Showing papers on "Kinetin published in 2009"

Characterization of OsIAA1 gene, a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis

Abstract: Aux/IAA and auxin response factor (ARF) are two important families that have been well recognized for their roles in auxin-mediated responses. Aux/IAA proteins are short-lived transcriptional regulators that mediate the auxin responses through interaction with ARF transcription factors. Although quite a few members of the Aux/IAA family have been functionally characterized in dicotyledonous plants such as Arabidopsis, but relatively limited information is available in important crops such as rice. This work focused on isolation and characterization of a member of Aux/IAA family in rice named OsIAA1. The results indicated that OsIAA1 was constitutively expressed in all the tissues and organs investigated. The expression of this gene was induced by various phytohormones including IAA, 2,4-D, kinetin, 24-epibrassinolide, and jasmonic acid. Over-expression of OsIAA1 in rice resulted in reduced inhibition of root elongation to auxin treatment, but increased sensitivity to 24-epiBL treatment. In addition, the OsIAA1-overexpression transgenic plants showed distinctive morphological changes such as decreased plant height and loose plant architecture. Protein interaction analysis suggested that OsIAA1 may act through interaction with OsARF1. T-DNA insertion mutant of OsARF1 showed reduced sensitivity to BR treatment, resembling the phenotype of OsIAA1-overexpression plants. In addition, expression patterns of some genes responsive to brassinosteroid and auxin were changed in the OsIAA1-overexpression plants. These data suggested that OsIAA1 may play important roles in the cross-talk of auxin and brassinosteroid signaling pathways and plant morphogenesis.

Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L.

Abstract: Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l−1 activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m−2 s−1). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m−2 s−1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m−2 s−1. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m−2 s−1) tested. Intercellular CO2 concentration (C i) and the ratio of intercellular CO2 concentration to ambient CO2 (C i/C a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Abstract: Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01-10 microM MeJA, either under root-inductive conditions, i.e., on medium containing 10 microM indole-3-butyric acid (IBA) and 0.1 microM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1-4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 microM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 microM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

Abstract: The major objectives of this study were to investigate an efficient rapid protocol for mass propagation of adventitious shoots of brahmi using semisolid and liquid cultures; and to assess the amount of bacoside A accumulated in the regenerated shoots. Leaf explants were grown in vitro on Murashige and Skoog semisolid medium supplemented with 0.5, 1.0, 1.5, 2.0 and 2.5 mg l−1 6-benzyladenine or kinetin (KN) or thidiazuron (TDZ) for 4 weeks. Adventitious shoots developed from leaf explants on all cytokinin supplemented media. After 4 weeks of incubation, leaf explants were split into two batches and one set was subcultured on semisolid medium and another set in liquid medium containing same concentration of cytokinins where they have come from. Highest rate of shoot regeneration was observed for explants cultured on medium with 2 mg l−1 KN. The fresh and dry weight of shoots was also highest with this treatment. Liquid cultures were found suitable for proliferation of shoots (155.6 shoots per explant) and they also favored highest biomass accumulation (8.60 g fresh and 0.35 g dry biomass). The bacoside A contents were determined in shoots using HPLC. Analysis revealed that, the contents were highest with shoots regenerated on medium supplemented with 2 mg l−1 KN. The amount of bacoside A was highest in the shoots regenerated in liquid medium (11.92 mg g−1 DW) and it was 2.2-fold higher compared to shoots grown on semisolid cultures.

Influence of growth regulators in biomass production and volatile profile of in vitro plantlets of Thymus vulgaris L.

Abstract: In vitro shoots of thyme (Thymus vulgaris L) were established, and the effects of the auxin indole-3-acetic (IAA) acid and the cytokinins benzyladenine (BA), zeatin (ZEA), and kinetin (KIN) at 10, 50, and 100 microM on rooting, biomass production, and volatile compounds production by these plants were investigated The volatiles were extracted by solid phase microextraction (SPME) and analyzed by gas chromatography The highest biomass shoot growth was obtained with BA at 50 microM, while IAA at all concentrations tested achieved 100% rooting frequency The three major compounds were gamma-terpinene (228-388%), p-cymene (138-279%), and thymol (65-290%) Quantitative changes of these compounds were observed in response to the effect of varying growth regulators concentrations in the culture medium Growing Thymus vulgaris L plants in media supplemented with IAA at 10 microM increased volatile compounds such as thymol by 315% Nevertheless, the same major compounds were produced in all treatments and no qualitative changes were observed in the volatile profile of thyme plants

Micropropagation of Swertia chirata Buch.-Hams. ex Wall.: a critically endangered medicinal herb

Abstract: An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA3 (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.

