GENETIC TRANSFORMATION OF SUGARCANE BY MICROPROJECTILE BOMBARDMENT OF YOUNG LEAF ROLLS

摘要

The introduction of foreign genes in sugarcane by microprojectile bombardment has, so far, been mainly effected using embryogenie callus as target expiants. Disadvantages of this method are that it can take several months to produce sufficient callusand regular subcultures are also required. An alternative method using bombardment of young leaf rolls was therefore attempted. About 3 mm thick leaf rolls of variety M 292/70 were dissected from surface-sterilised sugarcane tops and pre-cultured for sixto ten days on callus induction medium (CI3). They were bombarded with plasm id pAHC25 containing the gus and bar genes using the Bio-Rad PDS-1000/He Particle Delivery System. Six days after bombardment, the leaf rolls were transferred from CI3 medium onto regeneration medium supplemented with 1 mg/L bialaphos and they were subcultured onto fresh selective medium every two weeks. Out of 258 expiants bombarded, several developed shoot primordia but well-established shoots were regenerated from six of them. Twenty-two plants were transferred to the glasshouse and 17 established ones were sprayed with 0.1 % Basta four weeks later. Two plants were resistant to the herbicide and they were analysed for the presence of the bar gene by PGR. A band corresponding to the expected amplifiable fragment of the gene was detected in them. Gene integration in these plants was confirmed by Southern blot hybridisation. These two plants also expressed the gus gene.Our study has demonstrated the potential of using primary expiants of sugarcane variety M 292/70 as targets for microprojectile bombardment. This system will be further evaluated with other varieties.