Studies on genetic transformation of coffee by using electroporation and the biolistic method
1994
van Boxtel, J.H.J.
The present study aimed simultaneously at an improvement of coffee regeneration systems and at a definition of factors influencing the efficiency of direct gene transfer methods. The development of an improved regeneration system, based on high frequency somatic embryogenesis from leaf explants, passing through multiplication of embryogenic callus in liquid medium, is described. This method can contribute to the obtaining of high protoplast yields and offers perspectives for use in genetic transformation systems of coffee. Several factors affecting protoplast isolation, electroporation and regeneration were studied. This system appeared to be appropriate for transient expression studies but, due to difficulties with protoplast regeneration, less promising for achieving stable expression. Further expression studies were performed using particle gun bombardment on different tissues of several coffee genotypes. Best results were obtained using in vitro cultured leaves of Coffea arabica and plasmids carrying the EF1α-A1 promoter of Arabidopsis thaliana. The effect of tungsten particles on callus induction and the fate of GUS-expressing cells after bombardement on leaves has been described, and the consequences for their use are discussed. Studies on five selective agents showed best prospects of the herbicide glufosinate for detection of stably transformed coffee tissue. It was concluded that avoidance of polyphenolic oxidation, caused by tissue wounding, is of great importance for the development of a reliable genetic transformation method for coffee.
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