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1

Effect of nitrogen and carbon sources on in vitro shoot multiplication, root induction and withanolides content in Withania somnifera (L.) Dunal

100%
Sivanandhan G., Selvaraj N., Ganapathi A., Manickavasagam M.
Acta Physiologiae Plantarum
|
2015
|
tom 37
|
nr 02
Effect of nitrogen and carbon sources on multiple shoot regeneration and withanolides contents of Withania somnifera were evaluated. Inclusion of L-glutamine (20 mg/l) in medium fortified with the optimal levels of 1.5 mg/l 6-benzyladenine (BA) and 0.3 mg/l indole-3-acetic acid (IAA) resulted in the production of 58 shoots/explant. Sucrose at 4 % and 20 mg/l L-glutamine with ide ´al concentrations of BA and IAA improved shoot multiplication (62 shoots/explant) while 6 % sucrose enhanced withanolides contents in regenerated multiple shoots. All the shoots were rooted (26 roots/shoot) when cultured in MS medium amended with 15 mg/l ammonium nitrate, 2 mg/l IBA, and 2 % sucrose. The present study resulted in the production of 4.42-fold higher multiple shoots and 2.6- fold higher roots as well as enhancing the contents of all major withanolides (withaferin A, withanone, withanolide A and B) in regenerated plants when compared to previous reports on W. somnifera regeneration.
2

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

64%
Rajesh M., Sivanandhan G., Arun M., Vasudevan V., Theboral J., Girija S., Manickavasagam M., Selvaraj N., Ganapathi A.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 04
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.
3

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

64%
Mariashibu T. S., Subramanyam K., Arun M., Mayavan S., Rajesh M., Theboral J., Manickavasagam M., Ganapathi A.
Acta Physiologiae Plantarum
|
2013
|
tom 35
|
nr 01
For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacteriummediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l-1 citric acid, 100 lM acetosyringone, and 100 mg l-1 L-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l-1 hygromycin and the embryos were germinated in basal medium containing 20 mg l-1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacteriummediated transformation protocol for stable integration of foreign genes into soybean has been developed.
4

Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture

64%
Sivanandhan G., Rajesh M., Arun M., Jeyaraj M., Dev G. K., Arjunan A., Manickavasagam M., Muthuselvam M., Selvaraj N., Ganapathi A.
Acta Physiologiae Plantarum
|
2013
|
tom 35
|
nr 03
The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g 1-1 FW) with SA at 100 μM in the presence of 0.6 mg 1-1 BA and 20 mg 1-1 spermidine for 4 h exposure time at the 4th week in 20 m1 liquid medium recorded higher withanolides production (withanolides A [8.48 mg g-1 DW], withanolides B [15.47 mg g-1 DW], withaferin A [29.55 mg g-1 DW] and withanone [23.44 mg g-1 DW]), which were 1.14 to 1.18- fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.
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