μM Kn Research Articles

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023

Low cost callus regeneration for production of phenolics, flavonoids and antioxidant in Withania somnifera (L.) Dunal

The present investigation was carried out to develop an efficient protocol for low cost secondary metabolite production through in vitro callus culture of a medicinal herb Withania somnifera. The leaf explants, excised from the shoots collected from university campus, were cultured under different experimental conditions for in vitro callus induction and growth. The experimental conditions were optimized for reducing the cost of in vitro callusing medium. In the callusing medium double distilled water was replaced by reverse osmosis water, sucrose by table sugar and by lowering (0.7 % w/v) agar concentration instead of 0.8 %. Different concentration and combinations of 2,4-D and Kinetin were tested for callus induction and growth from leaf tissue. The leaf explant cultured on 0.7 % (w/v) agar gelled MS medium prepared with RO water and having 4.50 µM 2,4-D and 4.65 µM Kn were found best for callus induction. The cost of this medium was 11 times lower than the normal medium. The callus obtained on this medium was dried at room temperature and extraction of phytochemicals from dried callus was performed by using methanol as solvent. The methanolic extract of callus showed the presence of phenolic, flavonoid and antioxidant activity.

Kinetin mitigates Cd-induced damagesto growth, photosynthesis and PS II photochemistry of Trigonella seedlings by up-regulating ascorbate-glutathione cycle.

Cytokinins (CKs) plays a key role in plant adaptation over a range of different stress conditions. Here, we analyze the effects of a cytokinin (i.e., kinetin, KN) on the growth, photosynthesis (rate of O2 evolution), PS II photochemistry and AsA–GSH cycle in Trigonella seedlings grown under cadmium (Cd) stress. Trigonella seeds were sown in soil amended with 0, 3 and 9 mg Cd kg-1 soil, and after 15 days resultant seedlings were sprayed with three doses of KN, i.e.,10 μM (low, KNL), 50 μM (medium, KNM) and 100 μM (high, KNH); subsequent experiments were performed after 15 days of KN application, i.e., 30 days after sowing. Cadmium toxicity induced oxidative damage as shown by decreased seedling growth and photosynthetic pigment production (Chl a, Chl b and Car), rates of O2-evolution, and photochemistry of PS II of Trigonella seedlings, all accompanied by an increase in H2O2 accumulation. Supplementation with doses of KN at KNL and KNM significantly improved the growth and photosynthetic activity by reducing H2O2 accumulation through the up-regulation AsA–GSH cycle. Notably, KNL and KNM doses stimulated the rate of enzyme activities of APX, GR and DHAR, involved in the AsA–GSH cycle thereby efficiently regulates the level of AsA and GSH in Trigonella grown under Cd stress. The study concludes that KN can mitigate the damaging effects of Cd stress on plant growth by maintaining the redox status (>ratios: AsA/DHA and GSH/GSSG) of cells through the regulation of AsA-GSH cycle at 10 and 50 μM KN under Cd stress conditions. At 100 μM KN, the down-regulation of AsA-GSH cycle did not support the growth and PS II activity of the test seedlings.

Cost-Effective in vitro Multiplication and Phenolic Profile of an Important Medicinal Orchid, Satyrium nepalense D. Don

In this study we report a cost-effective and reproducible protocol for large-scale multiplication of an important medicinal orchid, Satyrium nepalense through a four-step protocol, involving asymbiotic seed germination, multiplication of protocorm-like bodies (PLBs), development and multiplication of shoots, and finally in vitro rooting of the developed shoots. Out of various media tested for seed germination, Mitra’s medium supplemented with 0.1 % peptone (BM) supported the highest (79.19 %) seed germination. Eight-weekold protocorms were sub-cultured on BM alone and supplemented with different concentrations (1-8 μM) of BAP, KN, or TDZ. The highest number of shoots was developed on the medium containing 4 μM KN. Here we also observed that the highest number of shoots were produced from protocorms cultured on a 2 % isubgol gelled medium out of the seven different low-cost gelling agents used. Further, optimal elongation of shoots was observed on BM alone and 2 % guar gum, among the tested gelling agents. Elongated shoots were transferred for rooting on BM containing 0-2 μM of IAA, IBA, or NAA. The best rhizogenic response was observed on BM fortified with 0.5 μM IBA and isubgol (3 %). The rooted plantlets showed a 76 % survival rate on acclimatization after transfer to the potting mixture of sand and vermiculite (1:1). HPLC analysis of therapeutically important phenolic acids of leaves and tubers of in vitro regenerated and in vivo plants revealed the presence of higher levels of selected phenolic acids in in vitro tissues than those from their native habitat. This protocol could facilitate the conservation and propagation of this important medicinal orchid.

