Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

Improvement of bioactive compound accumulation in adventitious root cultures of an endangered plant species, <Emphasis Type="Italic">Oplopanax elatus</Emphasis>

Efficiently culturing adventitious roots (ARs) has become an alternative route for the protection and utilization of endangered plant resources. In the present study, to improve accumulation of bioactive compounds (polysaccharides, phenolics, and flavonoids) in AR cultures of endangered plant species—Oplopanax elatus—effects of methyl jasmonate (MeJA) and salicylic acid (SA) were investigated. The optimal concentration of MeJA was 200 μM and SA was 100 μM for enhancement of polysaccharide, phenolic, and flavonoid contents. In addition, MeJA (200 μM) was more suitable than SA (100 μM) for polysaccharide and flavonoid production, but both elicitors were equally favorable for phenolic production. During AR bioreactor culture, MeJA was as an elicitor to study the effect of its addition time and contact time. Contents of polysaccharides, phenolics, and flavonoids increased when MeJA was added to culture medium after 40 days of culture, but the increased degree was lower and the AR biomass significantly inhibited. However, when MeJA was added to culture medium after 30 days of culture, polysaccharide, phenolic, and flavonoid contents dramatically increased without AR biomass decrease; the maximum productivity of three bioactive compounds was found on day 8 after the MeJA treatment. Therefore, a novel elicitation method during bioreactor culture of O. elatus ARs was established in the present study, the method could be applied to commercial production of O. elatus products in the future.     

Accumulation of anthocyanin in callus cultures of red-pod okra [<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m^??2 s^??1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47?±?0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m^??2 s^??1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.     

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae)

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.     

Accumulation of rosmarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L.

Eryngium planum L. cell and organ cultures were maintained on Murashige and Skoog media (MS), supplemented with exogenous hormones of different types and various concentrations for high biomass growth. The callus and cell suspension cultures were treated with increased sucrose concentration and/or elicited by methyl jasmonate for the enhancement of selected phenolic acids accumulation. Three phenolic acids, rosmarinic acid (RA), chlorogenic acid (CGA) and caffeic acid (CA), were detected by HPLC-DAD in those cultures. The sum of their content in the dry material was found to be higher in the shoot culture (3.95 mg g^?1), root culture (7.05 mg g^?1), callus (6.20 mg g^?1) and cell suspension (2.04 mg g^?1) than in the leaves (1.87 mg g^?1) and roots (0.76 mg g^?1) of intact plants. The major compound of in vitro cultures was always rosmarinic acid. The content of RA could be increased approximately threefold (16.24 mg g^?1) in the callus culture and approximately twofold (3.91 mg g^?1) in the cell suspension culture by elicitation with 100 μM methyl jasmonate (MeJA). The higher concentration of sucrose (S) in the medium (5, 6 %) led to over a twofold increase of CGA content in the callus culture (2.54 mg g^?1). The three mentioned phenolic acids have been found in E. planum undifferentiated and differentiated in vitro cultures for the first time.     

The effect of light and 2,4-D on anthocyanin production in Oxalis reclinata callus

In vitro cultures of O. reclinata accumulate red anthocyanin pigments. Two callus lines were established from O. reclinata, one red and the other non-pigmented. The red callus accumulated cyanidin-3-glucoside as a major pigment. Light irradiation induced anthocyanin synthesis in white callus, resulting in a heterogenous red callus line being formed. The incubation of red and white callus cultures in the dark or at low-light resulted in the repression of red pigment accumulation. The application of 2,4-D (1.0 mg l^-1) inhibited pigment production in the white callus and decreased anthocyanin accumulation in the red callus. The polypeptide composition of the red and white callus lines from O. reclinata were compared using two-dimensional electrophoresis. The red callus had a larger subset of neutral and acidic polypeptides.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.     

