Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

High frequency plant regeneration from immature embryos of an elite barley cultivar (Hordeum vulgare L. cv. Morex)

An efficient plant regeneration system was developed for Hordeum vulgare L. 'Morex' barley, an important United States malting cultivar. The protocol was based on a series of experiments involving the sizes of immature embryos and the culture media. We found that the embryo size is critical for the establishment of embryogenic callus. Smaller embryos (0.5-1.5 mm) showed a much higher ability to produce embryogenic callus capable of regenerating green plants with fewer albinos than did the larger embryos (1.6-3.0 mm). Either 3 mg/l 2,4-dichlorophenoxyacetic acid or dicamba in modified Murashige and Skoog's (MS) medium was optimum for the induction of embryogenic callus. The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium. Efficient shoot regeneration was obtained on modified MS medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BA). Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/l IBA. Plants were successfully transferred to soil and grown to maturity in the greenhouse. This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar.     

Improved Regeneration Efficiency from Mature Embryos of Barley Cultivars

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

Summary Genetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk. Mention of a trademark or proprietary product does not constitute a guarantce or warranty by the University of Wisconsin and does not imply its approval to the exclusion of other products that may be suitable.     

Effect of growth regulators concentrations on morphological development of meristem-tips in Dioscorea cayenensis-D. rotundata complex and D. praehensilis

The use of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d) has played an important role in the production and maintenance of totipotent cereal callus. However, 2,4-d has been implicated in the loss of totipotency from barley callus. To examine the effect of 2,4-d on barley callus, regenerability and karyotype were examined over time as influenced by cultivar differences and 2,4-d levels, during a period in which initially vigorous plant regeneration typically declines dramatically. Higher (20.4–27.1 M) versus lower (6.8–13.6 M) concentrations of 2,4-d were positively associated with the number of green plantlets recovered from calli maintained for 10 and 16 weeks before transfer to regeneration media, and with the longevity of regenerability. There was a positive relationship between 2,4-d concentration and normal karyotype. We also investigated the use of phenylacetic acid for the initiation of regenerable barley callus. Very poor callus growth and plant regeneration was supported by phenylacetic acid.Abbreviations PAA phenylacetic acid - SPDL(s) single plant-derived lines(s) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - MSO Murashige and Skoog medium lacking growth regulators     

Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales

Basal media and plant growth regulators were tested for the promotion of somatic embryogenesis from immature wheat-rye hybrid embryos. Influence of growth regulators and chilling on plant regeneration were tested on two media. A medium containing four amino acids-glutamine, arginine, glycine and aspartic acid-as the nitrogen source, promoted the production of, on average, twice as much embryogenic callus as the other media, and somatic embryos developed well. The growth regulator dicamba was significantly better than 2,4-dichlorophenoxyacetic acid in promoting somatic embryogenesis and subsequent plant regeneration. Germination of somatic embryos on both regeneration media was enhanced by cold treatment. Supplementing 190-2 plant regeneration medium with a combination of -naphthaleneacetic acid + benzyladenine, indole-3-acetic acid + kinetin or indole-3-acetic acid + zeatin resulted in equally high germination rates.Abbreviations 190-2 Plant regeneration medium of Chuang & Jia - 2,4-d 2,4d Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-o-anisic acid - AA Amino acid medium of Müller & Grafe - IAA Indole-3-acetic acid - BA Benzyladenine - NAA -Naphthaleneacetic acid     

河南省新育成8个小麦品种(系)幼胚培养再生性能评价

从新育成的小麦品种中筛选组织培养再生能力强的基因型,对于利用基因工程途径改良小麦具有重要意义。本研究以河南省近几年育成的8个小麦品种(系)的幼胚为材料,研究3种生长素2,4-D、Dicamba、Picloram对8个小麦品种(系)较大幼胚再生的影响,以及品种间再生能力的差异。结果发现培养基中添加Dicamba时,8个品种(系)的再生率达189%以上,其中郑麦1836和中育1439等6个品种(系)的再生率超过了400%;添加Picloram时,8个品种(系)的再生率均在210%以上,其中郑麦1860和郑麦5135等4个品种(系)的再生率超过了470%;添加2,4-D时,郑麦7698、郑麦0856和郑麦9023没有获得再生植株,郑麦1860和郑麦1836的再生率低于60%,中育1439再生率最高,其次为郑麦5135和郑麦1354。结果表明,小麦较大幼胚植株再生最适宜的生长素为Dicamba,其次为Picloram,不同小麦品种(系)适宜的生长素有所不同;在8个小麦品种(系)中,再生能力最强的基因型为中育1439,其次为郑麦5135、郑麦5135和郑麦1354。     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Influence of 2,4-D,TIBA and 3,5-D on the growth response of cultured maize embryos