Asymbiotic Seed Germination of Thunia alba, a Variable Orchid in an Isolated Karst Hill of Xishuangbanna, Yunnan, China

Asymbiotic seed germination of Thunia alba (Lindley) H.G. Reichenbach was tested on different media in order to propagate and preserve its genetic diversity. In addition different plant hormones were used in the similar germination experiments. Results from these experiments revealed that Knudson medium (KC) had the best germination ability but Mitra and modified Murashige and Skoog (MS) also showed good responses for growth and root formation in the seedling after eight weeks. Coconut water (CW) was the most efficient for promoting germination and growth, addition of 0.046 μM and 0.46 μM Kn also induced better germination rate and formation of leaves. The seedlings after eight months were transferred and acclimatized to grow in the garden on a medium containing bark and crushed concrete blocks. Although the species is amongst the rare orchid species that can successfully propagate asexually in the wild it is demanding for substrate and climatic conditions.
 Plant Tissue Cult. & Biotech. 30(2): 189-197, 2020 (December)

Improved protocol for micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cardamomum Maton)

Cardamom (Elettaria cardamomum Maton) is a highly valuable commercial spice crop, originating from the Western Ghats of southern India. It has importance both as a spice and for its medicinal properties. An efficient and reliable micropropagation protocol using shoot tips as explants is presented which can be utilized for development of elite planting material. Growth patterns of cardamom shoots were analyzed on 45 media treatments and best shoot proliferation was recorded on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.32 μM kinetin (Kn) with 5.83 shoots regenerated per explant. The longest shoots (6 cm) were observed on 0.44 μM BAP and 2.32 μM Kn containing medium. Regenerated shoots rooted well on full strength MS basal medium, generating 3.50 roots per explant with an average root length of 4.33 cm within 4 wk. Successful hardening with a field survival percentage of 80% was achieved after acclimatization of well-developed plantlets. Genetic stability of the micropropagated plants was confirmed using 13 ISSR markers. This study reports an improved micropropagation protocol for cardamom with an enhanced rate of shoot multiplication leading to the production of healthy shoots which can be easily rooted and successfully transferred to the field.

Combined Kinetin and Spermidine Treatments Ameliorate Growth and Photosynthetic Inhibition in Vigna angularis by Up-Regulating Antioxidant and Nitrogen Metabolism under Cadmium Stress.

Pot experiments were conducted to investigate the probable beneficial role of the individual as well as combined application of kinetin (50 μM Kn) and spermidine (200 μM Spd) on Vigna angularis under cadmium (Cd) stress. Cd treatment reduced growth by declining the content of chlorophylls and carotenoids, photosynthesis, and gas exchange parameters. Exogenously, Kn and Spd application enhanced the photosynthetic parameters and up-regulated the antioxidant system by improving the activities of antioxidant enzymes and the content of non-enzymatic components. In addition, the application of Kn and Spd resulted in significant improvement in the content of sugars, proline, and glycine betaine, ameliorating the decline in relative water content. Oxidative stress parameters including hydrogen peroxide, superoxide, lipid peroxidation, lipoxygenase activity, and electrolyte leakage increased due to Cd stress; however, the application of Kn and Spd imparted a significant decline in all these parameters. Further, reduced Cd uptake was also observed due to Kn and Spd application. Total phenols and flavonoids also increased due to Kn and Spd treatments under normal as well as Cd stress conditions, which may have further helped with the elimination of reactive oxygen species. Reduction in the activity of nitrate reductase and the content of nitrogen was ameliorated due to the exogenous application of Kn and Spd. Therefore, the exogenous application of Kn and Spd benefited Vigna angularis counteracting the damaging effects of Cd stress by up-regulating the tolerance mechanisms, including antioxidant and osmolyte metabolism.