Efficient micropropagation protocol of Spilanthes acmella L. possessing strong antimalarial activity

Abstract: Micropropagation has been achieved in a promising larvicidal asteraceous taxon Spilanthes acmella L. using seedling leaf explants. The explants were reared on a variety of growth regulators, namely 2,4-dichlorophenoxyacetic acid, 1-naphthalene acetic acid, Indole-3-butyric acid, N6-benzyladenine, and kinetin either alone or in combination on Murashige and Skoog’s (MS) medium. The best green and compact callus was obtained on 1 μM NAA and 10 μM benzyladenine (BA) in 15 d. The callus on subculture to the same but fresh medium after every 30 d differentiated an average of 12.90 ± 0.32 shoot buds in 50% cultures. Elongation in shoot buds occurred only if they were transferred to NAA lacking MS+BA medium. An average number of 4.22 ± 0.83 shoots and 15 ± 0.84 shoot buds per explant were obtained in 70.3% cultures on MS + 10 μM BA in 30 d. One hundred percent excised shoots rooted in MS(1/2) + 0.1 μM IBA within 2 wk. The plants were gradually hardened and established in soil where they flowered and set viable seeds. The regenerated plants were morphologically similar to the field grown plants and showed 100% larvicidal activity against malaria and filarial vectors.

In vitro organogenesis and antioxidant enzymes activity in Acanthophyllum sordidum

Abstract: The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 µM 2,4-dichlorophenoxyacetic acid + 4.65 µM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 µM 1-naphthalene acetic acid (NAA), 2.69 µM NAA + 4.54 µM thidiazuron and 2.46 µM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.

Direct plant regeneration from nodal explants of Balanites aegyptiaca L. (Del.): a valuable medicinal tree

Abstract: This report describes in vitro shoot induction and plant regeneration from nodal segments of Balanites aegyptiaca on Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA), thidiazuron (TDZ) and kinetin (Kin) (0.5–20.0 μM). MS medium supplemented with BA (12.5 μM) was the most effective in inducing bud break and growth and also in initiating multiple shoot proliferation. However, the optimal level of TDZ supplementation to the culture medium was 5.0 μM. Shoot cultures were established by repeatedly subculturing the original nodal explants on the same medium. Highest number of shoots (11.5 ± 0.7) and shoot length (5.0 ± 0.2 cm) were achieved when cultures were subcultured on MS medium supplemented with 12.5 μM BA and 1.0 μM α-naphthalene acetic acid (NAA). The shoots regenerated from TDZ supplemented medium when subcultured to hormone free MS basal medium considerably increased the rate of shoot multiplication and shoot length by the end of fifth subculture. Rooting of the shoots was achieved on MS medium augmented with 1.0 μM indole-3-butyric acid (IBA) plus 0.5% activated charcoal followed by their transfer to half strength MS basal medium. The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 70% survival rate. The results of this study provide the first successful report on in vitro direct plant regeneration of B. aegyptiaca.

In vitro manipulations in St. John’s wort ( Hypericum perforatum L.) for incessant and scale up micropropagation using adventitious roots in liquid medium and assessment of clonal fidelity using RAPD analysis

Abstract: The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l−1 Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l−1 IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l−1) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Abstract: Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l−1 NAA, 0.2 mg l−1 kinetin and casein hydrolysate 250 mg l−1. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l−1 yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l−1) by the 15th day.