Adventitious shoot regeneration from in vitro leaf explants of <Emphasis Type="Italic">Fraxinus nigra</Emphasis>

In order to establish an attractive method for the production of valuable medicinal alkaloids (galanthamine and lycorine), the plants of Leucojum aestivum and L. aestivum ‘Gravety Giant’ grown in bioreactor RITA® were subjected to various concentrations of methyl jasmonate (MeJA), salicylic acid (SA), 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (ethephon) at different times of culture. The application of MeJA showed a negative effect on L. aestivum and L. aestivum ‘Gravety Giant’ plant growth. We observed that the incubation of plants during 168 h with 100 µM of MeJA resulted above two times lower F.W. (fresh weight) increments compared with control. While SA showed an inhibitory effect only on the growth of L. aestivum cultures. ACC and ethephon had a positive effect on both types of culture. Treatment with 50 µM of MeJA during 168 h stimulated galanthamine and lycorine biosynthesis in L. aestivum and L. aestivum ‘Gravety Giant’ cultures. In addition, the accumulation of galanthamine was increased when 10 µM of ACC were added to both types of culture. 10 µM of ACC stimulated also lycorine biosynthesis by L. aestivum ‘Gravety Giant’. The addition of 10 µM of ethephon had a positive effect only on lycorine production in plants of L. aestivum. SA promoted galanthamine and lycorine biosynthesis in tested plants. Indeed the highest galanthamine (0.8 mg/g dry weight: D.W.) and lycorine (1.53 mg/g D.W.) concentrations were observed in L. aestivum ‘Gravety Giant’ plants treated with 5 µM of SA during 10 h.     

Effect of plant growth regulators and elicitors on rhinacanthin accumulation in hairy root cultures of Rhinacanthus nasutus (L.) Kurz

Several biologically active secondary metabolites like anthraquinones, sterols, triterpenes, flavonoids and naphthoquinones are present in Rhinacanthus nasutus. Naphthoquinones are important group of compounds generally known as rhinacanthin (RC) consists of 15 derivatives named RC A–D and G–Q of which RC-C, RC-D and RC-N have various medicinal properties. The individual role of two auxins i.e. indole-3-butyric acid (IBA) and α naphthalene acetic acid (NAA) and two elicitors i.e. methyl jasmonate (MJ) and salicylic acid (SA) in Murashige and Skoog medium on hairy root growth and RC (RC-C, RC-D and RC-N) accumulation was investigated in the present study. Time course study revealed that IBA and NAA at 2.5 μM showed maximum fresh weight (FW) and dry weight (DW) 4 weeks after culture. However, RC production was maximum after 6 weeks of culture on both media. A concentration-dependent response was observed when various concentrations of MJ (2.0, 5.0, 10 and 15 μM) and SA (10, 50, 100 and 150 μM) were supplemented in the medium. On MJ and SA media the FW and DW decreased as the concentration of elicitors increased. However, this decrease was more severe in MJ treated cultures. All the MJ and SA treated cultures showed significantly higher amount of RC-C, RC-D and RC-N in hairy roots harvested 7 days after elicitation as compared to control. Of the two elicitors, MJ was more efficient in inducing RC accumulation than SA. The highest RC content (6.3 mg/g DW RC-C; 1.1 mg/g DW RC-D and 0.61 mg/g DW RC-N) was observed after treatment with 10 μM MJ which was about 1.7-, 2.5- and 3.5-fold higher RC-C, RC-D and RC-N respectively than the control.     

Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture

The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g l^?l FW) with SA at 100 μM in the presence of 0.6 mg l^?l BA and 20 mg l^?l spermidine for 4 h exposure time at the 4th week in 20 ml liquid medium recorded higher withanolides production (withanolides A [8.48 mg g^?l DW], withanolides B [15.47 mg g^?l DW], withaferin A [29.55 mg g^?l DW] and withanone [23.44 mg g^?l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.     

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g^?1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g^?1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g^?1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.     

Accumulation of red elemental selenium nanoparticles and their biological effects in Nicotinia tabacum

The uptake, accumulation and biological effects of red nano-sized elemental selenium (nanoSe) in comparison to selenate were investigated in plant system at the first time. The data clearly indicated that red nanoSe was taken up by tobacco callus cultures and rooted tobacco plantlets. The roots of regenerated plantlets accumulated selenium in very high concentrations, 2,947 ± 99 mg/kg DW, from the medium containing 530 μM nanoSe. The biological effects of nanoSe were different from the selenate ion in plant tissue culture. NanoSe (265–530 μM concentration range) stimulated the organogenesis and the growth of root system significantly (~40 %) while selenate did not show these effects at any concentration moreover inhibited both callus growth and root regeneration totally in 265–530 μM concentrations.     

