Callogenesis and Agrobacterium-mediated genetic transformation of sugarcane (Saccharum officinarum L.) with a chitinase gene

The low cane and sugar yields due to fungal diseases have raised a very serious problem for sugarcane growers. Development of sugarcane plants with fungal resistant traits through genetic engineering is one way to overcome this problem. This study was therefore carried out to establish the Agrobacterium-mediated chitinase gene transformation system in sugarcane. Sugarcane calli formation were performed by inducing young leaf sheath of commercial sugarcane plants on Murashige and Skoog (MS) solid mediums supplemented with 3 mg/L 2,4-D and 10% coconut water. Calli obtained were then transferred to the same mediums supplemented with different concentrations of 6-benzylaminopurine (BAP) combined with indole-3-butyric acid (IBA) for shoot regeneration, and the maximum shoot regeneration was observed on mediums amended with 3 mg/L BAP combined with 0.5 mg/L IBA. Agrobacterium tumefaciens strain LBA 4404 (PBI 121), carrying a chitinase gene, 35S promoter and NOS terminator, was employed for the genetic transformation. Successful genetic transformation of sugarcane could be achieved by cocultivating sugarcane calli with A. tumefaciens for 30 min, followed by selection of transformed calli with 1,250 mg/L kanamycin. PCR and RT-PCR analyses confirmed the integration of the transgene.