In vitro propagation,clonal fidelity and phytochemical analysis of <Emphasis Type="Italic">Rhodiola imbricata</Emphasis> Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

Ex vitro rescue,physiochemical evaluation,secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of <Emphasis Type="Italic">Decalepis salicifolia</Emphasis> (Bedd. ex Hook.f.) Venter

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM)?+?α-naphthalene acetic acid (NAA) (0.5 µM)?+?adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97?±?0.01 shoots/explants with maximum shoot length of 6.46?±?0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10?±?0.07 roots/microshoot with average root length of 2.30?±?0.06 cm was obtained. As much as 90% plantlets survived when Soilrite^? was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml^?1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Development of reproducible regeneration and transformation system for Sesamum indicum

Thalictrum foliolosum is an endemic herb known for its medicinal properties and used for various clinical applications including ophthalmic, skin disease and dyspepsia. Due to its medicinal properties, the plants are uprooted hence can be prone to extinction. In the present study, a reproducible in vitro propagation protocol has been developed using axillary shoot buds and nodal segments. Seedling derived axillary shoot buds were cultured in Murashige and Skoog’s (MS) medium supplemented with 2.24 µmol of 6-benzylaminopurine (BAP) and readily produced maximum shoot (7.2?±?0.40) with the highest percentage of response (91.42%). Also, nodal explants (field-grown plant) developed maximum shoots (3.2?±?0.48) on MS medium containing 4.49 µmol BAP with a combination of 0.54 µmol α-naphthaleneacetic acid (NAA). Best growth and foliage development was achieved at 2.24 µmol BAP with 0.54 µmol NAA in presence of 0.3% activated charcoal and 113.4 µmol ascorbic acid. Micropropagated shoots showed maximum percentage (63.30%) of rooting in half-strength MS medium containing 1.23 µmol indole-3-butyric acid (IBA) and acclimatized in soilrite and leaf manure (2:1) during 4 weeks. Monomorphic bands developed by random amplification of polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers confirmed the genetic stability of in vitro established plants. Additionally, HPLC analysis showed higher benzylisoquinoline (BIQ) alkaloids content in in vitro established plant root extracts. The micropropagation protocol developed in this study provides an alternative strategy for germplasm conservation and protection which at the same time can also be exploits for the production of pharmacologically active compounds.

     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.     

Thidiazuron enhances shoot organogenesis from leaf explants of Saussurea involucrata Kar. et Kir

An efficient protocol for the in vitro micrpropagation of Saussurea involucrata Kar. et Kir, an endangered Chinese medicinal plant, was developed. Shoot organogenesis was obtained following culture of leaf explants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). After 28 d of culture, 15.6?±?1.4 shoots were regenerated per leaf explant on MS medium containing 0.5 ??M TDZ. After transfer of shoots to a medium containing 5.0 ??M indole-3-acetic acid, approximately 80% of the regenerated shoots formed roots and whole plantlets. After transfer of rooted shoots to the greenhouse, 83% of the regenerated plantlets survived and grew vigorously. The regeneration protocol developed in this study provides a basis for germplasm conservation and for the production of plant material necessary to study the medicinally active components of S. involucrata.     

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.     

Somatic embryogenesis,acclimatization and genetic homogeneity assessment of regenerated plantlets of <Emphasis Type="Italic">Anoectochilus elatus</Emphasis> Lindl., an endangered terrestrial jewel orchid

