Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album.

A hairy root line of Linum album LYR2i was obtained via the genetic transformation of a cotyledon segment of the plant by cocultivating it with Agrobacterium rhizogenes and culturing it in hormone-free B5 Gamborg liquid medium. Characteristic changes in root morphology associated with variations in biosynthetic capabilities were achieved by adding different combinations of phytohormones to the basal medium. A reversible system between hairy roots and its cultures with a diversified morphology was established by including and excluding phytohormones in the basal medium. The medium containing indole 3 acetic acid (IAA) at a concentration of 3 mg/l (MI3) induced thicker root tips and that containing 2,4-dichlorophenoxy acetic acid (2,4-D) at a concentration of 1 mg/l (MD1) induced compact green callus. Podophyllotoxin and 6-methoxypodophyllotoxin content increased by 1.86-fold and 1.45-fold as a result of adding IAA (MI3) and 2,4-D (MD1) to the basal medium, respectively. After regeneration, the hairy roots regained their original morphology and biosynthetic capability on hormone-free basal medium. The transformation status of the regenerated hairy roots was confirmed by PCR analysis.

Biotechnological aspects of the production of the anticancer drug podophyllotoxin.

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.