Review

114 KKU Science Journal Volume 39 Number 1 Research
Introduction
Chitosan is an N-deacetylated product of
chitin which is a natural carbohydrate polymer found
in crab and shrimp shells, which are waste products
from food-processing. Chitin is also found in wings
of some insects such as butterflies and ladybugs,
as well as in cell walls of yeast, mushroom and
other fungi (Devlieghere et al., 2004). Chitosan
production involves three steps: deproteination,
demineralization and deacetylation. Chitosan has been
applied in waste water treatment, biotechnology.
In agriculture, chitosan has been primarily use to
enhance plant growth and to boost the ability of
plant in defending against fungal infections (Hadwiger
et al., 2002; Barka et al., 2004). Phalaenopsis is a
dominantly epiphytic genus and most intensively
hybridized and propagated orchids. Phalaenopsis has
been known worldwide as the most valuable potted
plant. In Thailand, multiplication by tissue culture is
mostly done for mass-propagation (Young et al.,
2000). There have been many reports that chitosan
has ability to stimulate the differentiation of orchid
plant tissue (Limpanavech et al., 2003; Nge et al.,
2006; Pornpienpakdee et al., 2010; Kananont et al.,
2010). The objectives of the present study was to
determine the effect of chitosan, prepared from
cuttlebone, on micropropagation of P. cornucervi
(Breda) Blume & Rchb.f. This is the first attempt
to prepared chitosan from cuttlebone and use it to
enhance the growth of P. cornucervi (Breda) Blume
& Rchb.f.
Materials and Methods
Preparation of chitosan
Chitosan was prepared from cuttlebones
using the modified process of Alimunair and
Zainuddin (1992). Cuttlebones were obtained from
Teppitak Seafood Co.Ltd., Pattani, Thailand. They
(100 g) were cleaned with water and deproteinized
by treating with 4% sodium hydroxide for 4 h
and washed with distilled water to neutralize pH.
After this step, they were demineralized at room
temperature with 4% hydrochloric acid for 24 h,
washed with distilled water and then dried. The
obtained chitin was deacetylated using 50% sodium
hydroxide solution for 3 h. Samples were washed
with distilled water and dried in an oven at 80 ÌC.
Degree of Deacetylation (DD)
Chitosan was dissolved and stirred in 0.1
M acetic acid. After 24 h 10% sodium hydroxide
was added and stirred for 4 h. The mixture was
washed with distislled water to neutralize pH and
then wash with ethanol. The film of chitosan was
incubated in dessicator for 24 h and evaluated the
degree of deacetylation using Fourier Transform
Infrared Spectrometry, employing the method of
Baxter et al. (1992).
Plant materials
The mature pods of P. cornucervi (Breda)
Blume & Rchb.f. were surface sterilized with water
and then dipped in 95% ethanol and flamed in a
short period for 3 times. Seeds were scapen from
the placentae and put on Vacin and Went (VW)
(1949) medium and incubated at 25+2 ÌC in the
darkness, all germinated seeds were moved to light
condition for further development.
Effect of chitosan on protocorm growth
and development in liquid and semi-solid medium
The 12 week-old protocorms were used as
explant in VW liquid and solid medium. Protocorms
(0.6 g fresh weight/flask) were cultured in VW
liquid medium, supplemented with chitosan 5, 10,