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1

Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants

100%
Dandin V. S., Murthy H. N.
Acta Physiologiae Plantarum
|
2012
|
tom 34
|
nr 4
Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anticancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventitious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0 µM 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0 µM BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4 µM) and IAA (5.7 µM). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40 µmol m⁻² s⁻¹). The camptothecin content was determined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a considerable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction.
2

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

100%
Naik P. M., Murthy H. N.
Acta Physiologiae Plantarum
|
2010
|
tom 32
|
nr 1
Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 µM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 µM 2,4-D and 0.5 µM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 µM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 µM abscisic acid.
3

Withanolide A production from Withania somnifera hairy root cultures with improved growth by altering the concentrations of macro elements and nitrogen source in the medium

100%
Praveen N., Murthy H. N.
Acta Physiologiae Plantarum
|
2013
|
tom 35
|
nr 03
Withania somnifera is an important medicinal plant that contains withanolides as bioactive compounds. We have investigated the effects of macroelements and nitrogen source in hairy roots of W. somnifera with the aim of optimizing the production of biomass and withanolide A content. The effects of the macroelements NH4NO3, KNO3, CaCl2, MgSO4 and KH2PO4 at concentrations of 0, 0.5, 1.0, 1.5 and 2.09 strengths and of nitrogen source [NH4+/NO3- (0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/ 18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 mM)] in Murashige and Skoog medium were evaluated for biomass and withanolide A production. The highest accumulation of biomass (139.42 g 1-1 FW and 13.11 g 1-1 DW) was recorded in the medium with 2.09 concentration of KH2PO4, and the highest production of withanolide A was recorded with 2.09 KNO3 (15.27 mg g-1 DW). The NH4+/NO3- ratio also influenced root growth and withanolide A production, with both parameters being larger when the NO3- concentration was higher than that of NH4+. Maximum biomass growth (148.17 g 1-1 FW and 14.79 g 1-1 DW) was achieved at NH4+/NO3- ratio of 14.38/37.60 mM, while withanolide A production was greatest (14.68 mg g-1DW) when the NH4+/NO3 - ratio was 0.00/18.80 mM. The results are useful for the large scale cultivation of Withania hairy root culture for the production of withanolide A.
4

Production of withanolide-A from adventitious root cultures of Withania somnifera

100%
Praveen N., Murthy H. N.
Acta Physiologiae Plantarum
|
2010
|
tom 32
|
nr 5
Withanolides are biologically active secondary metabolites present in roots and leaves of Withania somnifera. In the present study, we have induced adventitious roots from leaf explants of W. somnifera for the production of withanolide-A, which is having pharmacological activities. Adventitious roots were induced directly from leaf segments of W. somnifera on half strength Murashige and Skoog (MS) semisolid medium (0.8% agar) with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) and 30 g l⁻¹ sucrose. Adventitious roots cultured in flasks using half strength MS liquid medium with 0.5 mg l⁻¹ IBA and 30 g l⁻¹ showed higher accumulation of biomass (108.48 g l⁻¹ FW and 10.76 g l⁻¹ DW) and withanolide-A content (8.8 ± 0.20 mg g⁻¹ DW) within five weeks. Nearly 11-fold increment of fresh biomass was evident in suspension cultures and adventitious root biomass produced in suspension cultures possessed 21-fold higher withanolide-A content when compared with the leaves of natural plants. An inoculum size of 10 g l⁻¹ FW favoured the biomass accumulation and withanolide-A production in the tested range of 2.5, 5.0, 10.0 and 20.0 g l⁻¹ FW. Among different media tested [Murashige and Skoog (MS), Gamborg’s (B5), Nitsch and Nitsch (NN) and Chu’s (N6)], MS medium favoured both biomass accumulation and withanolide-A production. Half strength MS medium favoured the biomass accumulation and withanolide-A production among the different strength MS medium tested (0.25, 0.5, 0.75, 1.0, 1.5 and 2.0). The current results showed great potentiality of adventitious roots cultures for the production of withanolide-A.
5

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of Bacopa monnieri (L.)

81%
Naik P. M., Manohar S. H., Murthy H. N.
Acta Physiologiae Plantarum
|
2011
|
tom 33
|
nr 4
The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄) and nitrogen source [NH₄⁺/NO₃⁻] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant⁻¹), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.09 strength of NH₄NO₃. The highest production of bacoside A content was also recorded in the medium of 2.09 NH₄NO₃, which produced 17.935 mg g⁻¹ DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO₃⁻ concentration was higher than that of NH₄⁺. Maximum number of shoots (70.00 shoots explant⁻¹), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g⁻¹ DW) were obtained at NH₄⁺/NO₃⁻ ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0 × NH₄NO₃ is recommended for most efficient bacoside A production.
6

Establisment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites

81%
Wu C.-H., Murthy H. N., Hahn E.-J., Paek K.-Y.
Acta Physiologiae Plantarum
|
2008
|
tom 30
|
nr 6
We have successfully established the co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions (5 g L⁻¹; 4:1, 3:2 and 2:1 ginseng and echinacea, respectively) in 5-L capacity airlift bioreactors containing 4 L Murashige and Skoog medium supplemented with 25 μM indole-3-butyric acid and 50 g sucrose L⁻¹ and maintained at 25C in the dark for 40 days. Results showed the negative effect of echinacea adventitious roots on the growth of ginseng roots, however, by limiting the inoculum density of echinacea, it was possible to establish the cocultures. To enhance the accumulation of secondary metabolites, co-cultures were treated with 200 μM methyl jasmonate after 30 days of culture initiation. Methyl jasmonate elicitation promoted the accumulation of ginsenosides in the co-cultures. It was possible to produce ginsenosides and caffeic acid derivatives in higher amounts by establishing co-cultures with higher inoculum proportion of ginseng to echinacea (4:1 and 3:2) followed by elicitation treatment. This work demonstrates the effectiveness of interspecies adventitious root co-cultures for the production of plant secondary metabolites.
7

The effect of light quality on the growth and development of in vitro cultured Doritaenopsis plants

71%
Shin K. S., Murthy H. N., Heo J. W., Hahn E. J., Paek K. Y.
Acta Physiologiae Plantarum
|
2008
|
tom 30
|
nr 3
The influence of light quality on growth and development of in vitro grown Doritaenopsis hort. (Orchidaceae) plants was investigated. Growth parameters like leaf and root fresh/dry mass and leaf area were highest with plants grown under red plus blue light emitting diodes (LEDs). Leaf length was greater with the plants grown under red LED. Carbohydrate (starch, sucrose, glucose and fructose) and leaf pigment (chlorophylls and carotenoids) biosynthesis of the plants was significantly increased in plants grown under red plus blue LEDs compared to red or blue LED and fluorescent light treatments. This study suggests that the production of quality Doritaenopsis plants is possible by culturing the plants in vitro under a mixture of blue plus red light sources.
8

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

71%
Praveen N., Naik P. M., Manohar S. H., Nayeem A., Murthy H. N.
Acta Physiologiae Plantarum
|
2009
|
tom 31
|
nr 4
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.
9

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

71%
Nagella P., Thiruvengadam M., Jung S. J., Murthy H. N., Chung I. M.
Acta Physiologiae Plantarum
|
2013
|
tom 35
|
nr 10
Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l-1 FM and 10.92 g l-1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g-1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MSbased liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.
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