EXPERIMENTAL METHOD 1 Plant material and the explant
Bogor, 21-22 October 2015
385 isorhamnetin, triterpenes e.g. sterols, organic acids, polyprenols including ginkgolic acid, and
tannic acid Zimmerman et al. 2002. The ginkgolide, a unique C
20
cage molecule, is a naturally occurring platelet-activating factor PAF antagonist Braquet 1987; Nunez et al. 1986. PAF is
a potent mediator of anaphylaxis and inflammation and is also implicated in shock, graft rejection, renal diseases, ovoimplantation, and certain disorders of the central nervous system
Kuster et al. 1986; Dive et al. 1989; Singh et al. 2013. Different types of PAF antagonists are available in present time, such as synthetic PAF antagonists and natural ones. Natural
compounds have less toxic effects compare to synthetic one as more and higher dose of natural compounds shows safer therapeutic margins Singh et al. 2013. Indeed, the growing
importance of PAF as a mediator of diverse pathologies increases the possible medicinal benefits that may be derived from ginkgolide, specific PAF antagonist Liu and Xia, 2006.
However, only trace amounts of ginkgolides are contained in the ginkgo leaves Nakanishi 1967; Teng 1988. In addition, location, climate, and seasonal variations have been affecting
the ginkgolides content. Thus the constant and continuous commercial-scale supply of the ginkgolides from the field-grown leaves appears to be quite uneconomically and questionable.
Tissue culture of the ginkgo leaves was conducted as an alternative way to solve the problem. This study was carried out to investigate the effect of growth hormone on biomass and
production of bilobalide, ginkgolides A and B in callus and cell suspension cultures from G. biloba leaves.
2. EXPERIMENTAL METHOD 2.1 Plant material and the explant
G. biloba leaves were collected from the education garden of Faculty of Agriculture, Ehime University, Japan, on September 2012 fall season. The plant material was washed using
neutral detergent two times and rinsing six to seven times with sterile distilled water. After surface sterilization with 70 ethanol EtOH for 1 min followed by a solution of 1 sodium
hypochlorite for 15 min, and cleanings in sterilized distilled-water for 10 min. Thus, Leaves have been already using as the explant material.
2.2 Optimization of culture condition Calluses were generated from leaves of G. biloba. The explant of leaves after pretreatment
describe above section was cut and transferred to Murashige and Skoog MS medium, containing sucrose 30 gL and gellan gum
2 gL, with pH 5.6. The growth calluses were sub-
culture every month and incubated at 24˚C in the dark. Callus growth was expressed as fresh weight gram and dry weight gram. Callus with 0.5-0.6 g of fresh weight was
transferred to MS medium supplemented with 1-naphthaleneacetic acid NAA, 2,4- dichlorophenoxyacetic acid 2,4-D and kinetin in certain combination, then the weight was
determined after 30, 60 and 90 days. Table 1: The design combination of growth hormone treatment in cell suspension cultures of
G. biloba
Kinetin mgL NAA mgL
2,4-D mgL 1.0