Cardiotonic glycosides from biomass of <Emphasis Type="Italic">Digitalis purpurea</Emphasis> L. cultured in temporary immersion systems

Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 μg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Change in total phenolic content and antibacterial activity in regenerants of Vitex negundo L.

A valuable medicinal plant, Vitex negundo L. has been investigated for its regeneration potential using shoot tip explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine, 2-isopentenyl adenine] used as supplement to Murashige and Skoog medium (MS), BA at 5.0 μM concentration proved best for multiple shoot induction yielding 3.60 ± 0.50 shoots after 8 weeks of culture. Inclusion of a low concentration of an auxin with optimal cytokinin concentration favoured shoot multiplication and the optimum response was observed on MS medium supplemented with BA (5.0 μM) along with α Naphthalene acetic acid (0.5 μM), where 65.0 ± 1.73 % cultures responded with a mean number of 4.80 ± 0.58 shoots per explants after 8 weeks of culture. Ex vitro rooting of in vitro derived microshoots was achieved upon dipping the cut ends of microshoots in 500 μM indole-3-butyric acid for 10 min followed by transfer to thermocol cups containing sterile soilrite. About 95 % of the plantlets survived the acclimatization procedure and were transferred to greenhouse and finally to field. Screening of the antibacterial activity and estimation of total phenolic content of ethanolic extracts of micropropagated plants were also carried out and compared with that of the mother plant.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Micropropagation and salt tolerance of in vitro grown Crithmum maritimum L.

Crithmum maritimum (Apiaceae), a perennial halophyte native in Greece, could be used as an alternative culture at problematic soils. It presents significant economical potentials as its essential oils are in high demand from the medicinal and cosmetic industry. The response of the species on in vitro conditions was studied. MS proved to be the most effective of the basal media tested for in vitro adventitious shoot production, resulting in significantly increased number of new microshoots/explant and higher shoots. 6-Benzyladenine (BA) at 2.5 μM increased shoot proliferation. The combination of α-Naphthaleneacetic acid (NAA) (1–2.5 μM) with BA (2.5 μM) had a positive influence at simultaneous proliferation and rooting resulting in high rooting percentage (82.5–95%) and increased number of roots. Rooting percentage reached 100% and number of roots increased significantly when 0.5 μM and 1 μM IBA was combined with ½MS and full strength MS. The in vitro response to salinity stress (0–300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher concentrations of NaCl but shoot height was enhanced. Acclimatization procedure was successful.     

Bud emergence and shoot growth from mature citrus nodal stem segments

Bud emergence and shoot growth from adult phase citrus nodal cultures were studied using Citrus mitis (calamondin), Citrus paradisi (grapefruit), and Citrus sinensis (sweet orange). The effects of 6-benzyladenine (BA), indole 3-acetic acid (IAA), and citrus type on shoot quality and growth of mature bud explants from greenhouse grown trees were determined using a 2-component mixture-amount × citrus type experiment. BA increased shoot number and IAA improved shoot growth. The best shoot quality (fewer shoots but large shoots) was obtained with 1 μM IAA for calamondin, 15.5 μM IAA for sweet orange, and 30 μM IAA for grapefruit. Grapefruit exhibited substantial leaf abscission compared to calamondin and sweet orange. Four factors (AgNO₃, silver thiosulphate (STS), CaNO₃, or gelling) were screened individually for their efficacy in reducing leaf abscission. Five factors (AgNO₃, gelling, MS ion concentration, plant growth regulator and venting) were investigated to identify potential combinations for reducing leaf abscission and maximizing shoot growth and bud emergence. The factor combination identified as most effective in minimizing leaf drop, promoting shoot growth, and maximizing bud emergence for grapefruit was 2 mg l^?1 AgNO_3, Gelrite, 1 × MS ion concentration, 30 μM IAA, and vented.     

Antifungal and high-performance liquid chromatography analysis of the crude extracts of Acorus calamus,Tinospora cordifolia and Celestrus paniculatus

