Establishment Of Cell Suspension Culture Of Hyoscyamus Niger L. For The Production Of Tropane Alkaloids
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Date
2012-06
Authors
Ishak, Rafidah
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Publisher
Universiti Sains Malaysia
Abstract
Aseptic seeds of Hyoscyamus niger were established via two-stage surface sterilization protocol using Clorox® solution coupled with Teepol with 95.6% of the seeds were aseptic. From these aseptic seeds, 79.8% of the seeds germinated and 100% of the seed-derived plantlets survived. Callus could be induced from the leaf, petiole and root explants of the in vitro plantlets on MS medium supplemented with 2.0 - 10.0 mg/L picloram. The leaf explants inoculated to MS medium supplemented with 2.0 mg/L picloram produced the best callus for cell suspension cultures. Even though the callus produced by the leaf and the petiole explants on the same medium was not significantly different (p ≤ 0.05) in terms of quantity, they are very different qualitatively. The callus produced by the leaf explants was more friable with tiny granules while the callus produced by the petiole explants was very compact. Small amount of compact and granular callus was produced from the root explants on the same medium. The leaf-derived callus subsequently sub-cultured on MS medium supplemented with 0.5 mg/L picloram became more friable with less granules but with higher growth rate. The cell suspension culture of H. niger was initiated by transferring 0.5 g of the most friable portion of the leaf-derived callus into 25 mL MS liquid medium supplemented with 2.0 mg/L picloram. After optimization, the best cell proliferation medium was MS medium supplemented with 0.5 mg/L picloram, 30 g/L sucrose and 0.1 mg/L myo-inositol, with the sub-culture time period fixed at fourteen days. The growth pattern of H. niger based on fresh and dried cell mass followed a typical sigmoid curve. Elicitations were carried out by adding varying amount of calcium chloride, yeast extract and casein hydrolysate into the medium. Lower concentration of calcium chloride induced higher cell growth as compared to higher concentration of calcium chloride.
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Keywords
Establishment of cell suspension culture , for the production of tropane alkaloids