after 20 days gave similar results. The dividing cells developed into microcalli in 50 to 70 days in MS medium with 1 mgl"1 NAA and 1 mgl"1 BAP. The protoplasts started cell division within 2 to 3 days and developed into microcalli in 50 to 70 days. So far, no reports are available on plant regeneration from protoplast-derived microcalli (Geetha et al., 2000).
The tremendous progress made during the last decade has demonstrated that refinement of routine in vitro techniques coupled with recombinant DNA technology and genetic engineering have opened up new vistas for plant improvement (Potrykus et al., 1985; Potrykus and Spangenberg, 1995). The Agrobacterium-mediated gene transfer is most successful in plants, but has limited applications in monocots due to host range limitations. Bombardment of intact plant cells with high-velocity, DNA-coated microprojectiles is another very effective method for production of transgenic plants (Sanford et al., 1987; Franks and Birch, 1991).
Nirmal Babu (1997) reported transient expression of GUS in ginger embryogenic calli (see Figure 4.3g) when it was bombarded with microprojectiles (1.6 ^m gold particles) using a BioRad PDS-1000/He gene gun at 900 and 1,100 psi helium pressure with the target distance of either 6 or 9 cm. The vector used was pAHC 25 containing GUS (^-glucuronidase) and BAR (phosphinothricin—acetyl transferase) as reporter and selectable marker genes respectively and carrying Ubi-1 (ubiquitin) promoter (Christensen and Quail, 1996). The best GUS score was obtained when the target distance was 9 cm with 900 psi helium pressure. The GUS score of 133 blue spots per square centimeter indicates not only the optimization and efficiency of the biolistic process, but also the ability of the ubiquitin promoter to drive the expression of the reporter gene (see Figure 4.3g).
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