An efficient in vitro plant regeneration system was established through somatic embryogenesis for Anoectochilus elatus Lindley, an endangered jewel orchid. Direct somatic embryogenesis was achieved from nodal explants (17.4 embryos per explant with 63.4% response) on Mitra medium supplemented with Morel vitamins, thidiazuron (4.54 µM) and ∞-naphthaleneacetic acid (2.69 µM). Simultaneously, a protocol was developed for indirect somatic embryogenesis from internodal explant, produced embryogenic calli and embryos (31.3 embryos with 76.4% response) on same medium amended with 50 mg/L peptone and 5% coconut water. Both types of embryogenic pathways, produced morphologically similar globular embryos in the form of protocorm like bodies and successfully germinated on hormone free Mitra medium supplemented with Morel vitamins. Morpho-histological investigation of the embryo revealed the initiation and developmental features of somatic embryos. In vitro regenerated plantlets were successfully established from heterotrophic to a photoautotrophic stage by reducing the nutrient content in culture media, adjusting temperature and humidity through three step method. During the process, no morphological and physiological abnormalities were observed. Hardened plantlets were successfully acclimatized at poly tunnel chamber with 95% of survival rate. Further, inter simple sequence repeats (ISSRs) molecular markers were used to analyse the genetic homogeneity of regenerated plants. Analysis with this method showed that the homogeneity is comparatively higher in direct somatic embryo regenerated plants (94.22%) as compared to plants elevated from an indirect somatic embryo (93.05%). The present study provides morpho-histological and genetically stable plants for germplasm conservation and further utility of this endangered jewel orchid.     

In Vitro Propagation Using Transverse Thin Cell Layer Culture and Homogeneity Assessment in Ceropegia bulbosa Roxb.

Ceropegia bulbosa is an endangered medicinal plant used traditionally in the treatment of various diseases. Our aim is to develop a rapid and a competent procedure for direct and indirect organogenesis from transverse thin cell layer (tTCL) explants of C. bulbosa. Optimum response to direct adventitious shoot bud induction from tTCLs was observed on medium augmented with 8.8 µM 6-benzyladenine (BA) producing 15.6 ± 0.31 shoots per responsive explant. Best callusing response (95 %) was observed with tTCL explants in medium containing 4.5 µM 2,4-dichlorophenoxyacetic acid and 2.2 µM BA. High frequency shoot regeneration (75 %) was observed from tTCL derived calli. Medium containing 8.8 µM BA and 0.27 µM α-naphthalene acetic acid produced 22.2 ± 0.64 shoots with shoots acquiring an average length of 4.6 ± 0.12 cm. In vitro rooting was recorded on ½ strength Murashige and Skoog medium, producing 10.9 ± 0.23 roots with a length of 4.24 ± 0.16 cm. Plants were successfully transferred to the field with a survival rate of 89 %. The clonal nature of the regenerants was assessed using Inter-simple sequence repeat markers.     

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of <Emphasis Type="Italic">Allamanda cathartica</Emphasis> L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00?±?1.30) and shoot length (7.50?±?0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50?±?0.16 mean roots/shoot with 4.05?±?0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.     

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.     

In vitro propagation of an endemic and endangered medicinal plant <Emphasis Type="Italic">Notopterygium incisum</Emphasis>

An efficient system in vitro propagation for Notopterygium incisum Ting ex H. T. Chang, an endemic and endangered medicinal plant, was established to address increased demand and germplasm conservation goals. Optimum response in callus induction (CI) was observed on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzylaminopurine (BAP), which the induction rate and growth of callus were 84.44% and 0.67 g respectively. The highest shoot regeneration frequency (76.97%) and maximum number of shoots (3.6 shoots per callus) were achieved on MS medium with 1.5 mg/l BAP and 0.2 mg/l naphthalene acetic acid (NAA). Half-strength MS medium supplemented with 0.6 mg/l indole-3-butyric acid (IBA) was determined to be the best rooting medium, resulting in the maximum number of roots (18.6 roots per shoot) and the highest rooting frequency (92.28%). An approximate 83.8% survival rate among the regenerated plantlets was recorded after they were transplanted in the field at an altitude of 3200 m. An HPLC analysis showed that the content of two main chemical constituents, notopterol and isoimperatorin, in the rhizomes of 3-year-old regenerated plantlets was higher (3.84 mg/g and 4.05 mg/g, respectively) than that in commercially marketed crude drugs. This first report of complete regeneration in vitro could provide an alternative method for the rapid, large-scale production and conservation of this valuable, rare, and endangered medicinal plant.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

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.     

