Effects of plant growth regulators on the rapid proliferation of shoots and root induction in the Chinese traditional medicinal plant Atractylodes macrocephala

We developed an efficient plant regeneration protocol for rapidly propagating Atractylodes macrocephala Koidz, an important traditional Chinese medicinal plant, via shoot organogenesis. Shoot multiplication was induced on Murashige-Skoog (MS) medium supplemented with various concentrations of N-phenyl-N-1, 2, 3-thidiazol-5-ylurea (TDZ), 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). Rooting was induced on half-strength MS medium supplemented with NAA and indolebutyric acid (IBA). The maximum mean number of shoots (5.61) was obtained from a single explant by the combined effect of 1.08 μmol/L NAA and 2.25 μmol/L TDZ. The longest roots and a minimum number of roots were produced when they were cultured in a medium without plant growth regulators. The shortest roots and the largest number of roots were observed in the medium supplemented with 2.7 μmol/L NAA.     

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

紫色大花矮牵牛组织培养与植株再生

矮牵牛叶片外植体在MS+6-BA 1.0mg/L+NAA 0.1mg/L培养基上培养3周后产生致密的浅绿色愈伤组织;转入芽分化培养基MS+6-BA 0.5mg/L+4-PU 0.5mg/L+NAA 0.1mg/L 1周后,从愈伤组织表面不断分化产生幼芽;待幼芽长至3cm时转接至生根培养基1/2MS+NAA 1.0 mg/L+GA₃0.5mg/L中生根,长成完整植株。     

High Frequency in vitro Multiplication of Centella asiatica: An Important Industrial Medicinal Herb

An efficient protocol was developed for rapid clonal propagation of the important medicinal plant, Centella asiatica (L.) Urban, using shoot tip culture. High frequency bud break (88 %) and multiple shoot formation (16.8 shoots/shoot tip) were induced from a shoot tip segment, which was cultured on MS medium supplemented with 6‐benzylaminopurine (BAP) (17.76 μM) and gibberillic acid (GA₃) (1.44 μM). Half‐strength Murashige and Skoog (MS) medium supplemented with naphthalen acetic acid (NAA) (10.74 μM) induced the maximum (27.66) number of roots. Plantlets with 3–4 fully expanded leaves and well‐developed roots were successfully transferred to potted soil which exhibited a 95 % survival. The protocol enables the harvest of more than 25,000 plantlets within 160 days starting from a single shoot tip explant.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

大果良种沙棘愈伤组织诱导及植株再生的研究

大果良种沙棘的幼嫩茎尖,茎段外植体接种在MS,1／2MS附加不同浓度配比的IAA,IBA,BA,NAA培养基上可诱导茎尖及腋芽生长,将诱导产生的无性系芽接种在MS或1／2MS附加BA0．3－0．5mg/L,NAA0．05mg/L的培养基上可形成丛生芽,同时在小叶片和嫩茎上诱导产生愈伤组织,继续培养愈伤组织表面形成大量的绿色突起,进一步分化成不定芽,在相同培养基上,不定芽上可直接产生不定芽,从而形成多达数百个的不定芽族,不定芽长至3cm时切下转至1／2MS附加IAA或IBA 0．2mg/L的培养基上可生根而形成完整 的再生植株。     

Plant regeneration from protoplasts of hydroxyproline resistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onobrychis viciaefolia was established.In SH medium supplemented with 1mg/L2,4-dichlorophenoxy-acetic acid(2,4-D),0.5mg/L kinetin(KT)and 0.2mg/L naphthalene acetic acid(NAA),the division frequency of protoplastderived cells reached up to over 60%,and microcalli were obtained in 5-6wk.Upon transferring them on agar solidified MS medium plus 2mg/L indole-3-acetic acid (IAA),shoots were induced.After cultivating them on MS medium with or without IAA,roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal(2n=28).The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant

Summary An efficient plant regeneration protocol has been developed from root explants of Psoralea corylifolia L., an endangered medicinally important herbaceous plant species belonging to the family Fabaceae. Nodular embryogenic callus was initiated from young root segments cultured on Murashige and Skoog (MS) medium (1962) supplemented with α-naphthaleneacetic acid (NAA; 2.68–13.42 μM) or 2,4-dichlorophenoxyacetic acid (2.4-D; 2.25–11.25 μM) in combination with 6-benzylaminopurine (BA: 2.2. μM). thiamine HCl (2.9 μM), L-glutamine (342.23 μM) and sucrose (3.0% w/v). The highest frequency (95.2%) of embryogenic calluses was obtained on MS medium supplemented with the growth regulators NAA (10.74 μM) and BA (2.2 μM). Development and maturation of somatic embryos was achieved after transfer of embryogenic calluses to MS medium supplemented with 1.34 μM NAA or 1.12 μM 2,4-D and 4.4–13.2 μM BA. The maximum number (13.8±1.34) of cotyledonary stage somatic embryos was obtained on MS medium containing 1.34 μM NAA and 13.2 μM BA. Germination of somatic embryos occurred on MS medium without any growth regulators and also on MS medium enriched with BA (1.1–8.8 μM), although the maximum germination frequency (76.1%) was obtained on 4.4 μM BA plus 1.45 μM gibberellic acid (GA₃). Plant regeneration without complete somatic embryo maturation was also achieved by transferring clumps of nodular embryogenic calluses onto MSO medium or MS medium supplemented with NAA (1.34 μM) and BA (2.2–8.8 μM). The highest frequency of plant regeneration (93.3%) and mean number of plantlets (15.4±0.88) were obtained on MS medium containing 1.34 μM NAA and 4.4 μM BA. Regenerated plants with well-developed root systems were transferred to pots where they grew vigorously, attained maturity and produced fertile seeds.     

