Scientia Horticulturae 124 (2010) 239–247

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids
(Asparagales: Orchidaceae)
Nungruthai Kananont a,b, Rath Pichyangkura b,c, Sermsiri Chanprame d, Supachitra Chadchawan a,b,1,*,
Patchra Limpanavech a,b
a
Environment and Plant Physiology Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b
Center of Chitin-Chitosan Biomaterial, Chulalongkorn University, Bangkok 10330, Thailand
c
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
d
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand

A R T I C L E I N F O A B S T R A C T

Article history: The effects of different types of chitosan on seed germination and protocorm development were
Received 15 May 2009 determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six
Received in revised form 22 November 2009 chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation
Accepted 27 November 2009
(P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10,
20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12
Keywords: weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the
Dendrobium
proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan.
Orchid
In vitro seed germination
In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum
Chitosan with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90,
were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or
20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is
concluded that chitosan responses in seed germination and protocorm development were somewhat
species and developmental stage dependent. Therefore, the appropriate chitosan application for each
plant species should be evaluated first before use.
ß 2009 Elsevier B.V. All rights reserved.

1. Introduction
The Orchidaceae is the largest family of flowering plants
(angiosperms) with 700–800 genera and 20,000–30,000 described
species, or approximately 10% of all known angiosperms, and is the
most highly evolved family amongst the monocotyledons (Arditti,
1979; Dressler, 1981, 1993). Orchids are a major floral crop
worldwide and represent over 8% of the global floriculture trade
(Griesbach, 2002). Globally, Dendrobium hybrids are considered to
be one of the most popular orchids. Indeed, besides the showy
flowers, the popularity of Dendrobium is increasing due to the
number of flowers per inflorescence and recurrent flowering
(Martin and Madassery, 2006), whilst the relatively short
production cycle from seedling to bloom for Dendrobium hybrids
increases their commercial value and interest.
* Corresponding author at: Environment and Plant Physiology Research Unit,
Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok
10330, Thailand. Tel.: +662 218 5485; fax: +662 252 8979.
E-mail addresses: s_chadchawan@hotmail.com (S. Chadchawan),
patchra.L@chula.ac.th (P. Limpanavech).
1
Tel.: +662 218 5485; fax: +662 252 8979.
While orchid seeds are typically small and without nutrient
storage, some orchids have been shown to have concentrated
nutrients stored in mature seeds (Rasmussen, 1995). To germinate
the seeds, they can be directly sown into the soil around the
mother plant in the hope that some seed will come into contact
with the right mycorrhizal fungus and germinate, or the isolated
mycorrhizal fungus can be added to the seeds germinating on the
culture medium. However, it has been shown that in asymbiotic in
vitro cultures the medium provides all the necessary nutrients,
vitamins and hormones to support germination and protocorm/
seedling development. This method is commonly used for the
epiphytic orchid species where the fungal association does not
play as important a role as with many of the terrestrial orchid
species (Wodrich, 1997). The ability to asymbiotically in vitro
culture orchid seeds serves to both increase the availability of the
plants to meet the increasing demand for them, and to serve as
reservoirs for restocking the wild populations which are declining
due to habitat destruction, fragmentation and over harvesting
(Anderson, 1990; Brumback, 1990). However, optimization of the
in vitro sowing method for orchid seed germination is still
required to improve the germination rate, growth and develop-
ment of the protocorms to plantlets. Seeds of two Dendrobium

