Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild type and hairy root cultures of Rhodiola kirilowii

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.     

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6′-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3^T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Comparative study of Rhodiola preparations on behavioral despair of rats.

The antidepressant-like activity of an extract of the roots of Rhodiola rosea (RR), its combination with piperine containing extract (RPE), pure substances isolated from Rhodiola, such as rhodioloside, rosavin, rosin, rosarin, tyrosol, cinnamic alcohol, cinnamaldehyde and cinnamic acid has been assessed in laboratory animals through application of the Porsolt behavioural despair assay. RR increased the swimming time of rats in a dose dependent manner (ED50=7 mg/kg) and, when administered at 20mg/kg, exhibited a stronger anti-depressant type effect than either imipramine (at 30 mg/kg) or an extract of Hypericum perforatum (at 20mg/kg). Rhodioloside, and tyrosol were identified as active principles of the extract, whereas rosavin, rosarin, rosin, cinnamic alcohol, cinnamaldehyde, cinnamic acid were inactive. A fixed combination of rhodioloside, rosavin, rosarin and rosin was more active than any of the individual components alone, indicating a synergistic effect of the ingredients in RR extract. Piperine in combination with Rhodiola (RPE) distorts pharmacological effect of Rhodiola most probably due to changes of pharmacokinetic profile of rhodioloside and rosavin. RPE cannot provide predictable therapeutic effect due to herb-herb interaction. Moreover, concomitant treatment of RPE with other drugs should also be excluded due to drug-piperine interaction.     

Vessel element formation in cultured carrot-root phloem slices

The effects of light, auxin and cytokinin on vessel elementformation in phloem slices of carrot root were examined. When slices of carrot cultivars, ‘Nakamura-senko-futo’and ‘Yamada-hyakunichisenk6- naga’, preculturedin the dark on modified Murashige and Skoog's medium for twodays were cultured on a medium containing 5x10^–6 M 2,4-Din the dark, no vessel element formation occurred. When preculturedslices were cultured in the light with 5x10^–6M 2,4-D,vessel element formation was remarkable. But when 5x10^–7Mkinetin, benzyladenine or zeatin was added, vessel elementswere readily formed even in the dark. When slices were cultured in the light, a cytokinin-like substance(s)that causes vessel element formation was produced in the slices,then was released to the medium. The substance(s) was fairlystable to heat. In slices of carrot cultivars, kuroda-gosun, ‘Kintoki’and ‘Kokubu-senk6-6naga’, a different result forvessel element formation was obtained. When slices of thesecultivars were cultured on a medium containing 5x10^–6M2,4-D in the dark, vessel element formation was remarkable.It seemed, therefore, that these cultivars contain enough ofa cytokinin-like substance(s) to form vessel elements. In fact,vessel element forming activity was found in the alcohol extractof carrot root phloem from these cultivars. (Received June 8, 1971; )     

Improvement of dioscorealide B production by elicitation in shoot cultures of Dioscorea membranacea Pierre ex Prain & Burkill

Dioscorealide B is an important secondary metabolite isolated from Dioscorea membranacea Pierre ex Prain & Burkill. The effect on secondary metabolite content of different concentrations of two elicitors [jasmonic acid (JA) and salicylic acid (SA)], and of medium status and JA exposure period were investigated. In the JA and SA concentration experiment, 6-week-old shoots were cultured on MS medium supplemented with 8.87 µM BA (6-benzyladenine) in combination with 100–500 µM JA or 50–200 µM SA for 3 weeks. MS medium supplemented only with 8.87 µM BA was used as a control. The highest dioscorealide B content was recorded in the 100 µM JA shoots. To determine the optimal medium status and JA exposure period, shoots were cultured on solid and in liquid MS media supplemented with 8.87 µM BA and 100 µM JA for 2, 3, 4 and 5 weeks. No interaction was found between the medium status and the elicitor exposure period in the dioscorealide B production. Shoots cultured on the solid MS medium supplemented with 100 µM JA had a higher dioscorealide B content (0.57 ± 0.35% w/w) than those cultured in liquid medium (0.36 ± 0.40% w/w) and 5-week JA exposure produced the highest dioscorealide B content of 1.05 ± 0.15% (w/w).     

Plant regeneration from in vitro leaf tissues of <Emphasis Type="Italic">Viburnum dentatum</Emphasis> L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.     

