ORIGINAL PAPER Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn Swati Jagga-Chugh • Sumita Kachhwaha • Manju Sharma • Aditi Kothari-Chajer • S. L. Kothari Received: 24 June 2011 / Accepted: 23 December 2011 / Published online: 8 January 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and b-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consid- eration for the assessment of parameters. Optimum con- ditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 lm size provided maximum transient GUS expression and trans- formation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced efficiency of particle bombard- ment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T 1 generation plants. Thus a successful genetic transformation system was developed using particle bom- bardment in E. coracana with 45.3% transformation effi- ciency. The protocol will be helpful for the introgression of desired genes into E. coracana. Keywords Finger millet  Microprojectile  Genetic transformation  Regeneration  Southern blot hybridization  Transgenic plants Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid GA 3 Gibberellic acid GUS b-Glucuronidase hptII Hygromycin phosphotransferase MS Murashige and Skoog medium Introduction Particle bombardment mediated genetic transformation provides an alternative method of gene transfer in those cases where other methods of gene transfer are not effi- cient. It facilitates DNA delivery into intact plant cells, simultaneous multiple gene transfers with no biological constraints or host limitations (Altpeter et al. 2005). Moreover, particle bombardment is also employed for DNA delivery in transient gene expression studies to investigate the plant gene expression and for its ability to introduce DNA directly into different tissues (Sivamani et al. 2009). Any kind of plant tissue could be used as an explant for microprojectile bombardment, which is an advantage over other methods. The common target tissue for particle bombardment is embryogenic somatic tissues such as immature embryo, isolated scutella, inflorescence S. Jagga-Chugh  S. Kachhwaha  S. L. Kothari (&) Centre for Converging Technologies, University of Rajasthan, Jaipur 302 004, India e-mail: slkothari@lycos.com; slkothari28@gmail.com S. Kachhwaha  M. Sharma  A. Kothari-Chajer  S. L. Kothari Experimental Morphogenesis and Plant Tissue Culture Laboratory, Department of Botany, University of Rajasthan, Jaipur 302 004, India 123 Plant Cell Tiss Organ Cult (2012) 109:401–410 DOI 10.1007/s11240-011-0104-7