0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2009.11.019
240 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247
species that are much in demand for commercialization as
flowering pot plants were used in this study. Dendrobium bigibbum
var. compactum C.T. White is a purple-flower bearing, miniature
wild orchid from Australia and Dendrobium formosum Roxb. ex
Lindl. is a native orchid species of Thailand, having a showy white
flower with a yellow-center lip.
Chitosan, a biopolymer that has been applied to various areas,
including agriculture (Babel and Kurniawan, 2003; Obara et al.,
2003; Wang et al., 2003; Worrell et al., 2002; Ohta et al., 1999), has
been shown to affect many plant responses. It has been considered
to elicit the induction of plant defence mechanisms where, for
example, it has been shown to induce phenylalanine ammonia-
lyase, which is involved in the synthesis of phenolic compounds
(Notsu et al., 1994; Vander et al., 1998). In addition, it can induce
phytoalexin biosynthesis in rice, Oryza sativa L. (Agrawal et al.,
2002), and acquired systemic resistance in Arachis hypogaea
(Sathiyabama and Balasubramanian, 1998). Moreover, it has also
been shown to decrease the stomatal aperture of tomato and
Commelina communis L. via the action of Ca2+ and H2O2 (Lee et al.,
1999), and potentially via this or other mechanism(s), to increase
the efficiency of water usage in Capsicum sp. (Bittelli et al., 2001).
Chitosan’s stimulating effect on plant growth has previously
been shown in Eustoma grandiflorum (Raf.) Shinn (Ohta et al.,
1999), Torenia fournieri Linden ex E. Fourn., Exacum affine Balf.,
Begonia hiemalis Fotsch, Sinningia speciosa (Lodd.), Lobelia erinus L.,
Mimulus hybridus hort. ex A. Siebert et Voss, Calceolaria herbeohy-
brida Voss and Campanula fragilis L. (Ohta et al., 2004). In orchids, it
was found to positively affect floral production in Dendrobium
‘Eiskul’, where a particular type of chitosan, oligomeric chitosan
with an 80% degree of deacetylation (O80), but not polymeric
chitosan or oligomeric preparations with different degrees of
acetylation, caused an increase in the chloroplast size and in the
number of inflorescences produced, suggesting also then the
specificity of the chitosan molecule and plant species that will
respond positively (Limpanavech et al., 2008).
Indeed, plants respond differently to various molecular types of
chitosan molecules. It was reported that chitosan of low molecular
weight was more effective at inducing a set of defence responses
than those of a higher molecular weight (Lin et al., 2005), including
the production of hydrogen peroxide (H2O2), increases in the
activities of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and
chitinase (CHI; EC 3.2.1.14), increases in transcription of the
defence-related genes beta-1,3-glucanase (glu) and chitinase (chi)
and accumulation of pathogen-related protein (PR1). The degree of
acetylation of chitosan was shown to differently elicit phenylala-
nine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin
deposition, and microscopically and macroscopically visible
necroses, when injected into the intercellular spaces of healthy,
non-wounded wheat (Triticum aestivum L.) leaves. Partially N-
acetylated, polymeric chitosans elicited both PAL and POD
activities, and maximum elicitation was observed with chitosans
having an intermediate degree of acetylation (35%) (Vander et al.,
1998).
Many complex organic additives, such as coconut water,
banana, potato extract, raisins, apple, tomato and V-8 juices, are
added to the in vitro germination media to enhance seed
germination and development (Sagawa, 1988). As chitosan has
been shown to enhance plant growth in some species (Ohta et al.,
2004), the effects on seed germination in native orchid species
should be investigated. The objectives of this research were to
examine the effect of chitosan types and concentrations on the in
vitro orchid seed germination so as to determine the appropriate
chitosan for improved asymbiotic in vitro orchid seed germination.
Two wild orchids from the genus Dendrobium, namely D. bigibbum
var. compactum C.T. White and D. formosum Roxb. ex Lindl, were
used for this study.
2. Materials and methods
2.1. Plant materials and seed extraction
Two months post-pollination, mature green seed capsules of D.
bigibbum var. compactum were collected from nursery grown
plants. For D. formosum, 1-year-old mature green seed capsules
were collected from hand pollinated plants in their natural habitat
at Ngao Waterfall National Park in the Ranong Province, Southern
Thailand. Due to the smaller size and lower seed content of D.
bigibbum var. compactum seed capsules, three seed capsules were
used for this species while only one capsule of D. formosum