Exogenous application of plant growth regulators increased the total flavonoid content in Taraxacum officinale Wigg

Abstract: The effects of plant growth regulators (PGRs) were studied on growth, total flavonoid, gibberellins (GA) and salicylic acid (SA) contents of Taraxacum officinale (dandelion), a widely used medicinal plant in Korea. All the four PGRs used; gibberellic acid (GA3), kinetin (Kn), salicylic acid (SA) and ethephon (2- chloroethylphosphonic acid) were applied at the rates of 0.5 and 1.0 mM. GA3 markedly enhanced fresh shoot weight, while 0.5 mM of kinetin application significantly enhanced dry root mass as compared to control. SA enhanced both shoot and root attributes, while ethephon decreased plant growth. Endogenous bioactive GA1 and GA4 content and SA content enhanced with the application of GA3, SA and kinetin, but declined with ethephon. The flavonoid content of dandelion significantly increased with SA treatment, but was not altered with the application of other PGRs. The current study demonstrated the favorable effect of GA3, kinetin and SA on growth, bioactive GAs, SA and flavonoid contents of dandelion. These investigations offered interesting information as PGRs were never tested for plant growth and development of dandelion. It also reports the presence of both early C-13 hydroxylation and non C-13 hydroxylation pathways of GA biosynthesis in dandelion for the first time.

Phenylpropanoid production in callus and cell suspension cultures of Buddleja cordata Kunth

Abstract: Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g−1 dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g−1 DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g−1 DW and 8.12 mg g−1 DW, respectively).

A micropropagation system for cloning of hemp (Cannabis Sativa L.) by shoot tip culture

Abstract: This study describes the standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from shoot tips of Hemp (Cannabis sativa L.). Hemp seedlings were germinated on half-strength 1/2 MS medium supplemented with 10 g·L -1 sucrose, 5.5 g·L -1 agar at a pH of 6.8 under light for 16 h per day. MS medium containing 0.2 mg·L -1 TDZ, 0.1 mg·L -1 NAA supported the maximal auxiliary bud multiplication rate of 3.22 per shoot tip. The proliferated buds were successfully rooted on MS medium supplemented with 0.1 mg·L -1 IBA and 0.05 mg·L -1 NAA resulting in 85% of the plantlets rooting. The procedure requires a 54 days cycle for the In vitro clonal propagation (14 days for shoot multiplication and 40 days for root induction) which includes 35-42 days for acclimatized plantlet production.

Role of phytohormones and nitrogen in somatic embryogenesis induction in cell culture derived from leaflets of Azadirachta indica

Abstract: A protocol for somatic embryogenesis in Azadirachta indica A Juss. has been standardized using in vivo leaflets. Experiments were carried out to examine the effect of various auxins, cytokinins, sucrose, inorganic and organic salts on subsequent somatic embryo induction and maturation. Embryogenic calli were induced on Murashige and Skoog (MS) medium supplemented with 1.5 mg dm−3 kinetin and 1.5 mg dm−3 indole-3-acetic acid and subsequently all the stages of somatic embryo development (globular, cordate, torpedo and cotyledonary) were observed. Maturation of these embryos was accomplished with the same growth regulators after three subcultures. The histological study of somatic embryos showed resembles to zygotic embryos. The matured somatic embryos were transferred onto half strength MS-medium devoid of growth regulators for their germination (82 %). Plantlets were acclimatized in the field with a survival rate of 80–83.5 %.

Shoot multiplication and plant regeneration of guava (Psidium guajava L.) from nodal explants of in vitro raised plantlets.

Abstract: A micropropagation method is described for guava (Psidium guajava L.) using nodal explants from somatic embryo-derived young and aseptic plantlets. Multiple shoots were induced from axillary buds on MS medium containing different concentrations of N 6 -benzylaminopurine (BAP), either alone or in combination with kinetin (Kn), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Medium containing 1 mg 1 -1 BAP was the most effective for shoot multiplication. In vitro regenerated shoots developed roots either on MS medium alone or on MS medium supplemented with indole-3-butyric acid (IBA). The rooted plantlets were successfully acclimatized.

