Establishment of an in vitro regeneration system for genetic transformation of selected sugarcane genotypes.

A good culture system provides considerable quantities of highly regenerable target tissues. Embryogenic callus cultures are ideal for micro-projectile-mediated transformation, because regenerable cells are not very stable. Effective exploitation of genetic transformation requires good regeneration systems. We selected three sugarcane genotypes for the establishment and optimization of good in vitro regeneration systems, viz., S-2003-us-359, S-2006-sp-30, and S-2003-us-165. Three callus induction media were investigated. These media were composed of Murashige and Skoog (MS) medium salt plus 1, 2, and 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Medium with 3 mg/L 2,4-D gave the greatest mass of embryogenic calli. The calli produced on the three callus induction media were transferred to 18 types of regeneration media (RM1-RM18). They varied with respect to plant growth regulators and sucrose levels but the basal medium was MS. Two levels of sucrose (30 and 40 g/L), three levels of 2,4-D (0.1, 0.25, 0.5 mg/L) and three levels of 6-benzylaminopurine (0, 0.25 and 0.5 mg/L) were studied in the regeneration media. The effects of callus age on regeneration were evaluated by transferring the calli to regeneration media after 15, 21, 28, and 35 days of culture. The 21-day-old callus of the genotype S-2003-us-359 on RM3 yielded the largest number of plants and was selected as the best for transformation. Six RAPD DNA primers were used to check genetic stability; this medium did not affect the sugarcane genomes.

Molecular characterization of wheat germplasm using microsatellite markers.

We investigated the genetic diversity of 63 wheat genotypes, composed of 48 accessions and 15 varieties, using 56 polymorphic simple sequence repeat primers. One hundred and eighty-six loci were found, with a mean of 131.26 alleles per locus. Cluster analysis based on microsatellite allelic diversity discriminated the accessions and varieties into different clusters; genetic diversity was the highest between variety Kohistan-97 and accession number 011512, giving a genetic similarity value of 0.4198. Accession numbers 011484 and 011356 gave a genetic similarity value of 0.9589, indicating that these accessions were 95.89% similar. We found that microsatellite markers could characterize and discriminate all of the genotypes; more primers could be used for saturation of different regions in further studies.