provided sufficient seeds for the experiment. The capsules of each
species were cleaned with soap and tap water, and then surface
sterilized by dipping in 95% (v/v) ethanol and flaming. The contents
of the seed capsule (D. formosum) or capsules (D. bigibbum var.
compactum) were then dispersed into 100 ml of sterilized water to
make a master seed suspension which was used as the seed source
for all the experiments reported herein. This stock suspension was
mixed well before aliquot removal.
2.2. Chitosan
Six types of chitosan molecules were prepared from crab shells
as previously described (Limpanavech et al., 2008). Polymer (P)
and Oligomer (O) forms of chitosan, each with a degree of
deacetylation of 70, 80 and 90% (designated P70, P80 and P90; and
O70, O80 and O90, respectively), were solubilised in 1N acetic acid.
Before application, the chitosan solution was adjusted to pH 5 with
NaOH.
2.3. Seed viability test
Seed viability was determined by the ability to reduce 2,3,5-
triphenyltetrazolium chloride (TTC) to the red coloured formazon
(Brewer, 1949). In this experiment, the orchid embryo either
turned red or stayed colourless after TCC staining, with no
ambiguous pale red stained seeds being observed. Therefore, seeds
with TTC reduction ability (red coloured) were scored as viable. For
each replicate, 1 ml of the D. bigibbum var. compactum seed
suspension, or 0.2 ml of the D. formosum seed suspension (see
above) were used, so as to result in approximately 1800 seeds in
each assay for each orchid species. The water was then removed
and replaced with 1 ml of 0.1% (v/v) TTC aqueous solution. The
seeds were kept in the dark for 24 h before viability determination
by inspection of their colour. Three replicates were performed, and
for each replicate, the seeds were sampled five times, approxi-
mately 180 seeds each time, to determine the seed viability, which
is reported as a percentage.
2.4. Seed sowing in vitro
For both orchid species, 300 ml of the respective seed suspension
(approximately 550 and 2700 seeds for D. bigibbum var. compactum
and D. formosum, respectively) were removed and inoculated onto
Petri dishes containing germinating medium supplemented with or
without the indicated chitosan. The germinating medium consisted
of the inorganic salts of Vacin and Went (VW) medium (Vacin and
Went, 1949), except for using FeSO4
7H2O and Na2EDTA, according
to the Murashige and Skoog (1962) formula, instead of ferric tartrate,
and was additionally supplemented with 15% (v/v) coconut water,
2% (w/v) sucrose and 0.8% (w/v) pharmaceutical grade agar. The
medium was adjusted to pH 4.8. Each of the six chitosan types, P70,
P80, P90, O70, O80 and O90 (Limpanavech et al., 2008), was used at a
final concentration of 0, 10, 20, 40 and 80 mg/L by direct
supplementation into the VW medium before autoclaving. These
 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247
25 treatments per orchid species (24 treatments of chitosan
supplementation plus a no chitosan control) were subjected to an
in factorial experiment in a completely randomized design (CRD)
with four replicates, and were independently repeated twice. All of
the cultures were incubated in the same culture room at 25 8C for 12
weeks under a cool white inflorescent light (35 mmol m 2 s 1) for
16 h each day.
2.5. Characterization of the chitosan effects on seed germination and
protocorm development
To study the effects of chitosan on orchid seed germination,
experimental data were collected every 2 weeks for 12 weeks by
counting the number of seeds identified to be in each develop-
mental stage and comparing this amongst the 25 treatments of
each orchid species. All 25 treatments were as indicated above in
the seed sowing experiment.
To determine whether the developmental stage of germinating
seeds correlated well with the developing seed sizes, seeds were
examined by light microscopy during the germination process for
distinct and reliably discriminatable morphologies. The four
defined morphologies were then used to define developmental
stages. At stage 1, the seed was imbibed, but no significant
development was detected. At stage 2, the embryo was enlarged
with an increased volume, becoming rather irregular oval in shape.
At stage 3, the protocorm stage, the visible development of the
embryo resulted in an irregular globular green structure, and
finally stage 4 is defined as when the protocorm has the first clear
visible signs of a developed leaf primordium (Fig. 1). Germination
in our experiment is defined as when the seed is swollen, becoming
somewhat irregular oval in shape, which is equivalent to stage 2 in
the criteria defined above.
The diameter of at least 100 samples in each developmental stage
was measured using light microscopy with a Nikon Coolpix 8700
