In Vitro Bioassay of Allelopathy in Robusta Coffee Callus Using Sandwich Method

Abstract

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.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method

“…We must focus on the existence of other candidate metabolites in coffee cells. In a previous study, we investigated the allelopathic activity of coffee nEC using the sandwich method [ 26 ]. The oven-dried 10 mg callus sample showed a strong inhibition, of more than 85% compared with the control, in the reduction of the elongation of lettuce hypocotyl and root.…”

“…C. canephora nEC (100 mg fresh weight) was extracted with 0.5 mL distilled water in a microcentrifuge tube, and the supernatant was subjected to a reverse-phase HPLC (column: Cosmosil Packed Column 5C18-MS-II, 4.6 ID × 150 mm (Nacalai Tesque, Inc., Kyoto, Japan); column oven: 40 °C; solvent: 20% methanol; flow rate: 0.6 mL/min; detection: 270 nm) [ 26 ].…”

Cellular Growth Dynamics Affects Allelopathic Activity in Coffee Cell Culture

“…We investigated the allelopathic activity of Robusta coffee callus (Cc) using a modified SW bioassay. Briefly, 10 mg oven-dried (60˚C) coffee callus tissue (4-week-old) and five lettuce seeds (Lactuca sativa L., Great Lakes 366) were placed on the test agar medium (0.5% w/v) in a well of six-well plate, as described previously [13]. The test plate was incubated at 20˚C for three days in the dark.…”

“…We established a unique coffee cell culture system, Robusta coffee (Coffea canephora) non-embryogenic calli (nEC), which showed a strong inhibitory effect on lettuce cell division [12] and seedling growth [13]. In this study, we evaluated the allelopathic activity of coffee calli using the SW method.…”

Picloram enhanced the callus induction, growth kinetics, antioxidant potentials, and secondary metabolites production of Zingiber officinale var. rubrum callus cultures

“…The present study displayed a lag phase of 20 days (Figure 4) accompanied by an insignificant increase of callus biomass in all the tested picloram concentrations (Table 2) due to the gradual adaptation of the callus to the environment. During the lag phase, cells synthesise and mobilise proteins and specific metabolites in the absence of cell multiplication (Asrori et al, 2020). However, at a longer incubation period, FW biomass increased distinctly in response to higher picloram concentration (Table 2).…”