Efficient plant regeneration via somatic embryogenesis and organogenesis from the explants of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

High-frequency plant regeneration of C. roseus cv. ‘little bright eye’ via somatic embryogenesis and organogenesis from five out of six explants was standardized. Two factors were found to be important for regeneration: (1) the type of explants, and (2) the combination and concentrations of plant growth regulators. The highest regeneration percentage through somatic embryogenesis was obtained from mature zygotic embryo in MS medium supplemented with 7.5 μM of thidiazuron (TDZ). The mature embryo also regenerated efficiently via organogenesis in MS medium supplemented with either 2.5 μM TDZ or 5.3 μM α-naphthalene acetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA). Hypocotyl and cotyledon did not induce somatic embryogenesis and organogenesis in TDZ-containing medium but gave a maximum percentage of shoots in MS medium supplemented with 5.3 μM NAA and 2.2 μM BA. Stem nodes and meristem tips showed better regeneration via organogenesis in the medium supplemented with NAA and BA and in lower concentrations of TDZ.     

花生幼叶为外植体的植株再生系统的建立

本文报道利用花生成熟胚幼叶为外植体获得高频植株再生的方法,为花生转基因提供有效的受体系统。通过诱导培养基TDZ、BA、NAA的浓度以及种子萌发时间、继代培养基种类五个因素不同水平的正交试验,筛选出了分化高频发生的最佳组合为：MS培养基中应含有TDZ 1.0 μmol/L、BA 0.4 μmol/L、NAA 5.0 μmol/L,种子萌发4 d,继代培养基为MS0。本研究表明,五因素中诱导培养基TDZ浓度为诱导花生幼叶分化的主要影响因素,其次为继代培养基、种子萌发时间,而诱导培养基中BA和NAA的浓度作用较小。试管苗生根后移栽田间,可正常开花结果。     

In vitro shoot tip multiplication of bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc.]

A rapid, simple and efficient protocol for direct in vitro multiple shoot induction and plantlet recovery was achieved from shoot tip explants of Bambara groundnut [Vigna subterranea (L.) Verdc.]. Shoot tips, were isolated from in vitro-grown seedlings and cultured on Murashige and Skoog basal medium (MS) containing Gamborg’ vitamins (B5) and supplemented with different concentrations of the plant growth regulators Thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), Kinetin (KIN) and Adenine sulfate (ADS). TDZ 0.45 μM was found to be best for shoot multiplication with a mean of 11shoots per explant. Among the carbon sources tested, the best response in terms of mean number of shoots per explant was obtained with sucrose (11). The mean number of shoots per explant induced among the studied landraces of Bambara groundnut varied from 9 to 13. Individual shoots, aseptically excised, produced normal roots within 2 weeks on the basal MS medium supplemented with Indole Butyric Acid (IBA) or Naphthalene acetic acid (NAA). The highest number of roots per shoot and the longest roots were obtained on MS medium with 2 mg/L IBA. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in the field conditions, with a recorded survival rate of 70 and 80?%, respectively. The transferred plants in the field were morphologically normal and fertile. This protocol can be efficiently used for mass propagation, germplasm preservation and probably also for gene transfer of Vigna subterranea (L.).     

An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Tylophora indica (Burm. f.) Merrill, via enhanced axillary bud proliferation from nodal explants collected from young shoots of a two-year-old plant. The physiological effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)], ascorbic acid (AA), different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.6 ± 0.71) of shoots and the maximum average shoot length (5.2 ± 0.31 cm) were recorded on MS medium supplemented with 2.5 μM BA, 0.5 μM NAA and 100 mg/l AA at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with 0.5 μM IBA. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with a 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long-term in vitro conservation of T. indica.     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens– a valuable medicinal plant

A rapid and efficient protocol for the large‐scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15‐day‐old aseptic seedlings is described. Of the three different cytokinins, 6‐benzyladenine (BA), kinetin (Kin) and 2‐isopentenyl adenine (2‐iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half‐strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α‐naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole‐3‐butyric acid (IBA). The in vitro‐raised plantlets with well‐developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.     

骆驼蓬的组织培养及植株再生

以骆驼蓬(Peganum harmala L)无菌苗下胚轴切段为材料,在不同的培养基上进行愈伤组织的诱导,发现在MS基本培养基附加2．0mg/L 2,4—D、0．5mg／L 6—BA和3％蔗糖时,可100％的诱导出愈伤组织。愈伤组织在附加2．0mg／L 6—BA、0．5mg／L NAA、500mg／L CH和3％蔗糖的MS培养基上诱导出丛生芽,进而发育成苗,苗的分化频率在30％左右。分化苗或其茎切断在附加0．2mg／L IBA、0．2mg/L NAA和3％蔗糖的l／2MS培养基上出现根的分化,分化频率在90％以上。再生植株经炼苗后移栽成活,成活率在80％以上。     

Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annum L.)

A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A₃ and the ethylene inhibitor AgNO₃. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - MES morpholinoethanesulfonic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls - 2,4D 2,4-dichlorophenoxyacetic acid