Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.
In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.
Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India; Molecular Genetics and Genomics Laboratory, Department of Horticulture, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305 764, South Korea.
Ganeshan
G
Sivanandhan
Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
Gnajothi
G
Kapil Dev
Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
Jeevaraj
J
Theboral
Plant Tissue Culture Laboratory, Department of Botany, Periyar E.V.R College (Autonomous), Tiruchirappalli 620 023, Tamil Nadu, India.
Natesan
N
Selvaraj
Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
Andy
A
Ganapathi
Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
Markandan
M
Manickavasagam
D060054:Agrobacterium / Q000235:genetics*; D013010:Sonication; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014170:Transformation, Genetic / Q000187:drug effects / Q000235:genetics*; D032341:Withania / Q000235:genetics* / Q000382:microbiology*
D013439:Sulfhydryl Reagents