The Response of Physiological Characteristics,Expression of OSC Genes,and Accumulation of Triterpenoids in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Sukto MeJA and SA Treatment

The pentacyclic triterpenoids from birch (Betula platyphylla suk) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we explored the effects of spraying 3-year-old white birch with different concentration of methyl jasmonate (MeJA) and salicylic acid (SA) on the expression of key genes in triterpenoid biosynthesis pathways and on the accumulation and physiological characteristics of triterpenoids in birch saplings. The results showed that spraying different concentration of MeJA and SA could obviously promote accumulation of total triterpenoids in 3-year-old white birch. The triterpenoid content in the stem bark was increased by 46.11 %, reaching 81.86 mg/g, after 1 day of treatment with 1 mmol·L^?1 MeJA (MJ2), and by 45.07 %, reaching 91.4 mg/g, after 14 days of treatment with 5 mmol·L^?1 SA (SA1). In addition, MeJA and SA treatment increased the contents of chlorophyll a and b, antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as photosynthetic performance, and affected the content of soluble sugar and soluble protein in birch leaf. Fluorescence quantitative polymerase chain reaction (qPCR) results showed that MeJA and SA treatment deferentially enhanced the key gene expression of cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and beta-amyrin synthase (BPY) in triterpenoid synthesis pathway in birch bark and leaves. The results showed that MeJA and SA induced triterpenoid synthesis of birch plant is closely related with not only the expression of key genes of triterpenoid synthesis pathway but also photosynthesis, anti-stress response and physiological indexes, suggesting that regulation of triterpenoid synthesis of birch by MeJA and SA may involve in more complex mechanisms at physiological and molecular levels.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)

Digitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.     

Anthocyanin production in a callus line of <Emphasis Type="Italic">Panax sikkimensis</Emphasis> Ban

A root-derived callus line of Panax sikkimensis that stably accumulates anthocyanins was established by small cell aggregate selection method. The selected line showed a growth index of 221.36 and an anthocyanin content of 2.76 mg/g fw (7.076% dw) in 50–60 d of growth on a modified MS medium containing 4.5 μM 2,4-dichlorophenoxy acetic acid and 1.2 μM kinetin under 16-h light and 8-h dark photoperiodic conditions. Incubation under continuous light increased the growth index to 435.57 but led to a marginal dilution of anthocyanin content to 2.192 mg/g fw (6.928% dw). The purple-red pigment had absorption maximum at 528 nm. The selected callus line has shown sustained growth and productivity for more than 6 yr now. Interestingly, pigment accumulation in the selected line did not hinder the ginsenoside production in the callus tissue (0.9–1.2% fw).     

Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.)

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100 %), and the combination of 5 μM α-naphthaleneacetic acid (NAA), 10 μM 6-benzyladenine (BA) and 0.1 μM gibberellic acid (GA₃) had the most significant effect on callus growth in term of weight (120.98 ± 22.56 mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10 μM NAA and 0.3 μM GA₃ for callus induction for 6 weeks followed by 2 μM NAA and 5 μM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) and the highest number of plantlets per explant (7.93 ± 1.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17 ± 9.80 %) and produced seeds (175.21 ± 28.01 firm seeds per plant).     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.     

Characterization of mercury-induced stress biomarkers in Fagopyrum tataricum plants

The effect of mercury stress on antioxidant enzymes, lipid peroxidation, photosynthetic pigments, hydrogen peroxide content, osmolytes, and growth parameters in Tartary buckwheat were investigated. The effect of Hg-exposure was found to be time (15 and 30 days) and concentration (0, 25, 50, and 75 μM) dependent. Hg was readily absorbed by seedlings with higher content in roots and it resulted in reduction of root and shoot length. The root and shoot Hg uptakes were significantly and directly correlated with each other. However, the fresh mass and biomass increased up to 50 μM Hg-treatment at both time periods. A significant positive correlation was observed between biomass accumulation with relative water content. Hg levels were positively correlated with the production of hydrogen peroxide in leaves as evidenced by 3, 3-diaminobenzidine (DAB)-mediated tissue fingerprinting. The osmolyte levels in general were elevated except for proline and protein which showed a decline at 75 μM Hg-treatment at 30-days. Amongst the photosynthetic pigments, chlorophyll showed a decline while as carotenoid and anthocyanin levels were elevated. The activity of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), Glutathione-s-transferase (GST) and superoxide dismutase (SOD) were positively correlated with Hg-treatment except SOD, which declined at 75 μM Hg-treatment in 30-days old seedlings. Catalase (CAT) activity showed a positive correlation up to 50 μM Hg-treatment but at 75 μM Hg-stress it decreases at both 15 and 30 days.