The antifungal activity of methanolic crude extracts of Acorus calamus, Tinospora cordifolia and Celestrus paniculatus was investigated against Alternaria solani, Curvularia lunata, Fusarium sp., Bipolaris sp. and Helminthosporium sp. at different concentrations (1000, 2000, 3000, 4000 and 5000 μg/ml). At 5000 μg/ml, crude extract of T. cordifolia is found to be highly effective against Helminthosporium sp. followed by A. calamus against A. solani. On the other hand, at 5000 μg/ml, C. paniculatus showed better activity against A. solani and Helminthosporium followed by A. calamus against A. solani at 4000 μg/ml. At 5000 μg/ml, all the three crude extracts showed least activity against fungus C. lunata and Fusarium sp. except A. calamus that showed better activity against C. lunata. The increase in the production of phenolic acid in the extract can be correlated with the induction of resistance in treated plants against phytopathogenic fungi. High-performance liquid chromatography analysis of the crude extract of medicinal plants showed six different phenolic acids (benzoic acid, cinnamic acid, caffeic acid, ferulic acid, gallic acid and tannic acid) present in varying amounts. The results of the study provide scientific basis for the use of the plant extract in the future development as antioxidant, antibacterial, antifungal and anti-inflammatory agent.     

Micropropagation,antioxidant properties and phytochemical assessment of Swertia corymbosa (Griseb.) Wight ex C. B. Clarke: a medicinal plant

Swertia corymbosa (Griseb.) Wight ex C. B. Clarke, a valuable medicinal plant, has been investigated for its regeneration potential using nodal explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyl adenine (2iP), thidiazuron (TDZ), and zeatin (Z)] used as supplements with MS, BA at 4.40 μM concentration proved best for multiple shoot induction yielding 26.50 ± 0.26 shoots after 12 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin BA (4.40 μM) favoured shoot multiplication. A mean number of 35.78 ± 0.81 shoots were produced per explant. Additive effect of BA (4.40 μM) in combination with Kn (4.64 μM) produced highest number of shoots (83.20 ± 4.29). Addition of GA₃ (1.4 μM) to the above medium not only favored shoot elongation but also enhanced the number of shoots (113.98 ± 3.80). The microshoots were rooted successfully on half-strength MS medium supplemented with 9.8 μM of IBA. The plantlets were successfully transferred to hardening medium containing vermiculite with 87 % survival rate. Screening of the antibacterial, antioxidant activity and estimation of total phenolic and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolic and flavonoid content than in vitro propagated plants. The content of secondary metabolites in wild-grown plants and in vitro propagated plants was determined by HPLC coupled with ESI-MS and the presence of loganic acid, swertiamarin, sweroside, gentiopicroside, isovitexin, amoroswertin, amarogentin, gentiacaulein, decussatin, and swertianin in the samples were confirmed. Gentiopicroside (40.726 mg/g) and swertianin (29.598 mg/g) were found to be the major compounds which may be responsible for the antimicrobial and antioxidant activities. The results of the present study confirmed the therapeutic potency of S. corymbosa used in the traditional medicine; in addition, the protocol for in vitro production developed in the present study could be applied for mass multiplication and for the conservation of germplasm.     

High frequency in vitro regeneration system for conservation of Coleus forskohlii: a threatened medicinal herb

Present study reports a high frequency regeneration system for in vitro propagation and conservation of an important and threatened medicinal herb Coleus forskohlii (Briq.). Shoot multiplication has been achieved through axillary bud development and direct adventitious shoot formation in nodal explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) (5 μM). Further shoot multiplication was recorded up to third subculture on MS medium containing BA (5 μM) in combination with 1-naphthleneacetic acid (NAA) (0.1 μM). Excised microshoots on transfer to root induction medium consisting of half-strength MS medium (1/2 MS) alone as well as in combination with various auxins, resulted in varied rooting pattern in terms of number, length, and type of roots. Rooted microshoots were acclimatized successfully in earthen pots containing garden manure, garden soil, and sand (1:2:1) as potting mix with survival rate of 70 %. Acclimatized plantlets were studied for the amount of chlorophyll and carotenoid content as well as the net photosynthetic rate (P_N) during subsequent weeks of transfer to ex vitro condition. Histological studies revealed the direct origin and development of shoot buds from basal swollen cut end of nodal explants.     

GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA₃) followed by incubation at dark, and (iv) immersion in 400 ppm GA₃ followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.     

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.–an important multipurpose plant of the Pacific traditional Medicine

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 μM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 μM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.     