digital camera equipped with an 8 zoom lens. Then, the digital files
were imported to Image Pro-Plus5.1 computer program for diameter
determination. The results were subjected to statistical analysis as
described below. If discrete diameter ranges for each developmental
stage were determined, they could then be used to classify the
developmental stage of the germinating seeds.
For determination of the relative proportions (percentage) of
each developmental stage in D. formosum cultures, four Petri dishes
in each treatment were divided into a 1 cm  1 cm grid, and the
diameters of seeds/embryos/protocorms in at least three indepen-
dent such areas in each plate were measured and recorded. In the
case of D. bigibbum var. compactum, due to the lower seed density,
the total numbers of all seeds/embryos/protocorms on each plate
were examined. Seed/embryo/protocorm diameter was measured
as outlined above.
Fig. 1. The four developmental stages of germinating D. bigibbum var. compactum
seeds. Stages 1–4 are indicated by the number listed above each figure. The dashed
line indicates the plain of measurement of the diameter.
241
To enable the logistics of having to distinguish each develop-
mental stage from the thousands of germinating seeds in this
research, the diameters of the seeds, embryos and protocorms at
each clear developmental stage, as defined above, were measured
(see Fig. 1 for the region for diameter determination) from at least 30
randomly selected samples of each developmental stage in each
species. Consequently, the size boundaries for each developmental
stage for both species were established and then used to identify and
count germinating seeds of various stages with the aid of the Image
Pro-Plus 5.1 computer program in all the subsequent experiments.
2.6. Statistical analysis
The statistical analysis to determine the effects of chitosan
types and concentrations on germination percentage and seedlings
development experiments were performed using factorial analysis,
with significant difference being accepted at the p < 0.05 level.
When a significant difference was found by factorial analysis, the
data was arcsine transformed to parametric assumptions and then
analysis of variance (ANOVA) was performed, whilst Duncan’s
Multiple Range Test (DMRT) was used for comparison of means
with the same probability threshold for acceptance of significant
differences between means.
For D. formosum seedling development, after 6 weeks on
germinating medium, the chi-squared analysis of goodness of fit
was performed in a pair-wise manner between the data derived
from each chitosan treatment and the non-chitosan control, to
indicate whether chitosan affected the frequency of the different
stages of development. The null hypothesis in each case was that
the ratios of the germinating seeds at the four different stages in
the chitosan supplemented medium were the same as that of the
non-chitosan supplemented medium. In case of D. bigibbum var.
compactum seed germination, similar analysis was done but after
an 8 week germination period, because at the next time point of
data collection (8 weeks after germination for D. formosum and 10
weeks after germination for D. bigibbum var. compactum), all
protocorms had developed to stage 4 but differed mainly in sizes.
3. Results and discussion
3.1. Seed viability in D. bigibbum var. compactum and D. formosum
For the samples evaluated here, their seed viability, as
determined by the ability to reduce TTC to the red coloured
formazon (Lauzer et al., 1994; Rasmussen, 1995), was found to be
low for D. bigibbum var. compactum at 32.6% viability. In some
support of this low seed viability is that most of the seeds were
clearly morphologically deformed with abortive and incompletely
developed embryos (Fig. 2C). In contrast, seeds from D. formosum
were found to be highly viable, with 94.1% viability, as determined
by positive formazon staining, and morphologically most seeds
appeared normal with fully mature embryos (Fig. 2E).
3.2. Germinating seed diameters are discrete and can represent four
early developmental stages
Four principally discrete and reliably distinguishable develop-
mental stages of germinating seeds could be classified according to
their morphology for both orchid species (Fig. 1).
Classification of the developmental stages, according to their
morphology for both orchid species as indicated above, revealed that
the diameters of D. bigibbum var. compactum and D. formosum
germinating seeds were discrete with significant differences
between the four stages (Table 1; Fig. 3). D. formosum germinates
at a significantly slower rate such that within the 12 weeks of the
experimental period, only development up to stage 3 was observed.
242 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247

