Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture

Abstract

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 μM 6-benzylaminopurine, 8.0 μM indole 3-acetic acid, and 50.0 mgl(-1) kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 μM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T0 transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant.

Establishment of a High-efficiency Genetic Transformation System of Cucumber (Cucumis sativus) using Csexpansin 10 (CsEXP10) Gene

“…150 mg L -1 of kanamycin was used to select transformants by Wang et al (2013), 200 mg L -1 of kanamycin was used by Oliveira et al (2011), Mishra et al (2013), and Alvarez and Ordás (2013). In present study, transformed cucumber lines were successfully selected at 100 mg L -1 of kanamycin.…”

“…Acetosyringone has been used to enhance Agrobacteriummediated genetic transformation (Mishra et al, 2013;Manickavasangam et al, 2013;Gupta and Rahman, 2015). In this invesitgation, we observed the effect of different concentrations of acetosyringone in the A. tumefaciens inoculum solution on the transformation efficiency of the cucumber cotyledonary node explants (Table 3).…”

“…Previously, co-cultivation for 2 d was reported by Wang et al (2013). In contrast, Mishra et al (2013) reported that explants co-cultivated for 5 d showed the maximum transformation efficiency in rice. The dissimilarity in cocultivation time may be due to the differences in type and age of the explants used.…”

“…Acetosyringone has been previously used to increase Agrobacterium-mediated transformation efficiency during infection (Mishra et al, 2013;Subramanyam et al, 2013). These studies reported that 20-200 µmol L -1 of acetosyringone were optimal for transformation and different concentrations of acetosyringone were used for different explants.…”

Preliminary assessment on Agrobacterium-mediated Transformation of Dendrobium Broga Giant orchids PLBs

“…7). Agrobacterium density is one of the key factors which affect the transformation frequency (Mishra et al, 2013). Optimal Agrobacterium tumefaciens density will enhance the T-DNA transfer capacity from the bacterium to the host.…”