Regeneration from long-term embryogenic callus of the <Emphasis Type="Italic">Rosa hybrida</Emphasis> cultivar kardinal

Summary Media components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Biotransformations of cinnamaldehyde,cinnamic acid and acetophenone with <Emphasis Type="Italic">Mucor</Emphasis>

A strain JX23 was isolated from soil and identified as a species of Mucor according to the morphological characteristics and the nuclear ribosomal internal transcribed spacer sequence and designated as Mucor sp. JX23. Biotransformations of cinnamaldehyde (CMD), cinnamic acid (CMA) and acetophenone (ACP) catalyzed by JX23 were investigated. After JX23 was cultured for 48 h, the substrates CMD, CMA and ACP were added to the growth medium respectively and the products were analyzed by GC–MS and HPLC. Mucor sp. JX23 exhibited considerable redox capability and different catalytic specificity to CMD, CMA and ACP. CMD was selectively hydrogenated to cinnamyl alcohol. CMA was biotransformed to ACP with α, β-oxidation like degradation, and ACP could not be reduced further by JX23. When ACP was added as substrate, it could be asymmetrically reduced to (S)-(−)-1-phenylethyl alcohol (S-PEA) with high stereoselectivity (90%). Further, the biotransformations of different binary mixture substrates with JX23 were also studied. The biocatalytic selectivity depended on the relationship between the binary mixtures in above-mentioned reaction.     

Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

‘Touriga Nacional’ is the most important Portuguese grapevine cultivar used for Port wine, table wine and varietal wine production. In order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, embryogenic cultures were induced from immature flowers of three Touriga Nacional selected clones. Gynoecia and anthers were cultured on Nitsch and Nitsch (Science 163:85–87, 1969) basal medium supplemented with four combinations of the growth regulators 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetyl-l-aspartic acid (IASP), at 28°C, in the dark. Primary callus, observed on anthers and gynoecia in all media, produced embryogenic callus when cultured on differentiation medium, at 24°C under light. The efficiency on induction of embryogenic callus ranged from 1.2 ± 4.7% to 7.9 ± 13.8% in anthers, and from 17.9 ± 24.9% to 25.3 ± 22.9% in gynoecia. Seven lines of embryogenic cultures were established from the three clones. Multiplication of embryogenic calluses was successfully obtained in maintenance medium, at 26°C, in the dark. These embryogenic calluses produced somatic embryos when subcultured on differentiation medium, under a 16 h photoperiod. Somatic embryos were isolated and cultured on germination medium to achieve conversion which ranged from 35.3 ± 48.5% to 72.7 ± 45.6%. The plantlets obtained were cultured in medium without growth regulators. Secondary embryogenesis was also frequently observed in the hypocotyl-root transition region of somatic embryos. Although some morphological variation occurred between somatic embryos, the regenerated plantlets had a normal phenotype. Maintenance of embryogenic cultures has been achieved since 2002.     

Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Membrane Potential of Cultured Carrot Cells in Relation to the Synthesis of Anthocyanin and Embryogenesis

Membrane potentials of cultured carrot cells in culture mediumwere about –40 mV and did not change with addition ofsalts of addition (or depletion) of 2,4-dichlorophenoxyaceticacid (2,4-D). When the measurement was performed in the testmedium (containing low concentration of salts), the values werewidely distributed (from –60 to –110mV) and changedlargely with external concentration of K⁺ but not Mg²⁺ nor Ca²⁺.When the cells were fractionated by Ficoll density gradientcentrifugation, the membrane potential of the cells of higherdensity (> 14% Ficoll) was about –150 mV in the testmedium and did not change during embryogenesis with depletionof 2,4-D. The membrane potential of cells of lower density (bandingbetween 6– 10% Ficoll) was less negative (– 60 to– 110 mV) in the test medium. When these cells were transferredand cultured in medium containing zeatin but lacking 2,4-D,the membrane potential was shifted negatively by about 15 mVprior to anthocyanin synthesis. When 2,4-D was added to anthocyanin-synthesizingcells in the medium containing zeatin, a transient hyperpolarizationand subsequent depolarization of the membrane were observedprior to the inhibition of anthocyanin synthesis. (Received October 22, 1987; Accepted April 20, 1988)     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea

A reliable method of plant regeneration has been achieved from decapitated mature embryo axes (DCMEA) explants. Shoots appear directly from explants of genotype T-15-15 when cultured on Maheswaran and Williams (EC₆) basal medium supplemented with N⁶-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) at various combinations. The shoots elongated on half strength Murashige and Skoog (MS) medium fortified with 3 μM gibberellic acid. Elongated shoots were rooted with 80 – 85 % efficiency on half strength MS medium with 0.5 μM indole-3-butyric acid. Survival of plants in the pots was 75 – 80 %. This protocol was used in Agrobacterium mediated transformation. The DCMEA explants were treated independently with two A. tumefaciens (LBA 4404) strains harbouring a binary vector carrying the green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter genes, respectively. Both the strains contained neomycin phosphotransferase selectable marker gene. After co-cultivation, the explants were cultured on EC₆ basal medium supplemented with 5 μM BAP and 1 μM IAA. The selection of putative transformants was on a medium containing 50 mg dm⁻³ kanamycin. Expression of GUS and GFP gene was confirmed by histochemical assay and fluorescence microscopy, respectively. The elongated shoots expressing GFP reporter gene were rooted and transferred to pots for hardening. The integration of GFP gene into the genome of putative transformants was confirmed by Southern blotting. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.