**4. Ex situ method of propagation**

Attempts to obtain podophyllotoxin through cell cultures or chemical synthesis techniques are still far from being economically feasible. The objective of the following study was to enhance the root formation and podophyllotoxin production of *P. hexandrum* cultivated in a glasshouse [37]. Two batches of plants grown for different time periods were obtained from two different regions and stored at 7–8°C in the dark to prevent the formation of shoot. They were later cultivated in glasshouse in the peat-perlite soil (2:1 w/w). For every condition and time point, 15 plants were randomly harvested. The root biomass and podophyllotoxin content of the plants of each temperature group were analyzed at the beginning. The plants were harvested for a period of 20 or 40 days, but all the plants were cultivated for a minimum period of 20 days before giving them the methyl jasmonate treatment. Fifteen plants were immediately harvested for baseline control, 30 plants were sprayed with water (control), and 30 plants were sprayed with 5 l of 1.5 mM methyl jasmonate. After 9 days, 15 plants from each group were harvested for analysis. The plants in the treatment group were sprayed again for 3 consecutive days with 5 l of 3 mM methyl jasmonate each day and harvested for analysis the next day. Roots from each plant were collected, rinsed with tap water, and dried for 18 h at 40°C. They were later pooled in groups of three, ground and stored at room temperature in closed containers in the dark. 10 ml of methanol was added to 1 g of the plant material. The sample was vortexed at 2500 rpm and incubated at 65°C in a water bath for 10 min. Then the mixture was centrifuged at 4°C at 2400 g for 10 min. The supernatant was separated and transferred to a fresh tube. This extraction process was performed five times. The podophyllotoxin content was determined by HPLC analysis and the samples were stored at 4°C before analysis. Podophyllotoxin is stable in the refrigerator at 4°C for at least 3 months and at 25°C. The results of the study showed a higher concentration of podophyllotoxin in MeJ-treated plants (30 mg/g) as compared to MeJ-deficient plants (18 mg/g).
