Showing papers by "Andy Ganapathi published in 2014"

Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

Abstract: The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

Abstract: A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N 6 –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA3), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA3 (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.

An efficient in planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting

Abstract: An efficient and reproducible Agrobacterium-mediated in planta transformation was developed in Jatropha curcas. The various factors affecting J. curcas in planta transformation were optimized, including decapitation, Agrobacterium strain, pin-pricking, vacuum infiltration duration and vacuum pressure. Simple vegetative in vivo cleft grafting method was adopted in the multiplication of transformants without the aid of tissue culture. Among the various parameters evaluated, decapitated plants on pin-pricking and vacuum infiltrated at 250 mmHg for 3 min with the Agrobacterium strain EHA 105 harbouring the binary vector pGA 492 was proved to be efficient in all terms with a transformation efficiency of 62.66 %. Transgene integration was evinced by the GUS histochemical analysis, and the GUS positive plants were subjected to grafting. Putatively transformed J. curcas served as "Scion" and the wild type J. curcas plant severed as "Stock". There was no occurrence of graft rejection and the plants were then confirmed by GUS histochemical analysis, polymerase chain reaction (PCR) and Southern hybridization. Genetic stability of the grafted plants was evaluated by using randomly amplified polymorphic DNA (RAPD), marker which showed 100 % genetic stability between mother and grafted plants. Thus, an efficient in planta transformation and grafting based multiplication of J. curcas was established.

Factors influencing podophyllotoxin production in adventitious root culture of Podophyllum hexandrum Royle

Abstract: To determine the possibility of generating the Podophyllotoxin accumulation using Podophyllum hexandrum adventitious roots derived from root segments, several nutrient constituents (carbon sources, media strength, initial medium pH, ammonium and nitrate proportion and phosphate ratio) were evaluated in culture. The maximum biomass accumulation was recorded in 0.50 MS medium containing 3 mg/l indole-3-butyric acid and 2 % sucrose, and the maximum accumulation of Podophyllotoxin was documented in the same strength of MS medium with 6 % of sucrose. When the initial medium pH was on 6 in the optimized MS medium, the biomass and Podophyllotoxin accumulations were highest. The lower concentration of ammonium (10 mM) in combination with a moderate concentration of nitrate (20 mM) was found ideal for maximum accumulations of biomass and Podophyllotoxin. Maximum Podophyllotoxin accumulation (6.4 mg/g dry weight) was recorded at the higher concentration of phosphate (2.25 mM), and lower concentration of phosphate (1.25 mM) showed highest growth accumulation. The outcome of the present work will be helpful for the large-scale cultivation of adventitious root for the production of Podophyllotoxin.

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

Abstract: A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g−1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g−1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g−1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle.

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.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

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.

An efficient hairy root culture system for Withania somnifera (L.) Dunal

Abstract: Withania somnifera is an important aromatic medicinal plant and possesses wide array of pharmacological properties. In the present investigation, an improved version of hairy root culture system was developed by optimizing various transformation parameters such as type of explant, concentration of acetosyringone, Agrobacterium types and co-cultivation period. Between the leaf and cotyledon explants and two Agrobacterium rhizgenes strains (R1000 and A4) tested, leaf explants infected with R1000 and cocultured for five days on MS basal half strength medium in the presence of acetosyringone (100 µM) attained a higher frequency (88%) of hairy root induction. By adopting this protocol, we could utilize the hairy root culture for industrial scale production of withanolides. Key words: Leaf explant, Agrobacterium rhizogenes, Withania somnifera, co-cultivation period, acetosyringone.

Improved production of withanolides in shoot suspension culture of Withania somnifera ( L .) Dunal by seaweed extracts

Abstract: The influence of Gracilaria edulis and Sargassum wightii extracts was investigated for the production of biomass and withanolides in the multiple shoot suspension culture of Withania somnifera. Supplementation of 40 % G. edulis extract in MS liquid medium for 24 h exposure time in the culture recorded the highest biomass accumulation [62.4 g fresh weight and 17.82 g dry weight (DW)] and withanolides production (withanolide A 0.76 mg/g DW; withanolide B 1.66 mg/g DW; withaferin A 2.80 mg/g DW and withanone 2.42 mg/g DW) after 5 weeks of culture, which were 1.45–1.58-fold higher than control culture. This naturally available G. edulis extract-treated multiple shoot suspension culture protocol offers a potential alternative for the optimum production of biomass and withanolides utilizing shake-flasks.

Agrobacterium -mediated genetic transformation of Withania somnifera using nodal explants

Abstract: Withania somnifera is an important medicinal plant and used to cure many diseases. Direct regeneration method was standardized for the nodal explants of W. somnifera. In this method, the maximum of 42.4 ± 2.68 shoots produced per explant was achieved at 1.5 mg l−1 BAP with 0.3 mg l−1 IAA in the second subculture. Transformation was performed in the nodal explants of W. somnifera via direct regeneration using Agrobacterium tumefaciens strain EHA105 that harbored a binary vector pGA492, which carrying kanamycin resistant (nptII), phosphinothricin resistant (bar) and an intron containing β-glucuronidase (gus-intron) genes. The sensitivity of nodal explants to kanamycin (300 mg l−1) was determined for the selection of transformed plants. Transformation was confirmed by histochemical β-glucuronidase (GUS) assay and amplification of the nptII gene by polymerase chain reaction (PCR). PCR and southern blot analyses confirmed the integration of nptII gene in the genome of W. somnifera and the transformation frequency of 3.16 % has been achieved. This is the first report on the genetic transformation of W. somnifera using nodal explants, which may aid in the transformation of any other character gene for improving therapeutic value.

Developing Rapid and Reliable RegenerationSystem Using Cotyledonary-Node Method inIndian Soybean Genotypes (Glycine Max L.)

Abstract: Regeneration through mature cotyledonary node has set rapid regeneration of plants directly from explants which is more time-saving and presented as an effective strategy so we have developed regeneration protocol through single shoot using cotyledonary node a rapid and efficient protocol for three Indian soybean cultivars. Two explants were collected from single cotyledonary node and cultured on medium containing N6-benzylaminopurine (BAP) for germination, BAP and indole-3-butyric acid (IBA) for shoot induction, Gibberellic acid (GA3) for shoot elongation and IBA for rooting of explants. The best combination of hormones for all genotypes were obtained as germination of seeds on half B5 medium supplemented with 1.0 mgl-1 of BAP, shoot induction on full B5 medium having BAP 1.0 mgl-1 and IBA 0.2 mgl-1 and shoot elongation on GA3 0.75 mgl-1 in full MS medium. Under these conditions, the plantlets could be raised within 40-45 days. It was observed that selection of proper medium for regeneration of soybean can overcome genotype associated problems. This regeneration system can be used for soybean transformation

Developing Rapid and Reliable Regeneration System Using Cotyledonary-Node Method in Indian Soybean Genotypes (Glycine Max L.)

Abstract: Regeneration through mature cotyledonary node has set rapid regeneration of plants directly from explants which is more time-saving and presented as an effective strategy so we have developed regeneration protocol through single shoot using cotyledonary node a rapid and efficient protocol for three Indian soybean cultivars. Two explants were collected from single cotyledonary node and cultured on medium containing N 6 -benzylaminopurine (BAP) for germination, BAP and indole-3-butyric acid (IBA) for shoot induction, Gibberellic acid (GA3) for shoot elongation and IBA for rooting of explants. The best combination of hormones for all genotypes were obtained as germination of seeds on half B5 medium supplemented with 1.0 mgl -1 of BAP, shoot induction on full B5 medium having BAP 1.0 mgl -1 and IBA 0.2 mgl -1 and shoot elongation on GA3 0.75 mgl -1 in full MS medium. Under these conditions, the plantlets could be raised within 40-45 days. It was observed that selection of proper medium for regeneration of soybean can overcome genotype associated problems. This regeneration system can be used for soybean transformation. Key word: Soybean genotype, transformation, regeneration, cotyledonary node. Abbreviations: BAP - 6-benzyl-aminopurine, IBA - Indolebutyric acid, GA3 - Gibberellic acid, MS - Murashige and Skoog.