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Abstract

The production of high quality protoplasts and the regeneration of plants from protoplasts are the major limitations to the application of electroporation and other direct gene transfer techniques for crop improvement in many plant species, especially in the Poaceae. A major aim of this thesis was to develop techniques for the establishment of a sugarcane protoplast regeneration system for a wide range of commercial Australian sugarcane cultivars so that direct gene transfer (electroporation) techniques could be applied for cultivar improvement. A second aim was to develop techniques for the establishment ofembryogenie callus and cell suspension cultures so that microprojectile bombardment gene transfer techniques could be applied to sugarcane. For 18 sugarcane cultivars, 4 distinct callus types developed on leaf explant tissue cultured on nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 13.6 µM), but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. High levels of endogenous ethylene were produced by Type 3 callus during the first weeks of culture. Regulating the rate of ethylene production by the addition to the culture medium of ethylene inhibitors (aminoethoxyvinylglycine, AVG; or salicylic acid) or a promoter (aminocyclopropane-1 -carboxylic acid, ACC) did not alter the rate of callus growth or plantlet regeneration at the concentrations tested. The addition of silver nitrate (inhibitor of the physiological action of ethylene; 25 to 200 µM) to the culture medium was unable to improve embryogenic callus growth and shoot production, and increased ethylene production. Cell suspension cultures were initiated from embryogenic callus from all 18 cultivars studied and developed in 3 stages. In stage 1 the callus adapted to the liquid nutrient medium. In stage 2 heterogeneous cell suspension cultures formed for 14 of the 18 cultivars after 5 to 6 weeks of culture. In stage 3 homogeneous cell suspension cultures developed for 6 of the 18 cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension cultures. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures but not from homogeneous cell suspension cultures.