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1

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

100%
Sivanandhan G., Mariashibu T. S., Arun M., Rajesh M., Kasthurirengan S., Selvaraj N., Ganapathi A.
Acta Physiologiae Plantarum
|
2011
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tom 33
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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.
2

Agrobacterium - mediated genetic transformation of Withania somnifera using nodal explants

84%
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
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2014
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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.
3

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

84%
Mariashibu T. S., Subramanyam K., Arun M., Mayavan S., Rajesh M., Theboral J., Manickavasagam M., Ganapathi A.
Acta Physiologiae Plantarum
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2013
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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.
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