Total phenolics,resveratrol content and antioxidant activity of seeds and calluses of pinto peanut (<Emphasis Type="Italic">Arachis pintoi</Emphasis> Krapov. &amp; W.C. Greg.)

Arachis pintoi is a peanut species native to Brazil, which is cultivated in many countries for animal forage, soil cover, landscaping, and recovery of degraded areas. Tissue culture studies for this species have been focused in plant production, whereas works on in vitro secondary metabolites production are scarce. The goal of the present work was to establish callus cultures from different seed explants of A. pintoi, aiming at evaluating the potential for metabolites production and antioxidant activity. Embryonic axes, leaflets, and cotyledons were cultured on solidified MS medium supplemented with picloram (PIC), 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), under light or dark conditions. Friable calluses with a high biomass (4.3?±?0.3 g FW per callus) were obtained from embryonic leaflets cultured on medium supplemented with 17.6 µM BA plus 5.4 µM NAA, in the dark. Cotyledons and embryonic axes cultured in the presence of 4.4 µM BA combined with 10.8 µM NAA formed heterogeneous calluses with a compact base and a large friable surface. Trans-resveratrol and other stilbenes that were not found in seeds were detected in callus extracts, especially those originated from cotyledons, although these materials showed lower total phenolic contents (TPC) when compared with seeds with and without testa, as well as cotyledons. Extracts from seeds with testa and from calluses derived from cotyledons and embryonic axes showed the highest EC₅₀ in DPPH assays. No correlation between TPC, trans-resveratrol and antioxidant activity was observed.     

Elicitor mediated enhancement of wedelolactone in cell suspension culture of <Emphasis Type="Italic">Eclipta alba</Emphasis> (L.) Hassk

The present research focused on enhancing the production of wedelolactone through cell suspension culture (CSC) in Eclipta alba (L.) Hassk. With an aim of attaining a sustainable CSC, various plant growth regulators, elicitors and agitation speed were examined. Nodal segments of in vitro propagated plantlets induced the maximum percentage (93.47?±?0.61%) of callus inoculated on Murashige and Skoog (MS) medium fortified with picloram (2 mg L^?1). The growth kinetics of CSC exhibited a sigmoid pattern with a lag phase (0–6 days), a log phase (6–18 days), a stationary phase (18–24 days) and then death phase thereafter. The highest biomass accumulation in CSC with 7.09?±?0.06 g 50 mL^?1 fresh weight, 1.52?±?0.02 g 50 mL^?1 dry cell weight, 1.34?±?0.01?×?10⁶ cell mL^?1 total cell count and 57.00?±?0.58% packed cell volume was obtained in the liquid MS medium supplemented with 1.5 mg L^?1 picloram plus 0.5 mg L^?1 kinetin at 120 rpm. High performance thin layer chromatography confirmed that yeast extract (biotic elicitor) at 150 mg L^?1 accumulated more CSC biomass with 1.22-fold increase in wedelolactone (288.97?±?1.94 µg g^?1 dry weight) content in comparison to the non-elicited CSC (237.78?±?0.04 µg g^?1 dry weight) after 120 h of incubation. Contrastingly, methyl jasmonate (abiotic elicitor) did not alter the biomass but increased the wedelolactone content (259.32?±?1.06 µg g^?1 dry weight) to an extent of 1.09-fold at 100 µM. Complete plantlet regeneration from CSC was possible on MS medium containing N⁶-benzyladenine (0.75 mg L^?1) and abscisic acid (0.5 mg L^?1). Thus, the establishment of protocol for CSC constitutes the bases for future biotechnological improvement studies in this crop.     

In vitro propagation and the acclimatization effect on the synthesis of 2-hydroxy-4-methoxy benzaldehyde in Decalepis hamiltonii Wight and Arn.

The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N₁) from the basal cut end similar to cytokinin–auxin combination (N₂). Between two types of nodular tissues, N₂ was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA₃). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.