Effect of different PGRs on in vitro organogenesis in Viola canescens Wall. ex. Roxb. from petiole callus culture

An efficient protocol for in-direct in vitro regeneration of Viola canescens is described using petiole derived callus. Proliferation of callus was achieved using 2,4-D, 2,4-D: Kn, 2-4D: BAP; IBA alone or in combination with Kn on Murashige and Skoog medium. The callus differentiated into multiple shooting buds on MS medium fortified with different concentrations of various cytokinins (Kn, BAP, Zeatine and Gibberellin). Maximum average number of shoots (19.93 ± 1.79) and maximum average length of petiole (5.41 ± 0.47) was achieved when medium was fortified with 22.20 µM BAP and 18.60 + 4.0 µM Kn + Gibberellin, respectively. The microshoots were separated and rooted on full and half strength MS medium fortified with different concentrations of various auxins (IBA and NAA). Maximum average number (14.30 ± 2.24) and length (3.98 ± 0.73) of root was recorded on half strength medium fortified with 4.92 µM IBA. The regenerated plantlets were phenotypically normal and successfully transplanted to soil conditions with 90% survival percentage.

In vitro Seed Germination, Protocorm Formation and Plantlet Regeneration in Aerides ringens Fisher

An attempt was made to in vitro seed germination and plantlet regeneration in Aerides ringens, a threatened, endemic and epiphytic orchid. Five basal media Knudson C (KC), Vacin and Went (VW), Burgeff’s (N3f), half strength MS, MS were examined for their effectiveness. Germination occurred in all the media. However, KC medium proved to be the best for seed germination (70.8%) and half strength MS was superior for the differentiation of protocorm like bodies (PLBs) into seedlings. The seeds were also cultured on various plant growth regulators (PGRs) to evaluate their effectiveness on seed germination and further differentiation. Among the PGRs tested, 0.57 μM IAA (IAA 89.3%), 4.64 μM Kn (88.6%) and 2.21 μM BAP (90.7%) and 0.57 μM IAA + 0.55 μM BAP (96.5%) were stimulated the germination and further differentiation of PLBs into seedlings. The concentrations of 2.21 μM BAP and combination of 0.57 μM IAA + 0.55 μM BAP stimulated the formation of MPLBs, which later differentiated into shoots and roots. Four potting substrates were evaluated for Ex vitro seedling survivability of which brick: charcoal: decaying litter: coconut husk (1 : 1 : 1 : 1) gave maximum survival rate (89.0%).
 Plant Tissue Cult. & Biotech. 29(1): 49-62, 2019 (June)

Production of bioactive compounds in cladode culture of Turbinicarpus valdezianus (H. Moeller) Glass & R. C. Foster

This study aimed to establish an in vitro propagation method for the large-scale cultivation of Turbinicarpus valdezianus and to examine the content of bioactive compounds, as well as the antioxidant and cytotoxic activities of cladodes regenerated in vitro. The ideal combination for multiple shoot production from the bottom, middle and top regions of the cladode explants was 2 μM KN with 1 μM IBA. The explant obtained from the middle part of the cladode produced a higher number of shoots (10.9) than the bottom (7.2) and top (6.8) regions. The highest ex vitro rooting percentage (96.1%) was observed on vermiculite moistened with MS basal salts solution containing 1.0 μM IBA. A total of nine major lipophilic metabolites were simultaneously determined in the cladode tissues of T. valdezianus by LC-MRM-MS. Seven fatty acids were identified in the cladode tissues of T. valdezianus by GC-FID and GC–MS. The most abundant lipophilic metabolite and fatty acid in the cladode tissues were α-tocopherol (31.51 μg g−1 FW) and linoleic acid (43.29%), respectively. The methanolic cladode extract of T. valdezianus exhibited strong antioxidant and antitumor activity. This in vitro organ culture method is an alternative method to obtain various bioactive compounds from rare and slow growing T. valdezianus.

Cold treatment prompts embryogenic callus and cytodifferentiation in the anthers of tea (Camellia sp.) genotypes

