We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

The allelopathic potential of various plants, including coffee species, has been studied. Although the allelopathic potential of coffee leaves, stems, and roots has been reported, there is no detailed information about coffee callus. Since this callus produces bioactive metabolites, we focused on investigating the allelopathic potential of Robusta coffee (Coffea canephora) callus as follows. The sandwich (SW) method, a small-scale bioassay using dried leaves of test plants and recipient lettuce seedlings, was adapted to identify the allelopathic activity of target coffee calli. Test samples were prepared as 10 mg of dried calli under five drying conditions: oven drying at 40 °C, 60 °C, 80 °C, silica gel drying, and freeze-drying. The appearance of each dried callus and chemical features of its water extract varied depending on drying conditions. However, all Robusta coffee calli showed a strong suppressive effect on the growth of hypocotyls and roots of lettuce seedlings. Based on this finding, we analyzed the endogenous level of caffeine (1,3,7-trimethylxanthine), a known allelochemical of coffee and tea plants, using HPLC. In this report, we discuss 1) technical points such as moisture content, drying method, and managing sample quality in the SW method for calli, and 2) the relationship between allelopathic potential and endogenous caffeine level of Robusta coffee calli.

Cellular growth dynamics and allelopathic activity in coffee cell cultures were examined as follows: First, we compared allelopathic activity of seven woody plant calli, Coffea canephora, Derris indica, Ficus carica L., Juniperus conferta, Prunus persica, Punica granatum, and Sonneratia ovata, using a modified "sandwich method bioassay" and found that coffee callus showed the strongest growth inhibition to lettuce seedling nearly 90% of hypocotyl and 96% of root. This coffee callus actively proliferated, with a 21-fold increase during five weeks of subculture, with a growth curve comprising two typical phases: a lag phase of 0 -2 weeks of culture and an exponential phase of 3 -5 weeks of culture. Allelopathic activity varied depending on the growth phase of the coffee callus. The strongest allelopathic activity was detected in 1 -2-week-old callus showing nearly 100% inhibitory effect on lettuce seedling growth. As the allelopathic activity of coffee calli is extremely high, beyond the natural level in coffee leaves and green beans, we focused on analyzing the allelopathic activity of its aqueous extracts using high-performance liquid chromatography. Several prominent peaks, including two reference alkaloids, theobromine and caffeine, which are known allelochemicals in coffee plants, and three distinct unknown peaks were identified at 270 nm in coffee calli during the lag phase (1 -2 weeks of culture). The higher value of the total phenolic content in the lag phase also suggested a key biosynthetic pathway in relation to the allelopathic activity of coffee callus will be activated in the lag phase.