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1

In vitro propagation and assessment of genetic stability of micropropagated Samanea saman (rain tree) using microsatellite markers

100%
Kasthurirengan S., Xie L., Li C. H., Fong Y. K., Hong Y.
Acta Physiologiae Plantarum
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2013
|
tom 35
|
nr 08
An efficient in vitro propagation of Samanea saman (rain tree) protocol has been successfully developed using nodal explants from a 20-year-old tree. Higher percentage (76 %) of explants produced up to five shoots per explant on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 6-benzyladenine (BA), 0.1 mg L-1 gibberellic acid (GA3) and 100 mg L-1 casein hydrolysate after 3 weeks of culture. When explants were subcultured to fresh medium after harvesting first batch of shoots, more shoots could be generated (another eight shoots per explant). Shoot elongation was achieved (3 cm) when shoots were cultured on MS medium supplemented with 0.25 mg L-1 BA and 0.75 mg L-1 GA3. In vitro generated shoots rooted on MS medium fortified with 0.75 mg L-1 indole-3-butyric acid plus 0.1 % of activated charcoal. A higher percentage of explant response and shoots per explant were obtained on MS medium with BA and GA3. Each responsive nodal explant yields an average of 15 rooted plants within a period of 10 weeks. Rooted plantlets were successfully acclimatized in green house with a survival rate of 90 %. Micropropagated plants were tested for genetic stability using simple sequence repeats (SSR) markers. Use of the 12 high-resolution SSR markers revealed the exact same genetic profile between the mother tree (donor) and micropropagated plants, suggesting the genetic fidelity of our micropropagation protocol. The same protocol was also used successfully in propagating a ‘‘Golden Rain Tree’’ although response of explant and efficiency of propagation was much lower. This protocol will be useful for germplasm preservation/large scale production of true-to-type clones of desirable genotypes.
2

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

86%
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
|
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.
3

Agrobacterium - mediated genetic transformation of Withania somnifera using nodal explants

72%
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.
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