In many functional genomic studies, cell suspension cultures have been very useful experimental tools. Experiments with grape cell suspensions are quick and convenient compared to studies on whole vines and berries, they make it easy to control the environment conditions and to manipulate with accuracy the experiment conditions, and it is possible to carry out experiments all year. Cell suspensions of ‘Cabernet Sauvignon’ have been set up in Gamborgs B5 liquid medium with minimal organics supplemented with 30 gL-1 sucrose, 0.25 gL-1 casein hydrolysate, 0.93 M kinetin and 0.54 M naphthaleneacetic acid by callus cultures established from young berries on the same medium solidified with and 0.8% (w/v) of agar. The cultures were incubated in darkness on an orbital shaker at 100 rpm. The cell suspensions were sub-cultured every 2 weeks using an initial packed cell volume (PCV) of 15%. In a set of preliminary experiments the effect of some elicitors (abscisic acid; epibrassinolide; ethephon; mannitol; jasmonic acid, methyl-jasmonate; polyethylene glycol; salicylic acid; sorbitol; sucrose) on induction of secondary metabolites in cell suspensions were tested. Among those methyl-jasmonate (MeJA) induced the production of at least 25 compounds with sesquiterpene-like mass spectra. Sesquiterpene production was assayed in the cell cultures at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 hours after the addition of 500 μM MeJA. To study the interaction between MeJA concentration and PCV at the moment of elicitor addition, the production of volatile compounds was assayed in cell suspensions induced with 500 μM of MeJA added at 0, 1, 2, 3, 4, 5 and 6 days after cell suspension subcultures were set up with an initial PCV of 28%. In a subsequent experiment, different concentrations of MeJA (0, 50, 100, 250, 500, 750, 1000, 1500, 2000, 3000 μM) were tested for their effect on volatile production when added to a cell suspension with a PCV of 35%. The production of sequiterpenes began 12 hours after the addition of MeJA, and reached a maximum 72 hours after MeJA addition. The production of sesquiterpenes quickly decreased with an increase of cell suspension PCV at the moment of MeJA addition, suggesting a strong interaction between PCV and MeJA concentration. The most effective concentration of MeJA in stimulating the production of sesquiterpenes, was found to be 500 μM if added when the cell suspensions had a PCV of 35%, and 1000 if added when the cell suspensions had a PCV of 70%. So the optimal concentration of MeJA for induction of sesquiterpenes appeared to be 1.43 μmol per ml of PCV. These results suggest that in the interaction elicitor/density of cell in suspension the real important parameter is the concentration of elicitor per PCV unit and not the concentration of elicitor per total cell suspension volume. The viability of cells drastically decreased after the addition of MeJA and this decrease depended of concentration of MeJA added. MeJA also induced a significantly greater amount of proanthocyanidins and stilbenes in the cell cultures. The analysis of gene expressions by real-time RT-PCR and microarray analysis revealed that the mechanism by which jasmonates induced the production of secondary metabolites in cultured grape cells varied depending on the pathway [1].
Optimization of aroma production in grape cell suspension induced by chemical elicitors
D'ONOFRIO, CLAUDIO;
2010-01-01
Abstract
In many functional genomic studies, cell suspension cultures have been very useful experimental tools. Experiments with grape cell suspensions are quick and convenient compared to studies on whole vines and berries, they make it easy to control the environment conditions and to manipulate with accuracy the experiment conditions, and it is possible to carry out experiments all year. Cell suspensions of ‘Cabernet Sauvignon’ have been set up in Gamborgs B5 liquid medium with minimal organics supplemented with 30 gL-1 sucrose, 0.25 gL-1 casein hydrolysate, 0.93 M kinetin and 0.54 M naphthaleneacetic acid by callus cultures established from young berries on the same medium solidified with and 0.8% (w/v) of agar. The cultures were incubated in darkness on an orbital shaker at 100 rpm. The cell suspensions were sub-cultured every 2 weeks using an initial packed cell volume (PCV) of 15%. In a set of preliminary experiments the effect of some elicitors (abscisic acid; epibrassinolide; ethephon; mannitol; jasmonic acid, methyl-jasmonate; polyethylene glycol; salicylic acid; sorbitol; sucrose) on induction of secondary metabolites in cell suspensions were tested. Among those methyl-jasmonate (MeJA) induced the production of at least 25 compounds with sesquiterpene-like mass spectra. Sesquiterpene production was assayed in the cell cultures at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 hours after the addition of 500 μM MeJA. To study the interaction between MeJA concentration and PCV at the moment of elicitor addition, the production of volatile compounds was assayed in cell suspensions induced with 500 μM of MeJA added at 0, 1, 2, 3, 4, 5 and 6 days after cell suspension subcultures were set up with an initial PCV of 28%. In a subsequent experiment, different concentrations of MeJA (0, 50, 100, 250, 500, 750, 1000, 1500, 2000, 3000 μM) were tested for their effect on volatile production when added to a cell suspension with a PCV of 35%. The production of sequiterpenes began 12 hours after the addition of MeJA, and reached a maximum 72 hours after MeJA addition. The production of sesquiterpenes quickly decreased with an increase of cell suspension PCV at the moment of MeJA addition, suggesting a strong interaction between PCV and MeJA concentration. The most effective concentration of MeJA in stimulating the production of sesquiterpenes, was found to be 500 μM if added when the cell suspensions had a PCV of 35%, and 1000 if added when the cell suspensions had a PCV of 70%. So the optimal concentration of MeJA for induction of sesquiterpenes appeared to be 1.43 μmol per ml of PCV. These results suggest that in the interaction elicitor/density of cell in suspension the real important parameter is the concentration of elicitor per PCV unit and not the concentration of elicitor per total cell suspension volume. The viability of cells drastically decreased after the addition of MeJA and this decrease depended of concentration of MeJA added. MeJA also induced a significantly greater amount of proanthocyanidins and stilbenes in the cell cultures. The analysis of gene expressions by real-time RT-PCR and microarray analysis revealed that the mechanism by which jasmonates induced the production of secondary metabolites in cultured grape cells varied depending on the pathway [1].I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
