An efficient protocol for somatic embryogenesis and subsequent
plant regeneration was developed for sugarcane variety Isd-16 using
leaf sheath explants of 3, 6 and 12-months-old field grown plants.
Explants from 6-month-old plant showed the best response and
produced highest percentage of calli on MS + 3.0 mg/l 2,4-D.
L-proline 25mg/l significantly enhanced somatic embryogenesis. The
embryos germinated well on half-strength MS and developed into
plantlets. Somatic embryo derived plants under field condition
showed considerable variation in morphological, agronomical and
biochemical characters. For genetic transformation the calli were
co-cultivated with A. tumefaciens strain LBA4404 harboring a binary
plasmid pCAMBIA1303 containing hpt (hygromycin phosphotransferase)
gene as a selectable marker and a -glucuronidase (gus) reporter
gene in the T-DNA region. The transient expression of gus in
hygromycin resistant calli and regenerated plants was confirmed by
GUS flurometric assay. PCR analysis of genomic DNA from regenerated
plants revealed that the hpt gene was integrated in the transgenic
plants."
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