High frequency somatic embryogenesis from coffee leaves - Factors influencing embryogenesis, and subsequent proliferation and regeneration in liquid medium

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called ''high frequency'' embryogenic callus of Coffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures. Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus l(-1) in a modified MS medium containing 4.5 mu M 2,4-dichlorophenoxyacetic acid, under 3 mu mol m(-2) s(-1) illumination, and subcultured every 7-10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250-1000 mu m in diameter) at an inoculum density of 5 g l(-1), with medium renewed every 3-4 weeks. Under these conditions, embryogenic potential of C. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g l(-1). Under these conditions of culture, 1 g of C. canephora or Arabusta callus produced 1.2 and 0.9 x 10(5) somatic embryos, respectively, after 8-10 weeks in liquid regeneration medium. This was an overall reduction of 4-6 months from explant to regenerant, when compared with other procedures.