The Optimisation Of Growth Regulators, Precursors And Elicitors Supplementation For Maximum Limonene And Linalool Accumulation In Cell Cultures Of Citrus Grandis (L.) Osbeck
Loading...
Date
2005-01
Authors
Zakaria, Zarina
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
The study on Citrus grandis (L.) Osbeck encompasses tissue culture production
and the extraction of limonene and linalool. The tissue culture methodology comprises
of sterilisation, induction and maintenance of callus on media favouring cell growth, to
favour production of limonene. Explants sterilisation attained high yields of sterile
explants (~ 90%) in treatment with 20% Chlorox® (I% sodium hypochloride). Callus
gro\\rth is favourable from explants originated from 5-week-old fruits and cultured on
modified MS media with 510 mg/1 phosphate, 3 mg/1 each 2,4-D and kinetin and 0.2
mg/1 ABA. Limonene was first detected from 7- month-old callus up to 10-month-old
cultures which yields the highest limonene concentration before slightly down at the
11th month. The amount of linalool extracted was highest from 7-month-old callus and
decreased as the age of callus cultures increased. Media 6 with supplementation of 51 0
mg/1 phosphate, 0.2 mg/1 ABA and 3 mg/1 2,4-D and kinetin respectively, gave
maximum concentration of limonene, which is 0.0031 mg/g fresh weight of callus. The
effects of supplementation various concentrations of exogenous mevalonic acid (MY A)
and linalool to C. grandis callus cultured at varying periods were studied. Callus grov,rth
was found to be proportional to the concentrations of MY A and linalc~_ol especially
during the first 6 weeks. Slow in callus gro\\rth was observed after week 7 in the
cultures. Limonene accumulation was detected as early as week 4 and continued to
increase until week I in added cultures, whereas no limonene was traced in cultures
which were no MYA or linalool added. The highest limonene accumulation, 0.0030
mg/g and 0.0032 mglg was obtained from cultures added with 0.077 mM MY A and
0.838 mM linalool respectively after the i 11 week of the culture period. Linalool
accumulation improved when 0.384 mM and higher concentrations of MYA was added
and reduced commencing from the 41
h to the ih week in the cultures. The consecutive
supplementation of MV A followed by linalool had shot up the limonene accumulation
to 0.0058 mg/g. Mevalonic acid supplied at 0.077 mM demonstrated an inverse
relationship between the callus growth and the limonene accumulation rates in the first
31 days, whereas cultures supplied with 0.384 mM MVA sho\ved a direct relationship
between the callus growth and the limonene accumulation rates throughout the culture
period. Supplementation of 0.559 mM linalool to the cultures increased the limonene
accumulation rate for the first 3 7 days and decreased thereafter. On the contrary, the
callus growth rate continuously increased throughout the culture period. With the
supplementation of 0.838 mM linalool, the limonene accumulation rate rapidly
increased while the callus growth rate dropped after 39 days. Observation on TEM
showed that there was no change at the ultrastructure level for added callus in
comparison to cultures without the supplementation of MV A or linalool. Yeast extract
which was used in the elicitation study revealed that a concentration of 1 00 mg/1
resulted in maximum callus growth. The elicitation effect of chitosan on the limonene
accumulation was based on media conductivity where chitosan was found effective only
when used between 0.5 to 1.0 mg per g fresh \Vt. callus. Yeast extract and chitosan as
elicitors enhanced the limonene accumulation in the callus cultures. Maximum limonene
accumulation at a concentration of 0.0028 mg/g and 0.0036 mg/g was achieved when
induced with 100 mg/1 of yeast extract and 1.0 mg/g fresh v..rt. chitosan respectively. A
high concentration of yeast extract and chitosan, 150 mg/1 and 2.0 mg/g fresh wt.
respectively, resulted in low linalool accumulation. Cultures left for a longer period of
more than 5 weeks with yeast extract and 2 hours with chitosan also accumulated low
linalool. Combined linalool-yeast extract cultures accumulated maximum limonene at
concentrations of0.0051 mg/g in the 51
h week ofthe culture period.
Description
Keywords
Distance Education , Optimisation Of Growth Regulators