Rapid micropropagation system in vitro and antioxidant activity of Scabiosa tschiliensis Grunning

A rapid micropropagation system for Scabiosa tschiliensis Grunning, an ethnic medicinal plant, has been developed. Calluses were induced from leaf and petiole explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg l^?1 thidiazuron and 0.5 mg l^?1 2,4-dicholorophenoxyacetic acid. In this medium, callus induction rate was about 94.05 %. Adventitious shoots developed from leaf (86.30 %) and petiole (83.33 %) calluses when cultured on MS medium containing 4.0 or 2.0 mg l^?1 N⁶-benzyladenine (BA), respectively. Up to 73.85 % of the regenerated shoots formed complete plantlets on MS medium supplemented with 2.0 mg l^?1 indole-3-butyric acid, with an average of 3.25 roots per shoot. Quantitative analysis of flavonoids showed that the phytochemical profiles of calluses and regenerated plants were similar to that of wild-type plants. The 2, 2-diphenyl-1-picrylhydrazyl assay revealed that the flavonoid extracts of calluses, adventitious shoots and wild-type plants had stronger antioxidant activities, the inhibitory concentrations being 23.944, 31.329 and 26.502 μg ml^?1, respectively, where 50 % of DPPH was scavenged (IC₅₀). Results showed that this perennial herb could be used as a potential source of new natural antioxidants.     

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L^?1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L^?1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L^?1 BAP and 0.4 mg L^?1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L^?1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers

An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l^?1 N ⁶-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l^?1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l^?1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered,high trade medicinal plant of Western Ghats

Nilgirianthus ciliatus is a globally endangered aromatic slender shrub of Western Ghats with extensive applications in Ayurveda. It is endangered due to its indiscriminate collection and overexploitation to meet the requirements of the pharmaceuticals. This study deals with the preservation of this endangered plant through in vitro nodal culture. Nodal explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) or 6-furfurylaminopurine individually for primary shoot induction. For multiple shoot induction, primary shoots were transferred onto MS medium containing BA individually or in combination with auxins. Clusters of multiple shoots (up to 24.3) occurred with highest frequency (93.2%) on MS medium fortified with BA (3 mg l^?1) and indole-3-acetic acid (0.1 mg l^?1). In vitro regenerated plantlets were rooted on half-strength MS medium with maximum rooting frequency (82.2%) obtained in the presence of indole-3-butyric acid (1.0 mg l^?1). The rooted plantlets were acclimatized to soil with 100% survival rate. Results of this study allowed us to develop an efficient regeneration system that will permit to carry out restoration programmes of N. ciliatus in Western Ghats. In future, this protocol will be an invaluable tool to produce synthetic seeds for cryopreservation and long-term conservation.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l^?1 benzyladenine (BA) and 0.1 mg l^?1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l^?1 BA along with 1.0 mg l^?1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l^?1 thidiazurone (TDZ) and 0.1 mg l^?1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l^?1 BA and 0.1 mg l^?1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.     

Assessment of the efficacy of amino acids and polyamines on regeneration of watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb.) and analysis of genetic fidelity of regenerated plants by SCoT and RAPD markers

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l^??1 N ⁶ –Benzyladenine (BA), 5 mg l^??1 leucine, and 10 mg l^??1 spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l^??1 indole-3-butyric acid (IBA), 5 mg l^??1 isoleucine, and 10 mg l^??1 putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants.     

Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems

Airlift bioreactors were programmed for continuous and temporary immersion culture to investigate factors that affect the rhizome proliferation, shoot formation, and plantlet regeneration of Cymbidium sinense. During rhizome proliferation, the continuous immersion bioreactor system was used to explore the effects of activated charcoal (AC) in the culture medium, inoculation density, and air volume on rhizome differentiation and growth. The optimum conditions for obtaining massive health rhizomes were 0.3 g l^?1 AC in the culture medium, 7.5 g l^?1 inoculation density, and 150 ml min^?1 air. In addition, the temporary immersion bioreactor system was used for both shoot formation and plantlet regeneration. Supplementing 4 mg l^?1 6-benzylaminopurine and 0.2 mg l^?1 naphthalene acetic acid (NAA) to the culture medium promoted shoot induction from the rhizome. Cutting the rhizome explants into 1 cm segments was better for massive shoot formation than cutting into 0.25 and 0.5 cm explant segments. NAA promoted plantlet regeneration and the rooting rate (94.7 %), with whole plantlets growing well in culture medium containing 1.0 mg l^?1 NAA. Therefore, applying bioreactors in C. sinense micropropagation is an efficient way for scaling up the production of propagules and whole plantlets for the industrial production of high-quality seedlings.     

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

We developed a micropropagation protocol for Cleome gynandra, a C₄ model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l^?1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l^?1 BA and 0.1 mg l^?1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l^?1 NH₄NO₃ and 950 mg l^?1 KNO₃) and 100 mg l^?1 (NH₄)₂SO₄. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l^?1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) + 100 mg l^?1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l^?1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.     

In vitro morphogenic responses of African yam bean (<Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> (Hochst ex. A. Rich.) Harms) accessions to plant growth regulators

Morphogenic responses of two accessions of African yam bean to different concentrations of plant growth regulator supplements to Murashige and Skoog basal medium was investigated to develop a more efficient regeneration system. Mature embryo explants were cultured on growth regulator-free and BAP + NAA supplemented media. Nodal cuttings excised from 4-week old shoots of the regenerated embryos were cultured on media containing varying concentrations and combinations of 6-benzyl aminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Growth regulator-free medium favored embryo regeneration and growth over supplemented media and both enhanced shoot regeneration and rooting, but could not induce multiple shoot formation on embryo explants. Multiple shoots were produced by nodal explants and the highest average number of shoots (5.3 ± 2.3), leaves (7.7 ± 3.6), roots (3.7 ± 2.9) and root length (3.1 ± 0.0 cm) were obtained on a medium with 0.6 mg l^?1 BAP + 0.03 mg l^?1 NAA for accession TSs154, while in TSs5, highest number of shoots (3.2 ± 2.5) and leaves (5.9 ± 1.5) were induced by 2.0 mg l^?1 Kinetin + 0.05 mg l^?1 NAA. Such differential morphogenic responses to culture media underline the genotypic control of in vitro propagation of this crop. Embryo and nodal explants rooted directly on shoot regeneration media, and regenerated plantlets were successfully acclimatized. The efficient regeneration system obtained will enhance genetic improvement of African yam bean by facilitating molecular genetic transformation for advanced breeding.     

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l^?1) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l^?1) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l^?1). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l^?1) and IAA (0.1 mg l^?1) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Light-induced production of antidepressant compounds in etiolated shoot cultures of Hypericum hookerianum Wight & Arn. (Hypericaceae)

Hypericum hookerianum is a lesser known ethnomedicinal plant having wound healing, antitumor and anti-HSV-1 properties. Isolated nodes of in vitro shoots sub-cultured in the dark for 4 weeks on half strength Murashige and Skoog medium solidified with Gelzan (1.5 g l^?1), and supplemented with 2.325 μM kinetin produced 8.0 ± 0.40 etiolated shoots of 5.0 ± 0.62 cm length at 74 % efficiency versus 9.2 ± 0.6 healthy shoots of 4.4 ± 0.5 cm obtained from nodes in light at 96 % efficiency. Low concentrations of hypericin were found in wild plant [0.35 ± 0.09 mg g^?1 dry weight (DW)] and control green shoot cultures (0.91 ± 0.03 mg g^?1 DW). Etiolated shoots exposed to a 12 h photoperiod (50 μmol m^?2 s^?1) through 1–25 days turned red incrementally due to synthesis and accumulation of 0.1–3.83 mg g^?1 DW hypericin in sub-epidermal cortical cells of the stem and varied shaped cells of the distorted mesophyll. Flavonoid and anthocyanin concentrations of the etiolated shoots subjected to the 12 h photoperiod were 3–5 fold higher than the control shoot cultures while total chlorophylls [1.97 ± 0.05 mg g^?1 fresh weight (FW)] of the light exposed shoots were significantly less compared to the control (2.86 ± 0.18 mg g^?1 FW) and natural plant (6.82 ± 0.29 mg g^?1 FW). HPLC analysis of shoot extracts revealed the presence of 0.14 ± 0.03, 0.16 ± 0.02 and 1.45 ± 0.16 mg g^?1 DW hyperforin in wild plant, control shoot cultures and etiolated shoot cultures illuminated for 25 days, respectively. Despite a reasonable presence in etiolated shoots (0.61 ± 0.15 g^?1 FW), total phenols did not increase significantly during illumination. The results indicate light induced synthesis of anti-depressant phenolic derivatives (hypericin, hyperforin and flavonoids) in etiolated shoot cultures of H. hookerianum.     

Optimization of culture conditions for the production of polysaccharides and kinsenoside from the rhizome cultures of <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis> (Wall.) Lindl.

The present study designed two sets of experiments by using the uniform design method and investigated the effects of medium components on the accumulation of bioactive compounds (polysaccharide and kinsenoside) in rhizomes, in order to select a suitable culture medium for the rhizome suspension culture of Anoectochilus roxburghii (Wall.) Lindl. Among the combinations of Murashige and Skoog (MS) medium strengths and plant growth regulator (benzylaminopurine, BA; kinetin, KT; and α-naphthaleneacetic acid, NAA) concentrations, and the combinations of nitrogen, phosphorus, and sucrose concentrations, the maximum yield of polysaccharides and kinsenoside was achieved with 0.75 × MS?+?2.0 mg L^?1 BA?+?0.2 mg L^?1 KT?+?0.5 mg L^?1 NAA and 45 mM nitrogen?+?0.93 mM phosphorus?+?35 g L^?1 sucrose, respectively. Therefore, the optimal rhizome suspension culture medium was 0.75 × MS medium supplemented with 2.0 mg L^?1 BA, 0.2 mg L^?1 KT, 0.5 mg L^?1 NAA, and 35 g L^?1 sucrose. Yeast extract (YE) enhanced bioactive compound accumulation in rhizomes. The polysaccharide and kinsenoside production was significantly improved when 75 mg L^?1 YE was added to the culture medium after 30 d of rhizome suspension culture; 8.3 g L^?1 of polysaccharide and 6.1 g L^?1 of kinsenoside were obtained after 4 d of YE treatment. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of YE-treated rhizomes was higher than that of YE-untreated rhizomes, demonstrating enhanced antioxidant activity of the treated bioreactor-cultured rhizomes.