Fig. 2. Flowers (A) and seeds (B and C) of D. bigibbum var. compactum, and flowers (D) and seeds (E) of D. formosum.

Consequently discrete size boundaries for each developmental stage
for both species were established as shown in Table 2.
3.3. The effects of chitosan type and concentration on the efficiency of
seed germination and protocorm development in both Dendrobium
species
Six types of chitosan molecules, namely chitosan polymer with
70, 80 and 90% deacetylation levels (P70, P80 and P90), and
chitosan oligomers with the same degree of deacetylation (O70,
O80 and O90), were each tested at concentrations of 0, 10, 20, 40
and 80 mg/L for the effects on seed germination in both orchid
species. The deacetylation of chitin/chitosan generates free amino
groups in the polymer chain. These amino groups can be
protonated in acidic conditions, which changes the solubility of
the chitin/chitosan polymer as well as its physical, chemical and
biological properties (Taghizadeh and Davari, 2006; Chang et al.,
2008; Kachanechai et al., 2008; Lin et al., 2009).

Table 1
The analysis of the difference in seed/seedling diameters of different developmental stages of D. bigibbum var. compactum and D. formosum.

Stage Diameter (mm) S.E. Subset for alpha = 0.05

1 2 3 4

D. bigibbum var. compactum

1 0.08913 0.00158 0.08913
2 0.14301 0.00307 0.14301
3 0.41195 0.01274 0.41195
4 0.9345 0.02724 0.9345

Sig. 1 1 1 1

Stage Diameter (mm) S.E. Subset for alpha = 0.05

1 2 3

D. formosum
1 0.11678 0.0018 0.11678
2 0.14221 0.00225 0.14221
3 0.21565 0.00312 0.21565
Sig. 1 1 1
 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247 243

Table 2
Diameter size ranges for each development stage.

Species Stage 1 Stage 2 Stage 3 Stage 4

(mm) (mm) (mm) (mm)

D. bigibbum var. 0.114 0.115–0.273 0.274–0.666 0.667

compactum
D. formosum 0.128 0.129–0.178 0.179 nd

The proportion of D. bigibbum var. compactum seeds that

Fig. 3. Scatter plots of the size of the four developmental stages of D. bigibbum var.
compactum (A) and of three developmental stages of D. formosum (B) showing the
degree of size overlap between each defined developmental stage.
germinated on VW semi-solid medium without chitosan was found
to be quite low at only 10.5%. Compared to the seed viability of 32.6%,
this represents a germination level of only approximately one-third
of the viable seeds. The addition of 70 or 80% deacetylated chitosan
caused a trend of decreasing germination rates with increasing
chitosan concentrations for both the polymer and oligomer forms,
but statistically this was only significantly different from the control
at the highest tested dose (80 mg/L) of P70, P80 and O70. In contrast,
both the polymer and oligomer forms of chitosan with 90%
deacetylation showed no significant effect upon the germination
rate at all four tested doses. Although numerically the addition of
10 mg/L of O70 or P80 chitosan resulted in an enhanced seed
germination rate when compared to the treatment without chitosan
addition, these were not statistically significant (Fig. 4A). Therefore,
none of the six types and four doses of chitosan tested were suitable
for enhancing the seed germination in D. bigibbum var. compactum.
Even after taking account of the low seed viability (32.6%), and thus
the potential near 1/3 dilution effect of any effective changes in the
seed germination rates, only P80 at 10 mg/L and O70 at both 10 and
20 mg/L would be likely to have any slight effect of increased
germination (ca. 10.5% to 13.7, 15.0 and 12.2%, respectively)
(Fig. 4A).
In contrast, a slight but statistically significant increase in the
germination ratios of D. formosum seeds was observed in response
to inclusion of all six types of chitosan at all four tested doses. O80