The simple and rapid protocol was developed to induce androgenic embryoids from immature anthers of tea genotypes. Anthers from unopened flower buds of ~ 5 to 6 mm size were subjected to two temperature treatments at 4 °C and 25 °C temperatures for 5 days and cultured onto two mediums, half strength MS liquid and 0.8% agar medium. The inoculated cultures were incubated under complete dark (24 h) and photoperiod (9:15 h) conditions. Anthers of nine tea clones (UPASI-1, UPASI-2, UPASI-3, UPASI-8, UPASI-10, UPASI-20, UPASI-28, and certain estate selections, ATK-1 and CR-6017) initially cultured onto half strength Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-dichlorophenoxy acetic acid (9.05 µM 2,4-D) combined with kinetin (2.44 µM Kn) and 6-benzylamino purine (2.22 µM BAP). The pre-treated anthers at 4 °C for 5 days of genotype UPASI-3 induced significantly higher percentage of callus induction when cultured onto half strength MS agar medium under both continuous dark and photoperiod (9:15 h) conditions. Percent of callus production was significantly (P < 0.001) influenced by the genotypes (G) and plant growth regulators (P) while it was not affected by culture conditions (Dark; D/Photoperiod: L). Irrespective of genotypes studied, medium supplemented with BAP is superior to Kn in inducing androgenic potential and promotes the emergence of embryoid structures in a short time span of 45 days and the emergence of morphologically prominent globular mature embryoid callus was identified for 55 days and subsequent cytodifferentiation were achieved only in medium fortified with 2,4-D along with BAP. The results found that cold treatment and culture medium accelerated the development of embryogenic calli. Histological evidences of embryoids revealed that in vitro cytodifferentiation rendering to cellular totipotency developed different meristematic cells and vascular elements. Anthers provided with cold treatment induced callus in the liquid growth medium. The liquid growth medium formulation used here is suitable for inducing androgenic potential and promoted callus formation from anthers in less time span for 10–15 days. Callus maturation and immature embryoid callus was induced for 45 days and morphologically prominent round mature embryoids were appeared by 55 days. And the histological studies reveal the trend of in vivo cytodifferentiation rendering to cellular totipotency while growing in vitro. Our experiment concludes the profuse callus induction and embryoid induction by using liquid growth medium formulation, which is difficult task in anther culture of tea because of its highly recalcitrant nature. Liquid medium hastens the androgenesis process in tea anthers and induced embryoids when cultured onto same solid agar medium.

Potential of exogenously sourced kinetin in protecting Solanum lycopersicum from NaCl-induced oxidative stress through up-regulation of the antioxidant system, ascorbate-glutathione cycle and glyoxalase system.

The protective role of exogenously applied kinetin (10 μM KN, a cytokinin) against the adverse effects caused by NaCl-induced (150 mM) stress in Solanum lycopersicum was investigated. Application of KN significantly enhanced growth and biomass production of normally grown plants (non-stressed) and also mitigated the adverse effect of NaCl on stressed plants to a considerable extent. Among the examined parameters, chlorophyll and carotenoid contents, photosynthetic parameters, components of the antioxidant system (both enzymatic and non-enzymatic), osmotica accumulation, and mineral uptake exhibited a significant increase following the application of KN. Furthermore, KN application reduced the generation of reactive free radical hydrogen peroxide, coupled with a significant reduction in lipid peroxidation and an increase in membrane stability. The activities of antioxidant enzymes, and glyoxylase system were found to be promoted in plants exposed to NaCl, and the activities were further promoted by KN application, thereby protecting S. lycopersicum plants against NaCl-induced oxidative damage. Further strengthening of the antioxidant system in KN supplied plants was ascribed to regulation of ascorbate-glutathione cycle, phenols and flavonoids in them. The levels of proline and glycine betaine increased considerably in KN-treated plants, thereby maintaining relative water content. Moreover, exogenous KN application reduced the inhibitory effects of NaCl on K+ and Ca2+ uptake, which resulted in a considerable reduction in tissue Na+/K+ ratio.

An improvised in vitro vegetative propagation technique for Bambusa tulda: influence of season, sterilization and hormones

Season and concentration of sterilizing agents play a significant role for establishment of aseptic in vitro shoot cultures and sprouting of nodal explants from field growing culms of bamboo species. In the present investigation the nodal segment explants of Bambusa tulda Roxb collected in different seasons and treated with various concentrations of HgCl2 showed significant variation in aseptic culture establishment and bud break. The rainy season (July–August) recorded with highest of 78% aseptic culture establishment whereas autumn recorded with lowest 46%. Summer and winter seasons emerged to be the best period, registering > 60% in vitro bud break. On the other hand, the autumn season had the lowest value for bud break, i.e. 42%. Among different doses of sterilizing agent tried, HgCl2 0.1% found to be suitable for maximum aseptic culture establishment (66%) as well as bud break (59%). However, among the interactions, summer season and the dose of 0.1% HgCl2 exhibited maximum of 73% response for both aseptic culture establishment and bud break. MS medium (liquid) enriched with 5.0 µM BA + 5.0 µM Kn [Kinetin (N6-Furfuryladenine)] with additional supplementation of 100 µM glutamine + 0.1 µM IAA supported a maximum in vitro shoot multiplication of 4.75 fold. The proliferated shoots were successfully rooted on MS medium (liquid) supplemented 40 µM coumarin. The plantlets transferred to the polythene bags showed 98% survival.

