Review

ß“π«‘®—¬
«“√ “√«‘∑¬“»“ µ√å ¡¢. ªï∑’Ë 39 ©∫—∫∑’Ë 1 115
15, 20 and 25 mg/l. Suspension of protocorms
were maintained at 25+2 ÌC, shaked at 110 rpm.
Protocorms were cultured on VW semi-solid
medium supplemented with chitosan 5, 10, 15, 20
and 25 mg/l (30 explants were used for each
treatments). Semi-solid culture medium were kept at
25+2 ÌC and provided photoperiods of 16 h per day
using cool white fluorescent. After 12 weeks, the
number of the leaves, roots, shoots and the fresh
weight (FW) of plant in semi-solid medium were
measured. Only the fresh weight of plant in liquid
medium was measured after 4 weeks of culturing.
Transplantation of plantlets
Plantlets of P. cornucervi (Breda) Blume
& Rchb.f. were dipped into fungicide solution for
15 min. They were then transferred to plastic basket
(size 27.5x13.2x12.3 cm) containing of charcoal
as planting material and watered with various
concentrations of chitosan solution at 5, 10, 15, 20
and 25 mg/l and kept under the plastic cover. The
cover were occasionally removed after the second
week to allow air circulation and to decrease the
internal humidity. After 4 weeks, the percentage
of survival was calculated using the formula as
followed:
% survival = number of plants survival x
100/total number of plants
Experimental design and data analysis
Each experiment contained 30 explants was
performed in a complete randomize design (CRD)
and repeated twice. All data were analyzed by
ANOVA and mean values were compared using
Duncanûs Multiple Range Test (DMRT) at 5%
significant level.
Results and discussion
Chitosan preparation
The chitosan was yielded from cuttlebone
in 20% (w/w) with 56% degree of deacetylation
(DD). The results showed that cuttlebone chitosan
had a lower degree of acetylation than brine shrimp
chitosan (67-74%) (Tajik et al., 2008). The DD
is an important parameter affecting solubility,
chemical reactivity and biodegradability. It may range
from 30-95% depending on the source and
preparation procedure (Martino et al., 2005).
Effect of chitosan on the growth of
protocorm in liquid and semi-solid medium
The results of chitosan on the growth of
protocorms in liquid were showed in Table 1 and
Figure 1. The protocorm (original weight 0.6 g) in
the medium supplemented with chitosan at various
concentrations; 5, 10, 15, 20 and 25 mg/l, were
produced more in higher weight than in the control
medium without chitosan. The effect of chitosan on
growth of protocorms cultured on the semi-solid
medium for 12 weeks was presented in Table 2.
The results showed that the highest number of leaves
(4.3 leaves), roots (2.5 roots) and shoots (1.5 shoots)
could obtain when cultured on the medium with 15
mg/l of chitosan. The result also showed that
concentration of chitosan higher than 15 mg/l
caused decreasing of growth and development of
protocorm. It was reported that chitosan stimulated
the development of Vitis vinifera L. (Barka et al.,
2004). Pornpienpakdee et al. (2010) also reported
that chitosan was found to enhance the in vitro
micropropagation of Dendrobium ùEiskulû.