High frequency somatic embryogenesis from coffee leaves

Abstract

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 1-1 in a modified MS medium containing 4.5 #M 2,4-dichlorophenoxyacetic acid, under 3 #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 #m in diameter) at an inoculum density of 5 g 1-1, 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 1-1. Under these conditions of culture, 1 g of C. canephora or Arabusta callus produced 1.2 and 0.9 × 105 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.

Coffee biotechnology

“…Browning of the tissues, caused by an excessive accumulation of phenolic compounds, is necessary for the somatic embryogenesis process in coffee (Quiroz-Figueroa et al, 2002). Similar observations have been reported by other authors (de García and Menéndez, 1987;Neuenschwander and Baumann, 1992;van Boxtel and Berthouly, 1996; Menéndez-Yuffá and de García, 1997).…”

“…Yields and germination rates of HFSE were markedly lower as compared to SCSE. The production of somatic embryos in liquid medium has been applied as an approach for a rapid mass propagation of coffee plants (Zamarripa et al, 1991;Ducos et al, 1993;Noriega and Söndahl, 1993;van Boxtel and Berthouly, 1996). Zamarripa et al (1991) reported that the production of somatic embryos is highly dependent on the inoculum density, the production and development of embryos being inhibited when the inoculum density is high.…”

Bioreactors in coffee micropropagation

“…Protocols describing the establishment of embryogenic suspensions for different coffee species, and their efficient regeneration, have been described by several teams. Embryogenic cell suspensions are the material most frequently used to inoculate bioreactors with a view to achieving mass production of somatic embryos (Zamarripa et al 1991, Noriega and Söndahl 1993, Van Boxtel and Berthouly 1996, Etienne et al, 1997. They provide large quantities of a very reactive material.…”

Metabolic engineering of caffeine production

“…The advantage of this method is that, since somatic embryos are directly induced from somatic tissues, the frequency of abnormities and somaclonal variations caused by callus formation could be suppressed to a minimum. The method also has advantages over published methods (Hatanaka et al 1991;van Boxtel and Berthouly 1996) in terms of efficiency and reproducibility. Second, we employed the RNAi technique, which was found to be highly efficient to suppress expression of target gene(s) in comparison with the anti-sense method.…”