Effects of culture conditions on multiple shoot induction from inflorescence and RAPD analysis of cloned plants in Limonium sinensis (Girard) Kuntze, var. Golden Diamond

The optimum physical and chemical microenvironment for micropropagation of Limonium sinensis (Girard) Kuntze, var. Golden Diamond was established from immature inflorescence segments as explant. The highest frequency (62 %) of axillary shoot induction was obtained on MS medium (Murashige and Skoog Physiol Plant 15:473–497, 1962) supplemented with 8.88 μM BA, 1.34 μM of NAA and two growth additives cysteine (142.33 μM), and glutamine (684.22 μM). In the subsequent culture maximum average number of shoots (11.13?±?0.34) were obtained from micro-shoot explant on MS medium supplemented with the same additives and 2.22 μM BA. During subcultures the problem of vitrification was mitigated through increasing agar concentration from 0.8 % to 1.0 % and providing better ventilation. The in vitro developed shoots were rooted on MS medium supplemented with 2.46 μM IBA and 0.88 μM BA. Rooted plants were acclimatized successfully in the greenhouse with 80 % survival rate. RAPD analysis using 15 random decamer primers generated monomorphic banding pattern in micropropagated plants and similar to those of mother plant revealing the genetic integrity of regenerants.     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.     

Antioxidant activity evaluation of essential oil and RAPD analysis of in vitro regenerated Blumea mollis (D. Don) Merr.

Essential oil obtained from the leaves of Blumea mollis (D. Don) Merr. was analyzed by gas chromatography and gas chromatography coupled with mass spectroscopy (GC and GC–MS). Bicyclic sesquiterpene, β-caryophyllene, was identified as a major compound which accounted for 24.54 %. Antioxidant activity of oil was significantly higher than that of methanol extract of callus. Murashige and Skoog (MS) medium supplemented with 4.4 μM BA showed multiple shoot induction after 8 weeks of culture. 4.6 μM Kin showed in vitro flowering and direct organogenesis was observed from the leaf explants on medium containing 4.4 μM BA and 5.4 μM NAA. Rooted plantlets developed on half strength MS medium fortified with 2.1 μM IBA were hardened and percentage survival was recorded up to 70. RAPD analysis revealed a little genetic variation in micropropagated plants.     

Cryptosin — a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem. & Schult

A steroidal glycoside of the digoxin type was isolated from fresh leaves and cultured cells of Cryptolepis buchanani Roem & Schult (Asclepiadaceae). Cultures were raised from leaf and shoot. Explants were cultured on MS medium containing 2,4-D, NAA, BA and coconut milk. Metabolically active calluses were proliferated and maintained on Gamborg's B₅ medium with 2,4-D and Kn. The new compound, named cryptosin, was characterised by infrared nmr and mass spectroscopy. Cryptosin was also screened for its pharmacological properties and its cardiotonic action was established.Abbreviations PVP Polyvinylpyrrolidone - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BA Benzyl adenine - Kn Kinetin - MS Murashige & Skoog - Na,K-ATPase Sodium, potassium — sensitive adenosine triphosphate     

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N ⁶ –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA₃), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA₃ (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.     

Production of biologically active phenolic acids in Aronia melanocarpa (Michx.) Elliott in vitro cultures cultivated on different variants of the Murashige and Skoog medium

Phenolic acids, both benzoic and cinnamic acid derivatives, are plant metabolites with high therapeutic and cosmetic values. Methanolic extracts from the biomass of shoot and callus cultures of Aronia melanocarpa growing on seven variants of the Murashige and Skoog (MS) medium with different concentrations of plant growth regulators, BA and NAA, ranging from 0.1 to 3.0 mg l^?1, were examined for the production of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. The extracts from the shoot and callus cultures were confirmed to contain five of the twelve compounds tested for: caffeic, p-coumaric, p-hydroxybenzoic, syringic and vanillic acids. The shoot extracts contained additionally salicylic acid. Both the total amounts and the amounts of individual compounds in either the shoot or callus extracts were dependent on the concentration of cytokinin and auxin in the MS medium variants. The total amounts in the shoot and callus cultures were in the range from 93.52 to 217.00 mg 100 g^?1 DW and from 47.11 to 83.83 mg 100 g^?1 DW, respectively. The amounts of individual compounds showed wide variation, from 1.31 to 91.86 mg 100 g^?1 DW in the shoot extracts, and from 2.58 to 40.16 mg 100 g^?1 DW in the callus extracts. Salicylic acid (max. 91.86 mg 100 g^?1 DW), p-coumaric acid (max. 62.39 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 50.66 mg 100 g^?1 DW) dominated in the shoot extracts, while syringic acid (max. 40.16 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 23.59 mg 100 g^?1 DW) were the main metabolites in the callus extracts. This is the first report on the quantitative analysis of benzoic and cinnamic acid derivatives in shoot and callus cultures of A. melanocarpa growing on MS-based media with different concentrations of selected plant growth regulators—BA and NAA. The obtained maximum amounts of some metabolites are of interest from a practical perspective.