Bruno Florin

Conversion of tryptamine to serotonin by cell suspension cultures of Peganum harmala

Abstract Biotransformation of tryptamine to serotonin by cell cultures of Peganum harmala was performed in 250 ml conical flaskes or 10 l bioreactor with high serotonin yields (2.5 g/l of culture and 20% of the biomass dry weight). The specific biotransformation rate reached more than 100 mg/g dry weight/day. The influence of pH, auxin concentration, and temperature were studied. Phenobarbital stimulated the reaction. Immobilized cells showed a lower biotransformation rate than cell suspensions. The stability of the cell line after cryostorage (growth and biotransformation capability) was established.

Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos

High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress.

Somatic embryogenesis and vegetative cutting capacity are under distinct genetic control in Coffea canephora Pierre

The purpose of the study was to evaluate the possible genetic effect on vegetative propagation of Coffea canephora. Diversity for somatic embryogenesis (SE) ability was observed not only among two groups of C. canephora Pierre (Congolese and Guinean), but also within these different genetic groups. The results therefore showed that, under given experimental conditions, SE ability is depending on genotype. Furthermore the detection of quantitative trait loci (QTLs) controlling the SE and cutting abilities of C. canephora was performed on a large number of clones including accessions from a core collection, three parental clones and their segregating progenies. On the one hand we detected eight QTLs determining SE. Six positive QTLs for SE ability, whatever the criteria used to quantify this ability, were localized on one single chromosome region of the consensus genetic map. Two negative QTLs for SE ability (frequency of micro calli without somatic embryo) were detected on another linkage group. Deep analysis of the six QTLs detected for SE ability came to the conclusion that they can be assimilated to one single QTL explaining 8.6–12.2% of the observed variation. On the other hand, two QTLs for average length of roots and length of the longest sprouts of cuttings were detected in two linkage groups. These QTLs detected for cutting ability are explaining 12–27% of the observed variation. These observations led to conclude that SE and cutting abilities of C. canephora Pierre appeared to be genetic dependent but through independent mechanisms.

A new approach for automation: Sorting and sowing dehydrated somatic embryos of Daucus and Coffea using seed technologies

Somatic embryos of Daucus carota L. and Coffea canephora L. (var. Robusta) were dehydrated under a 43 % relative humidity then placed in the hopper of a precision seeding system used in the transplant industry. The seeder was adjusted to distribute the embryos onto horticultural trays, each one containing 240 cells filled with soil. As a preliminary result, 72 % and 88 % of the individual cells received a single embryo, in Daucus and Coffea respectively. The embryo-to-plantlet conversion rate was not affected either by the vibration of the hopper or by the nozzles. In carrot 66 % of the embryos germinated after the use of the seeding system (62% germination for the control). Sorting methods traditionally used for the seeds (e.g. air column, vibrating table) can also be used. Such an approach, based on desiccation as a key step, has the potential for a complete automation of the large-scale handling and delivery of somatic embryos.