Agrobacterium-mediated genetic transformation of Populus deltoides marsh clone G48 with gus and npt-II genes
Saraswat A, Khan AA, Thakur AK*, Gaur A, Srivastava DK
Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh-173 230, India
*Corresponding author: Thakur AK, ICAR-Directorate of Rapeseed-Mustard Research, Bharatur-321 303, Rajasthan, India, Tel: 09649242662; E-mail: email@example.com
The present investigation had been carried out to standardize a protocol for Agrobacterium- mediated gene transfer in Populus deltoides Marsh. Clone G48 using petiole explants. Reproducibility of already standardized regeneration protocol for clone G48 had been evaluated. High frequency shoot regeneration (72%) from petiole explants was obtained on MS medium supplemented with 0.5 mg l−1 BAP, 0.2 mg l−1 IAA and 15 mg l−1 adenine sulphate. Root regeneration (100%) in in vitro developed shoots was obtained on MS medium supplemented with 0.1 mg l−1 IAA. Increasing concentrations of kanamycin (10-50 mg l−1) were given to find out the minimum dose of kanamycin required for the selection of putative transformed cells during genetic transformation. It was observed that a doze of 50 mg l−1 kanamycin inhibited callus formation and shoot regeneration and the explants turned brown and started dieing. This concentration of kanamycin would be the most useful for selection of npt-II gene transformed petiole cells/tissues. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector pBI 121 along with kanamycin resistance gene (npt-II) was used for genetic transformation experiments. Only the transformed cells/shoots were able to grow on selective shoot regeneration medium containing 50 mg l−1 kanamycin, whereas control explants did not survive. Regenerated putative transgenic shoots were subjected to transient gene expression analysis using spectrophotometric GUS assay and were found positive. This genetic transformation protocol will provide a platform for genetic manipulation of P. deltoides clone G48 for incorporation of genes governing various silviculturally important traits in future.