Callus induction, biomass growth, and plant regeneration in Digitalis lanata Ehrh.: influence of plant growth regulators and carbohydrates

Abstract: The effect of plant growth regulators (PGRs) and carbohydrate sources on callus induction, callus growth, and plant regeneration in foxglove was examined. Explants were transferred onto MS medium with various levels of PGRs and carbohydrates to determine the optimum explant and effective combinations of PGR treatment. For callus induction 6.0 mg L -1 of α-naphthalene acetic acid (NAA) and 3.0 mg L -1 of benzyl-aminopurine (BA) were very responsive. Addition of cytokinins (BA and kinetin) at 0.5-3.0 mg L -1 to media containing NAA enhanced callus growth. Shoot regeneration was best achieved in MS + 6.0 mg L -1 of BA. Adenine sulphate (Ade) and casein hydrolysate (Ch) were added to the medium as a nitrogen source to improve plant growth and maximum growth was obtained on medium supplemented with 1.5 mg L -1 of kinetin + 0.5 mg L -1 of IAA + 500 mg L -1 of Ch. Carbohydrates also influenced callus production and shoot regeneration potentiality. Among all the tested carbohydrates (sucrose, maltose, fructose, and glucose) and concentrations (3.0-6.0 g L -1 ), the optimum carbohydrate concentration was 3.0 g L -1 and was applied to all carbohydrate cases.

An improved micropropagation of Spilanthes acmella L. through transverse thin cell layer culture

Abstract: An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm−3 BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

Abstract: This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.

Kinetin in familial dysautonomia carriers: implications for a new therapeutic strategy targeting mRNA splicing.

Abstract: Familial dysautonomia (FD) is caused by an intronic splice mutation in the IκB kinase–associated protein gene (IKBKAP) that leads to partial skipping of exon 20 and tissue-specific reduction of IκB kinase–associated protein/elongator protein 1 (IKAP/ELP-1 protein). Kinetin increases IKBKAP mRNA and protein expression in FD cell lines. To determine whether oral kinetin alters IKBKAP splicing in vivo, we administered kinetin to 29 healthy carriers of the major FD mutation for 8 d. Adverse effects, kinetin, and IKBKAP mRNA levels were monitored. In the highest dosing cohorts (23.5 mg/kg/d), the target plasma kinetin level was achieved in 91% of subjects at 2 h. After 8 d, IKBKAP mRNA expression in leukocytes increased as kinetin levels increased. There is a linear association between log plasma kinetin level and corresponding log change from baseline in IKBKAP mRNA expression that allows estimation of IKBKAP mRNA levels because of kinetin ingestion. Adverse effects were transient and mild. This is the first report of in vivo IKBKAP splicing modification and strongly suggests kinetin's therapeutic potential in FD and perhaps in other splicing disorders. Furthermore, our findings support our hypothesis that treatments, which target a particular splicing mutation, can be successfully developed.

Mass production of an economically important medicinal plant Stevia rebaudiana using in vitro propagation techniques

Abstract: Experiments were conducted for standardization of in vitro culture technique ofstevia rebaudiana, an important non-caloric sweetening herb to explore its potential for micro-propagation and callus culture Nodal segments of the selected herb as explants were cultured for micro-propagation on MS medium containing 01 mg/l N6-benzyl amino purine for shoot initiation Maximum plantlets (832 ± 0445033) were found in MS medium treated with 35 mg/l N6-benzyl amino purine at multiplication stage Young leaves were placed on MS medium containing 2 mg/l 2-4- Dichlorophenoxyacetic acid +1 mg/1 Kinetin was given best result of callusing Higher regeneration of plantlets (38 plantlets/calli) was obtained by placing callus on MS medium with 5 mg/l BA + 1 mg/l NA Highest rooting average (111 ± 0264052) was recorded on 1/2MS medium with 100 mg/l activated charcoal The rooted plantlets were hardened in 1:1:1 ratio of sand: soil: vermicompost and successfully established in soil Key words: Stevia rebaudiana, in vitro propagation, regeneration of callus, non-caloric sweetener, medicinal plant, N6-benzyl amino purine, 2, 4-Dichlorophenoxyacetic acid

Effect of growth regulators on meristem tip development and in vitro multiplication of potato cultivar ‘Kufri Himalini’