Induction of embryogenic calli from immature zygotic embryos of maize requires the presence of 2,4-D or similar auxin-like growth regulators in the culture medium. Pulse-chase experiments with 2,4-D, using various concentrations of 2,4-D in the induction medium were tested in relation to induction of callus in the embryogenic inbred line A188 and the non-embryogenic inbred A632. Interactions of 2,4-D, 3,5-D and the auxin transport inhibitor TIBA were also studied. Pulse-chase experiments showed that exposure to 2,4-D influenced the culture response from 0.5 h onwards. After a pulse of 0.5 h, shoot and root elongation of the embryo was stimulated. A pulse of 16 h or longer induced outgrowths and callus formation at the basal side of the scutellum. Pulses of 7 days and longer resulted in the induction of friable embryogenic Type II callus in A188. Embryos were cultured at 2,4-D concentrations ranging from 0.002 to 2000 mg l⁻¹ and optimal concentration for the induction of embryogenic callus in A188 was 2 mg l⁻¹. At lower concentrations there was a transition between callus formation and germination; at increasing concentrations, callus induction was reduced and finally growth responses became blocked. When TIBA was added to medium without 2,4-D, root elongation decreased in a dose-dependent way suggesting the need of polar transport of endogenous auxins for root elongation. When added to medium with 2,4-D, TIBA caused suppression of callus formation, again pointing to the necessity of polar transport of 2,4-D. In combination with 2,4-D, cultures with 3,5-D resembled cultures at lower 2,4-D concentrations, pointing to a competitive interaction between 3,5-D and 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Effect of copper on shoot and root regeneration in wheat,triticale, rape and tobacco tissue cultures

CuSO₄ (0.1–100 M) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO₄ also stimulated root formation. When equal concentrations of CuSO₄ were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO₄ pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO₄, AgNO₃, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO₃ strongly increased morphogenesis, whereas CuSO₄ had no significant effect.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid     

In vitro culture of switchgrass: Influence of 2,4-d and picloram in combination with benzyladenine on callus initiation and regeneration

The objective of this study was to determine the types and concentrations of auxins and cytokinins that would result in effective culture initiation and plant regeneration of switchgrass (Panicum virgatum L.). Whole caryopses and young seedling explants were cultured on MS medium containing different concentrations of picloram or 2,4-dichlorophenoxyacetic acid (2,4-d) in combination with benzyladenine. Best results from mature caryopses were obtained with 11.3–45.0 M 2,4-d in combination with 15.0 or 45.0 M benzyladenine (BA). More than 300 plants were obtained per embryogenic callus from some treatments after the second transfer to regeneration medium (90 days after initiation of the cultures). Regeneration was obtained from young seedling explants with both auxins. However, picloram was more effective over a wider range of BA concentrations than 2,4-d. Protocols developed during this study were used to regenerate hundreds of plants which could easily be established in the field.     

High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.)

Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-D), 60 M 4amino-3, 5,6-picolinic acid (picloram), or 30 M 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 M dicamba plus 10 M 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 M dicamba, 20 M BA) resulted in regeneration of shoots from 45% of calli of the cultivar Baron. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,5,6-picolinic acid - BA 6-benzylaminopurine     

Growth and regenerability of long-term suspension cultures of the U.S. rice cultivar Mercury

Suspension cultures of the U.S. rice cultivar Mercury have been maintained in modified General Medium for more than 3 years. These suspensions have continued to have high and relatively stable regeneration rates. Two different explants, immature panicles and seeds, were compared during the development of these embryogenic suspensions. Initial formation of secondary embryogenic callus from immature panicles on induction medium was greater than that from seeds. Suspensions of these two cell lines, however, did not differ morphologically and maintained similar regeneration rates. After 5 months in culture the rates of regeneration began to decline. The suspensions were plated onto regeneration medium without growth regulators for 2 weeks and then embryogenic cells were manually selected and used to develop secondary suspensions. Through this simple rejuvenation procedure, the suspensions retained high and stable regeneration rates. Variability in suspension growth, however, was observed during the culture period. Slower growth occurred at weeks 13, 15, 27, and 29 and was associated with a decrease in regeneration rates. Reproductive fertility of regenerated plants remained high for 3.5 years but then declined.Abbreviations CH casein (acid hydrolysate) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog basal medium - SE standard error     

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.     

Modeling available 2,4-dichlorophenoxyacetic acid in a tissue culture medium containing activated carbon

Clonal propagation of high-value forest trees by somatic embryogenesis can help meet industry needs for uniform and high quality raw materials. Low embryogenic tissue initiation frequencies for loblolly pine (Pinus taeda L.) pose a limitation in work towards commercialization of this technology. At the time our research began most work on somatic embryo culture initiation in loblolly pine reported success in the range of 1–5%. Activated carbon (AC) has been reported to improve many tissue culture systems including embryogenic tissue initiation in Douglas-fir. To improve initiation frequencies in loblolly pine, the development of an AC-containing system was explored. In order to better understand the availability of 2,4-dichlorophenoxyacetic acid (2,4-D) in initiation medium, we tracked media surface concentrations of free or available 2,4-D. Media containing 1/2 modified P6 salts, 1.5% maltose, 2% myo-inositol, case amino acids, glutamine, vitamins, and 0.4% Gelrite were modified to include 0.625 – 2.5 g l^–1 of activated carbon (Sigma C-9157, acid washed) and 110 –440 mg l^–1 2,4-D. Adsorption and availability of 2,4-D in AC-containing medium was tracked by C¹⁴ labeled 2,4-D present in surface moisture absorbed into filter paper. High correlations were found between–available 2,4-D and time when AC and initial 2,4-D concentrations were held constant,–available 2,4-D and AC concentration when initial added 2,4-D and time were held constant, and–available 2,4-D and initial 2,4-D when AC and time were held constant.All of these relationships were exponential, not linear. Multiple regression models inputting initial 2,4-D added to medium in mg l^–1, activated carbon added to medium in %, and time in days, were able to explain 85–88% of the variability in available 2,4-D. These models can be used to achieve target levels of available 2,4-D by adjustment of initial 2,4-D levels or AC content.     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid