Callus induction, regeneration and transformation of sugarcane (Saccharum officinarum L.) with chitinase gene using particle bombardment

Abstract

This study was carried out to optimize the conditions for introducing a chitinase gene into the sugarcane cv. Phil 66-07 calli by particle bombardment. Young leaves were cultured on the modified Murashige and Skoog (MS) medium supplemented with varied concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), yeast extract and coconut water (CW). The maximum percentage of callus induction was obtained from the MS medium supplemented with 3 mg/l 2,4-D and 15% (v/v) coconut water. Multiple shoots were achieved by transferring sugarcane calli to the MS medium amended with 1 mg/l benzyl aminopurine (BA) and 0.5 mg/l indole-3-butyric acid (IBA). Additional experiments were also performed to determine the effect of antibiotics on regeneration of sugarcane. It was found that growth of sugarcane calli and plantlets were completely inhibited by hygromycin concentrations of 25 and 50 mg/l, respectively. The genetic transformation was achieved via particle bombardment with an optimal helium pressure of 900 psi and the stopping screen set at 9 cm. Sugarcane was transformed with either GUS or a chitinase gene and a gene for hygromycin selection. GUS transformed calli were produced to optimize the particle bombardment protocol. Using the optimized protocol, the chitinase gene was transformed into sugarcane and polymerase chain reaction (PCR) was used to verify the integration of a chitinase gene, 35S promoter and nitric oxide synthase (NOS) terminator in transgenic sugarcane.

Key words: Sugarcane, genetic transformation, particle bombardment, chitinase gene.