Plant Growth Regulator Effects on In Vitro Propagation and Stevioside Production in Stevia rebaudiana Bertoni

Stevia rebaudiana is of great importance due to its steviol glycosides (SGs) which are natural sweeteners used by the food industry as well as having medicinal purposes. In the present study, the effect of plant growth regulators (PGRs) and explant types on in vitro propagation and shoot growth of S. rebaudiana were studied, the effect of PGRs on SGs production was determined. For this purpose, nodal explants and shoot tip explants were cultured on woody plant medium (WPM) supplemented with cytokinins [6-benzyladenine (BA), kinetin (Kn), or thidiazuron (TDZ)] or cytokinins + auxins combinations [BA + indoleacetic acid (IAA); BA + naphthaleneacetic acid (NAA); Kn + IAA; Kn + NAA]. Although, the best shoot proliferation was obtained on WPM supplemented with BA + NAA combinations, shoots grown on PGR-containing media produced callus at the base of the shoots and showed chlorosis and necrosis. Additionally, shoots showed at all concentrations of TDZ, and at higher concentrations of BA, morphological changes such as malformed leaves and poor shoot growth. In contrast to PGR-containing media, on the PGR-free control medium, the development of shoots and roots occurred simultaneously and healthy and well-developed plantlets were obtained. Thus, we developed an economical viable means of in vitro propagation by minimizing the micropropagation steps and removing the requirement of PGRs. According to the high-performance liquid chromatography (HPLC) results, PGR-free control medium (WPM) led to considerably higher stevioside content in the leaves compared with the PGR(s)-containing media and the highest stevioside [34 mg·g−1 dry weight (DW)] and rebaudioside A content was only detected on the control medium without PGRs. Steviolbioside, rubusoside, and dulcoside A were detected qualitatively in the leaves of shoots grown on WPM supplemented with 2.27 μm TDZ, 4.54 μm TDZ, 2.22 μm BA + 2.69 μm NAA, 2.22 μm BA + 5.37 μm NAA, 2.32 μm Kn + 5.71 μm IAA, or 2.32 μm Kn + 2.69 μm NAA.

Indirect Organogenesis and Somatic Embryogenesis of Pineapple Induced by Dichlorophenoxy Acetic Acid

&lt;p&gt;This research aimed to study the effect of 2,4-D,&lt;br /&gt;AdS, and basal media to the regeneration of pineapple&lt;br /&gt;through indirect organogenesis and somatic embryogenesis,&lt;br /&gt;and to study the complete event of somatic embryogenesis.&lt;br /&gt;Callus formation was induced by 21, 41, and 62 μM 2,4-D&lt;br /&gt;with addition of 9 μM TDZ. The non embryogenic calli were&lt;br /&gt;transferred onto 4.65 μM Kn containing medium.&lt;br /&gt;Embryogenic callus formation was induced on MS or Bac&lt;br /&gt;basal media consisted of N-organic compounds with&lt;br /&gt;addition of AdS (0, 0.05 and 0.1 μM). The embryogenic calli&lt;br /&gt;were regenerated on modified MS medium with addition of&lt;br /&gt;0.9 μM IBA, 1.1 μM BA, 0.09 μM GA3 or MS medium&lt;br /&gt;supplemented with 0.018 mM BA. The result proved that the&lt;br /&gt;single auxin of 2,4-D was not enough to induce embryogenic&lt;br /&gt;cells. Therefore the non embryogenic calli were regenerated&lt;br /&gt;through organogenesis. The 21 μM 2,4-D yielded high level of&lt;br /&gt;callus formation (80%), higher fresh weight (0.2 g/explant)&lt;br /&gt;and higher number of shoot (25 shoots/explant in two&lt;br /&gt;months). Embryogenic calli were produced on N-organic&lt;br /&gt;compounds enriched media. The regeneration medium&lt;br /&gt;significantly affected the level of browning, where the MS&lt;br /&gt;medium with addition of 0.018 mM BA yielded lower level of&lt;br /&gt;browning. There was an interaction of embryogenic callus&lt;br /&gt;induction medium and regeneration medium to the number&lt;br /&gt;of mature somatic embryos. The embryogenic callus&lt;br /&gt;induction on MS medium enriched with N-organic&lt;br /&gt;compounds and 0.05 μM AdS followed by the regeneration&lt;br /&gt;of somatic embryos on MS medium with addition of 0.018&lt;br /&gt;mM BA was the best treatment which yielded 17 mature&lt;br /&gt;somatic embryos/explant.&lt;/p&gt;

