielding plant

Physiology and Molecular Biology of Plants - Bixa orellana L. (Bixaceae) is a multipurpose tree grown for the production of commercially important dyes. In the present study, an efficient,...     

Short Communication: Induction of systemic resistance and salicylic acid accumulation in Oryza sativa,L. in the biological suppression of rice blast caused by treatments with Pseudomonas spp.

Two strains of rice-associated pseudomonads Pseudomonas fluorescens 7-14 and P. putida V14i caused an induced systemic resistance (ISR) in three cultivars of rice. ISR response coincided with increased levels of salicylic acid and biological suppression (25%) of rice blast disease. When the strains of the bacteria with lacZY molecular marker tracking system were applied to rice seeds as a seed-coat, they persisted on rice roots for entire duration of the crop (110 days) but migrated to the rice shoot only up to 14 to 16 days. Bacteria applied as a root-dip did not migrate to aerial parts of the rice plant. Bacteria infiltrated into the rice stem survived for 20 days after the treatment and were not detected on the surface of the stem. It is suggested that the ISR triggered by these bacteria which were spatially separated from the rice blast pathogen, is an important mechanism for the biological suppression of rice blast.     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.