In vitro studies on catharanthus roseus [L.] G. [Don]: growth and maintenance of C. roseus calli and alkaloid production of shoot and root-derived calli grown on modified alkaloid production media

ABSTRACT

The aim of this work is studying the pattern of total alkaloids accumulation in callus derived from shoot and root explants of Catharanthus roseus in response to growth regulators, number of subcultures under two illumination conditions. In addition, this study aimed to increase both callus growth and alkaloids accumulation of C. roseus when transferring those calli [pretreated calli] onto modified alkaloid production medium [mAPM] under light or dark conditions. Shoot and root derived calli of C. roseus were initiated and then subcultured 15 times [each for 4 weeks duration] onto Gamborg's B5 medium containing NAA combined with Kin. The cultures were incubated under cycles of light/dark [16/8 hr] or in complete dark conditions. Fresh and dry weight of shoot or root derived calli increased progressively with increasing the number of subculture. Treatment of NAA at 0.5 mg/1 plus Kin at 0.05 mg/1 gave the best growth of shoot-calli, while treatment of NAA at 2mg/l plus Kin at 0.05 mg/1 showed the highest value of the growth of root-calli. The highest content of total alkaloids of C. roseus calli derived from shoot or root explants were obtained from that medium supplemented with NAA at 2.0 mg/1 plus Kin at 0.2 mg/1. Total alkaloids content reached 17.4 and 15.3 mg/g dry weight in shoot callus tissues under photoperiod [light/dark;16/8 hr] and complete dark conditions, respectively. On the other hand, total alkaloids content obtained from root calli reached 10.0 and 7.5 mg/g dry weight under photoperiod and dark conditions, respectively. A new composition of alkaloid production medium [mAPM] was obtained. Modified alkaloid production medium contained mineral salts, Na-EDTA and vitamins of MS medium, 5 percent sucrose, indoleacetic acid [IAA] at 10 M, benzylaminopurine [BA] at 5 x 10 M, and L-tryptophan at 0.05 percent as a precursor of C. roseus alkaloids. Shoot and root derived calli of Catharanthus roseus were affected by both of the pretreatments transferring to modified alkaloid production media mAPM and transferring to modified alkaloid production media mAPM. The pretreatment 0.5 mg/1 NAA plus 0.2 mg/1 kin resulted the greatest increase of fresh and dry weights of shoot derived calli under light or dark conditions, when cultures were transferred to mAPM. The pretreatment 2.0 mg/1 NAA plus 0.1 mg/1 kin produced the greatest increase of fresh and dry weights of root derived calli under light or dark conditions, after transferring to mAPM. Dark condition was more favorable for fresh and dry weight of shoot and root derived calli cultured on mAPM. The highest total alkaloid content in the shoot-derived calli was 15.86 mg/g dry wt and in the root-derived calli was 18.18 mg/g dry wt on mAPM for those calli were initiated onto pretreatment NAA at 20 mg/1 plus 0.2 mg/1 kin under light condition