μM Adenine Sulphate Research Articles

High-frequency clonal propagation of Curcuma angustifolia 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.

High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in Gymnema sylvestre

The present study describes the first attempt of exploiting encapsulation technology for high plantlet recovery, short-term storage and conservation of Gymnema sylvestre—an antidiabetic liana. Indole-3-butyric acid (IBA) pretreated nodal segments (NS) were encapsulated in sodium alginate (Na2-alginate) matrix and the optimal culture conditions were evaluated in terms of maximum conversion capacity of synseeds into complete plantlets. Highest conversion frequency of synseeds was obtained on Murashige and Skoog’s (MS) medium supplemented with 5.0 µM 6-benzyladenine (BA). Augmentation of Na2-alginate matrix with plant growth regulators (PGRs) and additive further improved in vitro conversion rates and the synthetic endosperm composed of 3% Na2-alginate in MS + 2.5 µM BA + 2.5 µM gibberellic acid (GA3) + 50 µM adenine sulphate (AdS) stimulated maximum recovery (88.2 ± 0.48%) of complete plantlets from synseeds. Studies on short term cold storage of synseeds showed that nutrient encapsulation maintains the viability of NS for a storage period of 8 weeks. Ex-vitro conversion of synseeds was also carried out on soilrite and vermicompost (3:1) mixture under culture room conditions. Monomorphic DNA profiles produced through Inter-Simple Sequence Repeat (ISSR) markers confirmed the genetic uniformity between synseed derived and mother plantlets.

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers

ABSTRACTThis study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.

Enhanced multiplication and improved ex vitro acclimatization of Decalepis arayalpathra

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite™ with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant-1), dry mass (0.35 g plant-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm-3(root extract) were recorded after 8 weeks of acclimatization.

Micropropagation of Clerodendrum phlomidis L.F.

Abstract Clerodendrum phlomidis L. f. is an important medicinal plant of the Lamiaceae family, particularly its roots, which are used for various therapeutic purposes in a pulverized form. The objective of this study was to develop a standard protocol for axillary shoot proliferation and rooting of C. phlomidis for its propagation and conservation. Nodal explants were inoculated on Murashige and Skoog (MS) medium that was supplemented with one of six cytokinins: 6-benzyladenine, kinetin, thidiazuron, N 6-(2-isopentenyl) adenine (2iP), trans-zeatin (Zea) and meta-topolin. Callus induction, which was prolific at all concentrations, formed at the base of nodal explants and hindered shoot multiplication and elongation. To avoid or reduce callus formation with the objective of increasing shoot formation, the same six cytokinins were combined with 4 μM 2,3,5-tri-iodobenzoic acid (TIBA) alone or in combination with 270 μM adenine sulphate (AdS). Nodal explants that were cultured on the medium supplemented with 9.12 μM Zea, 4 μM TIBA and 270 μM AdS produced significantly more and longer shoots than on medium without TIBA and AdS. Half-strength MS medium supplemented with 8.05 μM α-naphthaleneacetic acid was the best medium for root formation. Most (75%) in vitro rooted plantlets were successfully acclimatized under natural conditions.

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species.

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88μM BAP with 108.6μM adenine sulphate and 11.84μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84μM IBA, with 81.1% rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.

High-frequency clonal propagation, encapsulation of nodal segments for short-term storage and germplasm exchange of Ficus carica L.

The study describes an efficient regeneration system and encapsulation of nodal segments in F. carica accompanied with frequent root formation in microshoots under the influence of salicylic acid. The present study describes an improved protocol for clonal propagation and a short-term conservation strategy using an encapsulation technology for Ficus carica. Nodal segments were more responsive than shoot tips for shoot multiplication. Among the cytokinins tested, Murashige and Skoog’s (MS) medium augmented with 7.5 µM 6-benzyladenine (BA) was the best, but the leaves exhibited a rudimentary structure. Cytokinin–auxin combinations enhanced the multiplication rate; but leaf growth did not improve. However, the supplementation of 50 µM adenine sulphate (Ads) to MS + 7.5 µM BA + 0.5 µM α-naphthalene acetic acid (NAA) significantly enhanced mean shoot number per explant and overall growth of shoots and induced a maximum of 26.8 shoots per nodal segment and 15 shoots per shoot tip with mean shoot length of 8 and 4.3 cm, respectively, after 4 weeks of culture. Microshoots rooted on half-strength MS medium containing 2.5 µM salicylic acid (SA) either in semi-solid (mean of 4.6 roots per shoot) or liquid medium (mean of 5 roots per shoot). A gelling matrix of 4 % sodium alginate (Na-alginate) and 100 mM calcium chloride (CaCl2·2H2O) was used for the encapsulation of nodal segments. Maximum shoot growth (95.6 %) was recorded on MS medium supplemented with 7.5 µM BA + 0.5 µM NAA + 50 µM Ads. Rooting was induced upon transfer of sprouted microshoots to semi-solid half-strength MS medium supplemented with 2.5 µM SA. To examine the retention of encapsulated and non-encapsulated nodal segments’ viability, a low-temperature storage (4 °C) experiment was carried out. Encapsulated nodal segments stored at 4 °C for 1–6 weeks sprouted at variable frequencies in successive weeks of transfer. Plantlets were acclimatized and established in field where they grew without any detectable variations after 6 months of transfer.

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.

Regeneración de plantas a partir de cultivos de callos de <i>Vitex trifolia</i> (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn.

An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl2·2H2O). A gelling matrix of 4 % Na-alginate and 100 mM CaCl2·2H2O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00 ± 2.09 %) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 30.0 μM adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5 μM α-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.

In vitro propagation of Eulophia nuda Lindl., an endangered orchid

An in vitro propagation and tuberization method was successfully established for Eulophia nuda Lindl., an endangered orchid of Aravalli hills. Shoot buds were induced from tuber explants (1cm cut pieces) on Murashige and Skoog (MS) medium containing 44.4μM 6-benzyladenine (BA) and additives (283.8μM ascorbic acid, 67.8μM adenine sulphate, 143.5μM arginine and 118.9μM citric acid). After excision of newly formed shoots, explants were successively transferred to MS medium with 22.2μM BA and additives for shoot multiplication up to three passages. Further shoot multiplication and tuberization was achieved, by subculturing of in vitro-formed shoots, on MS medium containing 8.88μM BA, 4.68μM Kinetin (Kn) and additives. Shoot multiplication was influenced by cytokinin type, concentration and combinations. Shoots with tubers were successfully rooted in vitro and ex vitro. Shoots were treated with 2.46mM indole-3-butyric acid (IBA) for 4min for ex vitro rooting. Plantlets were acclimatized in the greenhouse conditions in bottles containing soilrite. Acclimatized plantlets were transferred to the earthen pots containing soil as a mixture of clay, sand and vermicompost (1:1:1). In vitro tuber formation of E. nuda was high compared to the ex vitro/natural conditions. This is the first report for in vitro propagation and tuberization of E. nuda. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species.

Micropropagation of Dendrocalamus asper {Schult. & Schult. F.} Backer ex k. Heyne): an exotic edible bamboo

Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February–April) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS + 15 μM BAP. For shoot multiplication, MS medium supplemented with 10 μM BAP and 75 μM Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ½ strength MS medium supplemented with 5 μM each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6 months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.

Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L. (Scrophulariaceae)–an ethnomedicinal herb

Objective To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis (S. dulcis) L. Methods Explants were inoculated on MS basal medium supplemented with kinetin and 6-benzylaminopurine for shoot bud induction. To enhance the shoot induction, various auxins like 3-indoleacetic acid or 3-indolebutyric acid or α-naphthylacetic acid were tested along with 2.32 M KI and 4.44 μM BAP. The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA, IBA or NAA. After roots were developed, the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%–80% humidity under 16 h photoperiod. After acclimatization, the plantlets were transferred to the garden and survival percentage was calculated. Data were statistically analyzed and means were compared using Duncan's multiple range test ( P < 0.05). Results An in vitro method was developed to induce high frequency shoots regeneration from stem, mature leaf and young leaf explants of S. dulcis. Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins (2.32 μM KI and 4.44 μM BAP) 2.85 μM IAA, 10% CM and 1 483.79 μM adenine sulfate. A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP. Conclusions Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S. dulcis.

Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella

To induce variation through chromosome doubling in Gerbera jamesonii Bolus cv. Sciella, two-week-old in vitro grown shoots were treated with various concentrations of colchicine (0.01, 0.05, 0.10, 0.50 or 1% w/v) for 2, 4 or 8 h. Treated shoots were then cultured on Murashige and Skoog (MS) medium supplemented with 8.8 μM 6-benzyladenine (BA) and 155 μM adenine sulphate (ADS), and subsequently transferred to fresh MS medium containing 2.85 μM indole-3 acetic acid (IAA) for rooting. When shoots were treated with 0.1% colchicine for 8 h, 64% of recovered plantlets were tetraploid. Ploidy of plantlets was confirmed by flow cytometry, stomatal analysis, and morphological characters. Tetraploid plantlets displayed slower proliferation along with higher vigor and thickened broad leaves. Moreover, tetraploid plants developed larger flowers, longer stalks, and have improved vase-life, all contributing to higher ornamental value of gerbera.

Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA3), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO3, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.

High Frequency Micropropagation of Aloe vera L. Burm. f. as a Low Cost Option Towards Commercialization

An efficient micropropagation protocol has been developed using shoot apical meristem as explants in a high barbaloin content 'bitter' cultivar of Aloe vera L. The protocol involves induction, multiplication and in vitro rooting of the regenerated shoots and their acclimation under ex vitro conditions. 35.5 µM BAP and 9.8 µM IBA in combination with 81.4 µM adenine sulphate proved optimum for shoot bud induction. Combination of 8.87 µM BAP and 2.46 µM IBA produced highest number of shoot buds (22.0 ± 0.14) and enhanced bud proliferation within one - two weeks after first subculture. For induction of in vitro rooting, Aloe gel as an alternative to conventional rooting medium used for the first time resulted in 100% rooting and highest number of roots per culture (10.90 ± 0.17). The plantlets were successfully hardened. Cent per cent plants survived in the field condition. Chromosomal analysis of the regenerated plantlets established a stable germplasm with 2n = 14 bimodal chromosomes. The cost effectiveness and economic viability of the protocol has also been evaluated. Key words: Aloe vera, bitter cultivar, micropropagation, cost analysis D.O.I. 10.3329/ptcb.v20i1.5962 Plant Tissue Cult. &amp; Biotech. 20(1): 29-35, 2010 (June)

Micropropagation of Costus speciosus (Koen.) Sm. Using Nodal Segment Culture

Nodal segments of Costus speciosus (Koen.) Sm. containing single axillary buds were cultured on Murashige and Skoog medium (MS medium) supplemented with plant growth regulators for inducing plantlets. For breaking of axillary bud dormancy, nodal segments were cultured on 40-70gl-1 sucrose or 1-13 μM adenine sulphate (AdS) supplemented MS basal medium containing 5 μM 6-benzylaminopurine (BAP) and 1μM α-naphthalene acetic acid (NAA). The nodal segments cultured on 1-13 μM AdS, 5 μM BAP, 1 μM NAA and 50gl-1 sucrose showed simultaneous production of shoots and roots while those cultured on 5 μM BAP, 1 μM NAA and 40-70gl-1 sucrose produced shoots only. The most effective media for breaking axillary bud dormancy was 5 μM BAP, 1 μM NAA, 50 gl-1 sucrose and 10 μM AdS supplemented medium. The propagules from 40-70gl-1 sucrose produced roots in shoot multiplication medium, i.e.,10 μM AdS, 1 μM NAA, 50gl-1 sucrose and 3-11 μM BAP supplemented medium. The best response for shoot multiplication was on 10 μM AdS, 1 μM NAA, 50gl-1 sucrose and 7 μM BAP. The well-rooted shoots were hardened and transferred to the soil where they showed 95% survival rate. Results show that axillary bud can be used for micropropagation of Costus speciosus.

An efficient somatic embryogenesis system for velvet bean [Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug

The Mucuna pruriens Linn. is an important medicinal legume cover crop. Almost all the parts of the plant are reported to contain l-3,4-dihydroxy phenylalanine (l-DOPA), a non-protein amino acid that acts as precursor for the neurotransmitter dopamine. Here we report a rapid and reliable protocol for high fidelity regeneration of M. pruriens plants via somatic embryogenesis. Embryogenic callus was induced from cotyledon segments of in vitro grown seedlings on Murashige and Skoog medium supplemented with 6.7 μM 2,4-dichlorophenoxy-acetic acid (2,4-D). High-frequency somatic embryogenesis was achieved after transfer of embryogenic callus clumps to MS medium supplemented with 2.3 μM Kinetin (Kn) and 5.4 mM α-naphthaleneacetic acid (NAA) supplemented with 13.6 μM adenine sulphate. The maximum number of cotyledonary-stage embryos (60.5 ± 12.7) was obtained after 10 weeks. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. The protocol should provide an efficient means for large-scale cultivation and in vitro manipulation of M. pruriens, an important green manure cover-crop with medicinal properties.

Rapid in vitro multiplication of Melia azedarach L. (a multipurpose woody tree)

An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l−1 ammonium sulphate, (NH4)2SO4, and 100 mg l−1 K2SO4, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About 90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully acclimatized first under culture room conditions, then to green house with 85% survival rate.

In vitro propagation of a high value medicinal plant: Asparagus racemosus Willd.

Asparagus racemosus Willd. is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in traditional systems of medicine such as Ayurveda, Unani, and Siddha. The multiple uses of this species have increased its commercial demand, resulting in over-exploitation. Because of destructive harvesting, the natural population of A. racemosus is rapidly disappearing, and it is recognized as ‘vulnerable’ (Warner et al., Some important medicinal plants of the Western Ghats, India: a profile. International Development Research Centre, Artstock, New Delhi, India, 15 pp, 2001). The development of an efficient micropropagation protocol will play a significant role in meeting the requirements for commercial cultivation, thereby conserving the species in its natural habitat. In the present study, in vitro shoot proliferation was obtained by culturing single node segments in Murashige and Skoog’s (MS) medium supplemented with 3.69 µM 2-isopentyl adenine and 3% sucrose with a multiplication rate of 3.5. For proper root formation, the in vitro-formed shoot clusters were cultured on half strength (major salts reduced to half) MS medium with 1.61 µM 1-naphthalene acetic acid, 0.46 µM kinetin, 98.91 µM adenine sulfate, 500 mg/l malt extract, 198.25 µM phloroglucinol, and 3% sucrose. On this medium, 85% rooting was observed within 20 d. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%.