An efficient multiple shoot induction and genetic fidelity assessment of Exacum bicolor Roxb., an endemic and endangered medicinal plant

Exacum bicolor, an endemic and endangered medicinal plant, belongs to the family Gentianaceae. A rapid protocol has been developed for efficient multiple shoot induction by testing nodal explants on Murashige and Skoog (MS) medium supplemented with various cytokinins. The cytokinins 6-benzyladenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyladenine (2-iP) and zeatin (Zn) were used individually and in combination at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 μM). The maximum number of shoots (19.33 ± 1.09 per explant) as well as their fresh weight (5.1 ± 0.68 g) and dry weight (216.83 ± 2.84 mg) were obtained with 10.0 μM BA + 2.0 μM Kn. After 4 wk, the multiple shoots from agar culture were subcultured into liquid medium containing the same growth regulator combinations. After 8 wk of liquid culture, the best treatment had about tenfold increase in shoot number (199.5 ± 1.14 per explant). The fresh weight (13.76 ± 0.14 g) and dry weight (909.33 ± 1.92 mg) were highest with full strength MS medium supplemented with 3% sucrose and containing 10.0 μM BA + 2.0 μM Kn. Maximum root development was observed after 30 d with 0.5 μM indole-3-butyric acid (IBA) supplementation. Regenerated plants were successfully transferred to pots containing coco peat:perlite mixture and showed a 75% survival rate. Genetic fidelity of in vitro plantlets compared to mother plant were assessed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. All the regenerated plants were genetically identical to their mother plant, showing no detectable genetic variation in the regenerated plantlets. Thus, this protocol could be successfully used for mass multiplication and germplasm conservation of E. bicolor.

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation.

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.

Micropropagation of Anacyclus pyrethrum and chemical profiling of the regenerated plants for pellitorine, the active principle

The paper reports the development of a protocol for large-scale micropropagation of Anacyclus pyrethrum, an invaluable medicinal plant, and the presence of pellitorine, the active principle, in leaves and roots of regenerated plants. Three explants—cotyledonary nodes, hypocotyls and cotyledons—were compared for their shoot development/differentiation ability on Murashige and Skoog’s (MS, Physiol Plant 15:473–497, 1962) medium supplemented with 1–10 µM of 6-benzylaminopurine (BAP) or kinetin (Kn). The best response (8.88 ± 0.28 shoots per explant) was elicited from the cotyledonary nodal explants on MS + 2.5 µM Kn. The in vitro regenerated shoots continued to multiply on being sub-cultured on the same medium. Elongated shoots were cultured on MS medium augmented with different concentrations (0.2–15 µM) of 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for induction of roots. The media augmented with 5 or 10 µM IAA or 5 µM NAA were the most effective for the initiation and proliferation of roots. The plantlets were successfully transferred to the following three potting mixtures: garden soil, vermiculite and garden soil (1:1), vermiculite, garden soil and perlite (1:1:1). Among these, the maximum number (75 %) of the transferred plants survived in the garden soil. The high performance liquid chromatographic analyses of roots and leaves of the regenerated plants as well as those from the native habitat revealed the presence of pellitorine in all, with the content being higher in the roots than the corresponding leaves. The developed protocol would be of use for production of A. pyrethrum plants throughout the year, and thus could become a perennial source of the herb and its active principle.

Effect of Physiological Age and Growth Regulators on Micropropagation of Aloe vera Followed by Genetic Stability Assessment

Effect of physiological age and growth regulators on micropropagation of Aloe vera was investigated to develop an efficient protocol for in vitro propagation using shoot bud as explant. Maximum per cent establishment of 72.22 % was observed on MS medium supplemented with 10 µM BA, 5.0 µM Kn and 1.50 µM NAA. The best medium for the multiplication of microshoots was MS medium supplemented with 10 µM BA and 7.5 µM Kn followed by subculturing on same medium for six times at an interval of 4 weeks. Increase in number of microshoots per explant was observed with the increase in subculturing passages. After sixth subculturing 22.70 number of microshoots were obtained from the single explant which was four times more as compared to the first subculturing. The percent root induction was 77.78 % in the sixth subculturing as compared to 36.11 % in the first subculturing on MS medium supplemented with 0.04 % activated charcoal. Genetic stability of the in vitro grown plants was analysed by RAPD which indicated negligible variation.

Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. &amp; Fernandez

Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 μM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88–26.6 μM) was significantly effective on shoot multiplication, while Kn alone (8.88–26.6 μM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. The most suitable combination was 8.88 μM BAP + 8.88 μM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm.