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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

RNAi-mediated resistance to yellow mosaic viruses in soybean targeting coat protein gene

76%
Kumari A., Hada A., Subramanyam K., Theboral J., Misra S., Ganapathi A., Malathi V. G.
Acta Physiologiae Plantarum
|
2018
|
tom 40
|
nr 02
In this genomic era, soybean has entrenched genomic database which offer an extensive scope for improvement through genetic manipulation, although demand for transgenics soybean with better production and enhanced quality has been handicapped due to Mungbean yellow mosaic India virus (MYMIV) belonging to the genus Begomovirus. MYMIV is a causative agent of yellow mosaic disease that has been emerged as a threat to the cultivation of bean family in India. In this study, transgenic soybean plants were generated using the intron-spliced hairpin construct encoding the coat protein sequence of MYMIV in the control of 35S promoter and ocs terminator. Integration of coat protein gene in independently transformed plants was confirmed by PCR and Southern hybridization where one transgenic line of coat protein-event A, two transgenic lines of coat protein-event B, and two transgenic lines from the coat protein-event C showed gene hybridization. Inoculation was performed on T₁ seedlings of transgenic and non-transgenic plants where the viral replicative DNA level was assessed for ten plants and a quality concentration of viral replicative form was seen in the transgenic lines. Northern blot analysis detects siRNA in the transgenic line 2 of event A, line 5 and 6 of event B, as well as line 9 and 10 from event C inoculated with viruliferous whiteflies and a high level of siRNA (21–22 nt) was observed in the transgenic line 2 and line 10 which corroborated by the non-detectable level of viral replicative DNA and low concentration of viral transcript for replication as estimated in qRT-PCR. Results obtained in this study confirmed the transgene construct can be used to develop resistance against begomoviruses in soybean and other crops, as it targets the most conserved domain governing whitefly transmission.
3

The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants

76%
Sivanandhan G., Mariashibu T. S., Arun M., Rajesh M., Kasthurirengan S., Selvaraj N., Ganapathi A.
Acta Physiologiae Plantarum
|
2011
|
tom 33
|
nr 6
An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5 mg L⁻¹], indole-3-acetic acid (IAA) [0.3 mg L⁻¹] and with the addition of polyamine, spermidine (20 mg L⁻¹) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L⁻¹) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.
4

Agrobacterium - mediated genetic transformation of Withania somnifera using nodal explants

64%
Udayakumar R., Kasthurirengan S., Mariashibu T. S., Sahaya Rayan J. J., Ganapathi A., Kim S. C., Kim J. J., Choi C. W.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 08
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.
5

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.
6

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.
7

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.
8

High-efficient Agrobacterium-mediated in planta transformation in black gram (Vigna mungo (L.) Hepper)

52%
Kapildev G., Chnnathambi A., Sivanandhan G., Rajesh M., Vasudevan V., Mayavan V., Arun M., Jeyaraj M., Alharbi S. A., Selvaraj N., Ganapathi A.
Acta Physiologiae Plantarum
|
2016
|
tom 38
|
nr 08
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