Effect of Methyl Jasmonate on Allixin production in Callus of Garlic (Allium sativum L.)

Abstract

Allixin (3-hydroxy-5-methoxy-6-methy1-2-penthy1-4H-pyran-4-one) is a nonsulfur containing phytoalexin with red-brown color. This compound showed several unique biological properties such as anti-oxidative, anti-microbial, anti-skin tumor promoting activity in vivo, radical scavenging effect, inhibition of aflatoxin B, binding to DNA and neurotrophic effects. Allixin is found accumulating on the surface of garlic (Allium sativum L.) bulbs that have been stored long-term, about 9 months to 2 years. However, allixin is absent in fresh garlic and may possibly be produced via plant tissue culture technique that is highly efficient for producing plant secondary metabolites. The experiment began by employing sterile techniques to obtain germ-free garlic explants. Garlic cloves were surface sterilized with 0.1% (w/v) mercuric chloride with 2 drops of tween 20 for 15 min and then soaking with 70% ethanol for 30 sec, following by rinsing in sterile distilled water for 15 min 3 times prior to culturing in Murashige and Skoog (MS) medium supplemented with 1.5 mg/L of 2,4-dichloropheoxyacetic acid and 5.0 mg/L of kinetin. Callus appeared clearly on a new shoot around cut wound within 28 days. Next, the callus of garlic were induced with different concentrations of methyl jasmonate (25, 50, 100 and 200 µM) for 49 days. Fresh weight was recorded every week for a total of 7 weeks. Fresh weight of callus decreased every week indicating the state of secondary metabolite production. In addition, treatment of callus by methyl jasmonate for a period of 28 days resulted in red-brown pigment formation on the callus. Extraction of allixin was carried out by grinding elicited garlic callus in methanol. This suspension was centrifuged at 9,000 xg at 4 °C for 20 min. The supernatant was analyzed by High Performance Liquid Chromatography (HPLC) in comparison with commercial allixin standard. Callus of garlic was elicited with 100 µM of methyl jasmonate for a period of 28 days provided the highest allixin content at 3.202 ± 0.240 mg/g fresh weight and 11.793 ± 0.335 mg(g dry weight, respectively. On the other hand, allixin was not detected in the control group of garlic callus. Callus elicited with 100 µM of methyl jasmonate for 28 days was used for isolation of allixin by column chromatography and identification by Nuclear Magnetic Resonance spectroscopy (NMR). Results confirmed that the elicited callus contained allixin. Hence, elicited garlic callus is a newfound source of allixin. Which can produce higher amounts in shorter period of time.