Abstract: In the present study meristem tips of potato (Solanum tuberosum) were cultured on Murashige and Skoog (MS) medium, supplemented with different hormonal combinations i.e. MSGN1 (0.25 mg/l GA3 and 0.01 mg/l NAA), MSGN2 (0.25 mg/l GA3 and 0.03 mg/l NAA), MSGN3 (0.25 mg/l GA3 and 0.04 mg/l NAA), MSKN1 (0.01 mg/l Kinetin and 0.1 mg/l NAA), MSKN2 (0.001 mg/l Kinetin and 0.1 mg/l NAA) and MSKN3 (1 mg/l Kinetin and 0.1 mg/l NAA), which affected in vitro propagation of potato. After 35-40 days of culture shoot height, number of nods, root length, shoot and root fresh weight were measured. Shoot height in M.S. medium with GA3 and NAA combination showed better result in comparison to M.S. medium with Kinetin and NAA. Shoot height in MSGN1 combination reached 8.28 (±0.5) cm. with 11.9 (±1.1) cm. root length and 9.4 (±1.0) nodes while in MSKN1 shoot height reached 6.4 cm. (±0.6) with 8.2 (±0.5) cm. root length and 5.0 (±0.7) nods. MSKN2 and MSKN3 reached low shoot height respectively 5.3 cm. (±1.2) with 4.2 (±0.8) nods and 4.0 cm. (±0.6) with 2.7 (±0.7) nods in comparison to all combinations. MSGN2 and MSGN3 combinations reached respectively 7.15 (±0.5) cm. with 8.2 (±1.0) nodes and 6.15 (±0.6) cm. with 6.3 (±0.9) nodes. Result showed that lower concentration of auxin (0.01 mg/l NAA) with Gibberelic Acid (0.25 mg/l GA3) is best for development of complete plantlets and multiplication from meristem tips. [Nature and Science, 2009;7(9):31-34]

Effect of growth regulators on microspore embryogenesis in coconut anthers

Abstract: The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y3 medium containing 66 μM 2,4-D), followed by maturation medium (Y3 medium without growth regulators) and germination medium (Y3 medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA3), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).

Micropropagation of Pueraria tuberosa (Roxb. Ex Willd.) and determination of puerarin content in different tissues

Abstract: Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring conservation In this study, fresh shoot sprouts of P tuberosa, produced by tubers, were used as explants for in vitro micropropagation Surface-sterilized nodal shoots were incubated on Murashige and Skoog (MS) medium supplemented with 888 μM benzyladenine (BA), 50 mg l−1 ascorbic acid, and 25 mg l−1 of each of citric acid and adenine sulphate Cut ends of nodal stem segments rapidly turned brown, and cultures failed to establish When 100 mg l−1 ascorbic acid (ABA) and 250 mg l−1 polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established Bud-breaking of nodal stem explants resulted in multiple shoot formation Shoots proliferated (35–40 shoots per culture vessel) on MS medium as described above, but supplemented with 444 μM BA and 057 μM indole acetic acid (IAA) and additives After 4–5 passages, proliferating shoots exhibited tip-browning and decline in growth and multiplication However, when shoots were transferred to fresh shoot proliferation medium supplemented with 232 μM kinetin (Kn), sustained growth and high rate of shoot proliferation (50–60 shoots per culture vessel) was observed Shoots rooted when transferred to medium consisting of half- strength MS medium with 984 μM indole butyric acid (IBA) and 002% activated charcoal Alternatively, individual shoots were pulsed with 9840 μM IBA and transferred to glass bottles containing sterile and moistened soilrite These shoots rooted ex-vitro and were acclimatized in the greenhouse Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin

Rapid clonal multiplication through in vitro axillary shoot proliferation of Centella asiatica L.

Abstract: Single nodal explants isolated from field grown plants of Centella asiatica, an important medicinal plant, when cultured for 4 wks on Murashige and Skoog’s (MS) medium containing different concentrations and combinations of BAP and Kn produced multiple shoots. A maximum of 15.24 shoots/node were produced after 30 d of culture in the presence of 2.0 mg/L BAP. Individual shoots (2-5 cm), when transferred onto full strength MS medium containing 1.5 mg/L IBA induced maximum number of roots (12.6). The rooted plants were successfully established in greenhouse condition after hardening.