Pembentukan kalus dan ampaian sel citrus grandis osbeck secara in vitro dan pengekstrakan limonena dan linalool dengan kaedah bendalir lampau genting - karbon dioksida
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Date
2001-06
Authors
Jenimar
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Abstract
The study on Citnts grandis Osbeck encompasses tissue culture production and the
extraction of limonene and llnalool using supercritical fluid (SFE-CO:.!) with carbon
dioxide as solvent. The tissue culture component comprises of induction and
maintenance of callus on medium favouring cell growth, and suspension cell medium
manipulation, to favour production of secondary metabolite, limonene.
Effect of explant size, subculturing, varying concentrations of abscisic acid, kinetin,
2,4- dichlorophenoxy acetic acid (2,4-0), phosphate and nitrate in Murashige and
Skoog (MS) medium were studied in callus induction. The calli obtained were
extracted for limonene and linalool using SFE-C02. Callus induction is favourable from
explants originated from fruits of smaller diameter and cultured on modified MS
medium with 5700 mg/1 phosphate, 0.2 mg!J ABA and 3 mg/l each 2,4-0 and kinetin.
Limonene extracted with SFE-C02 at 2000 psi and 50°C from 7 months old calli gave
concentration of 0.009 mg limonena/g callus. Further extraction up to 10 months old
callus cultures yields the highest limonene concentration of0.043 mg/g. The amount of
limonene dwindled in callus exceeding 10 months old. The amount of linalool extracted
was highest (0.067 mg/g) from 7 months old callus, however linalool decreased as the
age of the callus culture increased. Egher limonene content of 0.043 mg/g was from
~xplants from fruits 6 em diameter arJ. those from fruits 4 em diamet~r yield hmonene
concentration of 0.015 mg/g. Linal0\.1i yidd displayed a converse pattern to those of
hmonene.
Limonene could be detected in the suspension cell culture as early as 5 days after
culturing. In the control test using unmanipulated MS mediwn, the maximum limonene
concentration of 11.5 mg/1 was detect~d between 20 to 25 days corresponding to the end
of the exponential stage and beginning of the stationary phase. Linalool content was
higher than limonene even at 5 days after culture and decreased subsequently with age
of culture up to 25 days. In medium 2, vrith three times phosphate concentration of the
original MS medium, limonene reached its highest concentration of 12 mg/1 at 25 days
while linalool was found to be as high as 30 mg I at 5 days in culture. Linalool
concentration though reduced slightly, remained higher than limonene.
Medium 3 with addition of three times phosphate and nitrate gave maxtmum
concentration of 1 imonene, 19 mg/l as early as 15 days which reduced to ll mg/l at 25
days. The concentration of linalool in trus medium was found to be consist~ntly lower
than limonene even in 5 day old culture. The amount oflimonene produce in medium
4 with three times nitrate remained between 10-15 mg/1 for the duration ofthe study (25
days). Conversely, the linalool level dropped but continued to be higher than limonene.
The favourable medium for accelerating and accumulating limonene contained three
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times phosphate and nitrate concentration as the basic MS media with addition of 0.2
mg/1 ABA and 3 mg/1 each of 2,4-D and kinetin.
Supercrit1cal lluid extraction using CO:! can be used to extract limonene and linalool
from both callus and suspension cells cultures at 2000 psi and 50 ''C.
Description
Keywords
Ampaian sel citrus grandis osbeck , Pengekstrakan limonena , Kaedah bendalir lampau genting