Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

Rapid and efficient plant regeneration from hypocotyl protoplasts of Brassica carinata

Protoplasts were isolated from hypocotyls of 7-d-old seedlings of three genotypes of Brassica carinata after enzymatic digestion in cellulase R-10 (0.5%) and pectolyase Y-23 (0.025%). The protoplasts were stabilized with 0.4 M mannitol used as osmoticum, and were cultured in darkness in Kao's liquid medium containing 0.4 M glucose and the growth regulators 2,4-D (1.0 mg/l), NAA (0.1 mg/l) and zeatin riboside (0.5 mg/l). Protoplasts were transferred to 16 h photoperiod conditions after 3 d of dark culture, and the medium was diluted to reduce the osmoticum on the seventh and tenth days of culture. Microcolonies were thus obtained which, upon transfer to MS agarose medium with 2,4-D (0.1 mg/l), BAP (1 mg/l) and 0.1 M sucrose, proliferated further to produce callus clumps. The plating efficiency of the three genotypes varied from 1 to 2%. Calli 2–3 mm in diameter were transferred to MS agarose plates with zeatin (2 mg/l) where they produced shoot buds and shoots with frequencies ranging from 22.5 to 74.2% for the three genotypes. The shoots were rooted in medium with IBA (1 mg/l) and were then established in soil. The time required for protoplast to plant development was 8 to 10 weeks.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -naphthalene acetic acid - BAP 6-Benzylaminopurine - KN Kinetin - 2IP 6-(Gamma, gamma-dimethylallyl-amino)purine     

Genetic tranformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8× 10^–6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefaciens pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 mg/l) containing shoot induction medium. Approximately 15–19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - hpt hygromycin phosphotransferase - IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.Abbreviations BAP 6-Benzylaminopurine - Kn Kinetin - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - ATP Adenosine triphosphate - MS Murashige and Skoog (1962) medium     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Induction and differentiation of callus from embryos of Cocos nucifera L. by IAA-conjugates

Calli from young embryos of Cocos nucifera L. were induced on B5 medium supplemented with IAA-conjugates (IAA-asp or IAA-ala) at a concentration of 2.0 mg/1 and callusing was increased by about 10% if both IAA-conjugates, IAA-asp and IAA-ala were added together. Differentiation of shoots and roots was achieved by transferring calli to B5 medium supplemented with either IAA-asp (2.0 mg/1)+Kn(2.0 mg/1) or NAA (2.0 mg/1). Complete plantlets were obtained on B5 medium supplemented with NAA (0.5 mg/1)+BAP (2.0 mg/1)+PVP (1.0 g/1).Abbreviations IAA Indole-3-acetic acid - IAA-ala Indole acetyl-L-alanine - IAA-asp Indole acetyl-L-aspartic acid - Kn Kinetin - BAP N⁶-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - PVP Polyvinylpyrolidone     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson

Prolific shoot bud differentiation was induced in callus and suspension cultures of hypocotyl origin in Brassica campestris cv. Yellow Sarson on MS medium supplemented with K (13.9–23.2 M) or BA (13.3–22.1 M). Plantlets were obtained by rooting the in vitro differentiated shoots. Histological studies revealed a unique mode of meristemoid formation.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA Benzyladenine - IAA Indole-3-acetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthalene acetic acid     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Factors affecting somatic embryogenesis in <Emphasis Type="Italic">Prunus incisa</Emphasis> cv. February Pink

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 M 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 M IBA produced somatic embryos on medium containing 10 M 2,4-D and 3% sucrose.Abbreviations BA 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

Plant regeneration from protoplasts isolated from cell suspension cultures of the wild tomato,Lycopersicon chilense Dun.

A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l^-1 zeatin and 0.1 mg l^-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.Abbreviations BAP 6-benzylaminopurine - IAA indole acetic acid - IBA indole butyric acid - MES-2 (N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Micropropagation of mature siberian elm in two steps

A simple and reliable two-step micropropagation system was developed for mature Siberian elm (Ulmus pumila L.). Shoot-tip cultures were initiated and multiplied on a modified Murashige and Skoog medium supplemented with 1 M benzyladenine, B5 vitamins, solidified with 0.22% Gelrite, and subcultured weekly. Proliferated shoots 2–3 cm in length rooted successfully in sterile and non-sterile soil mix at 100% efficiency. Healthy plants were acclimatized to the ambient environment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IBA indole-3-butyric acid - MS Murashige & Skoog medium (1962)     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis

Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/l benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/l BA, 500 mg/l polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4–6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - mWP modified Woody Plant medium Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station.     

Cloning of Maytenus emarginata (Willd.) Ding Hou — a tree of the Indian Desert,through tissue culture

Summary An in vitro method for cloning and mass multiplication of Maytenus emarginata, a highly drought resistant tree of the Indian Desert, has been developed. Shoot segments harvested from a plus tree (30-year-old) were cultured to produce multiple shoots (10–15 shoots/explant) on MS medium containing 0.1 mgl^–1 IAA and 2.5mgl^–1 BAP. In vitro produced shoots were cut into segments and cultured on shoot proliferation medium but with only 1.0 mgl^–1 of BAP to further multiply the shoots. Isolated individual shoots were cultured on a filter paper bridge in half strength MS liquid medium containing 25 mgl^–1 of IBA for 72 h in the dark at 28±2⁰ C for induction of root(s). About 70–80 percent of shoots rooted. The treelets developed were hardened and transferred to pots. Around 20,000 plants can be obtained from a single explant within a period of 6 months. The protocol is highly reproducible and efficient.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthalene acetic acid - NOA -naphthoxy acetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - B5 Gamborg et al. (1968) medium - MS Murashige and Skoog (1962) medium     

In vitro propagation of cashewnut

In vitro plant propagation was developed for seedling shoot tips, leaf axils, and cotyledonary nodes of cashew, Anacardium occidentale. Factors affecting multiplication rate included age of explant source, explant type, medium composition, light requirements, and transfer frequency. Cotyledonary nodes produced more buds than other explant types. Nodes had a 90% viability when transferred daily to fresh medium containing activated charcoal for 7 d while exposed to continuous dark. Cultures were then exposed to low light illumination with weekly transfers. The phytohormone composition producing the most buds was 2.32 M kinetin, 9.12 M zeatin and 4.40 M BA. The highest frequency of rooted shoots was obtained by treating shoots with the bacterium, Agrobacterium rhizogenes. Plants also were recovered by induction of roots using auxin treatment on propagated shoots.Abbreviations Kn Kinetin - Zn Zeatin - BA N6-Benzyladenine - 2iP (2-Isopentenyl) adenine - BPA n-Benzyl -9 (2-tetrahydro-pyrany l) adenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - TIBA 2,3,5-Tri-iodo-benzoic acid