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1.
Protoplasma ; 251(5): 1231-43, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24633328

ABSTRACT

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.


Subject(s)
Plant Somatic Embryogenesis Techniques , Podophyllotoxin/biosynthesis , Podophyllum/embryology , Podophyllum/metabolism , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Endangered Species , Plant Shoots , Regeneration , Seeds/growth & development
2.
Appl Biochem Biotechnol ; 172(4): 1763-76, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24258793

ABSTRACT

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.


Subject(s)
Gene Expression Regulation, Plant/genetics , Glycine max/enzymology , Glycine max/metabolism , Methyltransferases/metabolism , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/metabolism , Tocopherols/metabolism , Methyltransferases/genetics , Plants, Genetically Modified/genetics , Promoter Regions, Genetic/genetics , Glycine max/genetics
3.
Indian J Exp Biol ; 51(10): 849-59, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24266110

ABSTRACT

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 microM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 microM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 microM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.


Subject(s)
Glycine max/embryology , Plant Somatic Embryogenesis Techniques/methods , Acclimatization/drug effects , Acclimatization/physiology , Amino Acids/pharmacology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cotyledon/drug effects , Cotyledon/growth & development , Cotyledon/physiology , Desiccation , Germination/drug effects , Germination/physiology , Plant Growth Regulators/pharmacology , Glycine max/drug effects , Glycine max/growth & development , Glycine max/physiology
4.
Appl Biochem Biotechnol ; 171(2): 450-68, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23852797

ABSTRACT

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 µM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).


Subject(s)
Agrobacterium/genetics , Genetic Engineering/methods , Seeds/genetics , Solanum melongena/genetics , Transformation, Genetic , Acetophenones/pharmacology , Coculture Techniques , Genotype , Germination , Glucuronidase/genetics , Glucuronidase/metabolism , Seeds/growth & development , Solanum melongena/drug effects , Solanum melongena/enzymology , Solanum melongena/growth & development , Sonication , Surface-Active Agents/pharmacology , Time Factors , Transformation, Genetic/drug effects , Transgenes/genetics , Vacuum
5.
Plant Cell Rep ; 32(10): 1557-74, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23749098

ABSTRACT

KEY MESSAGE: An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant. Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA(®) and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.


Subject(s)
Agrobacterium tumefaciens , Genetic Engineering/methods , Saccharum/genetics , Seeds/genetics , Acetophenones/chemistry , DNA, Plant/genetics , Gene Transfer Techniques , Genes, Reporter , Genotype , Plants, Genetically Modified/genetics , Sonication , Surface-Active Agents/chemistry , Transformation, Genetic
6.
Colloids Surf B Biointerfaces ; 102: 708-17, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23117153

ABSTRACT

Now-a-days synthesis and characterization of silver nanoparticles (AgNPs) through biological entity is quite interesting to employ AgNPs for various biomedical applications in general and treatment of cancer in particular. This paper presents the green synthesis of AgNPs using leaf extract of Podophyllum hexandrum Royle and optimized with various parameters such as pH, temperature, reaction time, volume of extract and metal ion concentration for synthesis of AgNPs. TEM, XRD and FTIR were adopted for characterization. The synthesized nanoparticles were found to be spherical shaped with average size of 14 nm. Effects of AgNPs were analyzed against human cervical carcinoma cells by MTT Assay, quantification of ROS, RT-PCR and western blotting techniques. The overall result indicates that AgNPs can selectively inhibit the cellular mechanism of HeLa by DNA damage and caspase mediated cell death. This biological procedure for synthesis of AgNPs and selective inhibition of cancerous cells gives an alternative avenue to treat human cancer effectively.


Subject(s)
Caspases/metabolism , Plant Extracts/chemistry , Podophyllum/chemistry , Silver/chemistry , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , DNA Damage/drug effects , Female , Humans , Hydrogen-Ion Concentration , Metal Nanoparticles/adverse effects , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Polymerase Chain Reaction , Spectroscopy, Fourier Transform Infrared
7.
Protoplasma ; 250(4): 885-98, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23247920

ABSTRACT

Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.


Subject(s)
Biomass , Cell Culture Techniques/methods , Withania/cytology , Withania/growth & development , Withanolides/metabolism , Carbon/metabolism , Cells, Cultured , Culture Media , Withania/metabolism
8.
Nat Prod Commun ; 7(10): 1271-2, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23156987

ABSTRACT

This study optimized carbon sources in half MS liquid medium for maximum biomass accumulation and withanolides production in hairy root culture of Withania somnifera. The highest production of withaferin A and withanone was achieved when sucrose and sucrose+glucose were used individually as carbon sources. The hairy root suspension culture supplemented with a lower level of sucrose (2%) favored hairy root biomass accumulation (1.41 g DW) followed by sucrose+glucose (2+1) when compared with other carbon sources in half MS liquid medium after 40 days of culture. The hairy roots grown on sucrose (4%) enriched half MS liquid medium stimulated higher production of withaferin A (2.21 mg/g DW) and withanone (2.41 mg/g DW) on the 40th day of culture, followed by sucrose+glucose (4+1%) compared with glucose, fructose, maltose and other combinations tested.


Subject(s)
Antineoplastic Agents, Phytogenic/biosynthesis , Plant Roots/growth & development , Plant Roots/metabolism , Triterpenes/metabolism , Withania/chemistry , Withanolides/metabolism , Biomass , Carbohydrates/pharmacology , Carbon/metabolism , Chromatography, High Pressure Liquid , Culture Media , Sucrose/metabolism , Triterpenes/chemistry , Withanolides/chemistry
9.
Planta ; 236(6): 1909-25, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22936305

ABSTRACT

Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T(1)) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T(1) soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T(1) soybean plants maintained higher net photosynthetic rate, CO(2) assimilation, and stomatal conductance than NT plants. The three T(1) soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean--Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T(1) soybean plants produced 32-35 soybean pods/plant containing 10.3-12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.


Subject(s)
Antifungal Agents/metabolism , Glycine max/physiology , Nicotiana/genetics , Plant Diseases/immunology , Plant Proteins/metabolism , Ascomycota/physiology , Basidiomycota/physiology , Carbon Dioxide/metabolism , Chlorophyll/metabolism , Disease Resistance , Gene Expression Regulation, Plant , Germination , Photosynthesis , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/physiology , Plant Proteins/genetics , Plant Somatic Embryogenesis Techniques , Plant Stomata/genetics , Plant Stomata/immunology , Plant Stomata/physiology , Plant Transpiration , Plants, Genetically Modified , Proline/metabolism , Salinity , Salt Tolerance , Seeds/genetics , Seeds/immunology , Seeds/physiology , Sodium Chloride/pharmacology , Glycine max/genetics , Glycine max/microbiology , Stress, Physiological , Nicotiana/metabolism
10.
Appl Biochem Biotechnol ; 168(3): 681-96, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22843063

ABSTRACT

Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70 g FW) on 30-day-old adventitious root cultures with 150 µM SA for 4 h elicitor exposure period resulted in the production of 64.65 mg g(-l) dry weight (DW) withanolide A (48-fold), 33.74 mg g(-l) DW withanolide B (29-fold), 17.47 mg g(-l) DW withaferin A (20-fold), 42.88 mg g(-l) DW withanone (37-fold), 5.34 mg g(-l) DW 12-deoxy withastramonolide (nine fold), 7.23 mg g(-l) DW withanoside V (seven fold), and 9.45 mg g(-l) DW withanoside IV (nine fold) after 10 days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.


Subject(s)
Acetates/metabolism , Cell Culture Techniques/methods , Cyclopentanes/metabolism , Oxylipins/metabolism , Plant Roots/metabolism , Salicylic Acid/metabolism , Withania/metabolism , Withanolides/metabolism , Withania/growth & development
11.
Int J Mol Sci ; 10(5): 2367-2382, 2009 May 20.
Article in English | MEDLINE | ID: mdl-19564954

ABSTRACT

Withania somnifera is an important medicinal plant, which is used in traditional medicine to cure many diseases. Flavonoids were determined in the extracts of W. somnifera root (WSREt) and leaf (WSLEt). The amounts of total flavonoids found in WSREt and WSLEt were 530 and 520 mg/100 g dry weight (DW), respectively. Hypoglycaemic and hypolipidaemic effects of WSREt and WSLEt were also investigated in alloxan-induced diabetic rats. WSREt and WSLEt and the standard drug glibenclamide were orally administered daily to diabetic rats for eight weeks. After the treatment period, urine sugar, blood glucose, haemoglobin (Hb), glycosylated haemoglobin (HbA1C), liver glycogen, serum and tissues lipids, serum and tissues proteins, liver glucose-6-phosphatase (G6P) and serum enzymes like aspartate transaminase (AST), alanine transaminase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) levels were determined. The levels of urine sugar, blood glucose, HbA1C, G6P, AST, ALT, ACP, ALP, serum lipids except high density lipoprotein-bound cholesterol (HDL-c) and tissues like liver, kidney and heart lipids were significantly (p < 0.05) increased, however Hb, total protein, albumin, albumin:globulin (A:G) ratio, tissues protein and glycogen were significantly (p < 0.05) decreased in alloxan-induced diabetic rats. Treatment of the diabetic rats with WSREt, WSLEt and glibenclamide restored the changes of the above parameters to their normal level after eight weeks of treatment, indicating that WSREt and WSLEt possess hypoglycaemic and hypolipidaemic activities in alloxan-induced diabetes mellitus (DM) rats.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Glyburide/pharmacology , Liver/drug effects , Plant Extracts/pharmacology , Withania/metabolism , Alloxan , Animals , Blood Glucose/drug effects , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/chemically induced , Glycosuria/metabolism , Hemoglobins/drug effects , Hypoglycemia/chemically induced , Hypoglycemic Agents/pharmacology , Hypolipidemic Agents/pharmacology , Hypolipoproteinemias/chemically induced , Lipids/blood , Liver/enzymology , Liver Glycogen/metabolism , Male , Plant Leaves/metabolism , Plant Roots/metabolism , Rats , Rats, Wistar
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