Fig. 4. The proportion of seeds that had germinated for D. bigibbum var. compactum after 12 weeks (A), and for D. formosum after 4 weeks (B), when sown in modified VW
medium supplemented with the indicated various chitosan types and concentrations. (C) Represents the control experiment without chitosan addition. Data are shown as the
mean  1 S.E. and are derived from four independent replications. The columns marked with different letters are significantly different (P < 0.05; DMRT).
244 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247
revealed a decreasing level of enhanced germination with
increasing doses, resulting in the lowest rate seen in all the
chitosans tested at the highest dose of 80 mg/L, but this was still
significantly higher than that attained without chitosan. However,
both O90 and P90 showed no chitosan dose-dependency to their
induced enhancement of the germination within the tested range,
whilst P70 showed a weak bell-like dose response with the
maximal enhancement of seed germination being attained at
20 mg/L. Taking into account, the seed viability of 94% for these D.
formosum seeds, then the germination levels seen from O70 at
80 mg/L and O80 at 10 mg/L treatment at 90.6 and 91.2%,
respectively, would be nearly maximal compared with that
attained in the absence of chitosan at 67.8% (Fig. 4B).
These results of the chitosan-mediated enhanced seed germi-
nation in D. formosum outlined here are consistent with the
reported effect of chitosan in other studies, such as in the prior
treatment of wheat seeds infected by the plant pathogenic fungus,
Fig. 5. Developmental stage distribution of D. bigibbum var. compactum after 8
weeks of germination. Data are shown as the mean  1 SE. Means with an asterisk are
significantly different (P < 0.05).
Fusarium graminearumwit, with a chitosan solution (2–8 mg/ml)
for 15 min to improve the seed germination capacity (Bhaskara
Reddy et al., 1999), and the ability of the chitosan formulation
ElexaTM to increase the proportion of germinating seeds when used
at a 20-fold dilution (Sharathchandra et al., 2004). Although it is
plausible that in these studies the chitosan removed or controlled
the plant pathogens and induced plant phenolic responses via the
induction of phenylalanine ammonia-lyase (PAL) gene expression
(Vander et al., 1998; Lin et al., 2005), or any other unknown
mechanism(s) that enhances germination, it is not clear whether
the same broad mechanism is operating here at these lower
chitosan doses inducing D. formosum seed germination. In contrast,
chitosan application failed to enhance seed germination in D.
bigibbum var. compactum, and rather, in four of the six tested
chitosans (O70, O80, P70 and P80), a significant dose-dependent
decrease in the seed germination was found. Again, whether this is
due to chelation of essential molecules or the overdose signalling
of chitosan action at a molecular level remains to be evaluated.
What is clear is that this effect is partially chitosan specific in terms
of both oligomer/polymer composition and, especially, the
deacetylation ratio. Taking into account the observed seed viability
with the germination ratios seen, the data is consistent with, but
not conclusive for, the notion that chitosan cannot rescue the pre-
existing deformation of non-viable seeds, but acts as a growth
stimulator/inhibitor on the normal or dormant mature embryos, as
would be expected and predicted.
Fig. 6. Developmental stage distribution of D. formosum after 6 weeks of
germination. Data are shown as the mean  1 SE. Means with an asterisk are
significantly different (P < 0.05).
 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247
After 8 weeks of seed germination, four various stages of
development were found in D. bigibbum var. compactum seed
germination. Over 80% of germinating seeds on all media were at
developmental stage 1. However, the percentage of other
developmental stages found on the media containing 10 mg/L of
P70, P80, P90 or O70, 20 mg/L of O70 or O80, or 40 ppm of O90
were significantly higher than that found on the same medium
without chitosan addition. Each of these seven specific chitosan–
dose combinations enhanced protocorm development to the
fourth stage within 8 weeks, while only three stages of protocorms
were found in the control medium (Fig. 5). In the absence of any
significant mortality during this culture period, except for the high
dose (80 mg/L) of chitosan treatment (range of 33.0–82.2% across
chitosan treatments compared to 57.8% in the control), these data
represent true differences in development rather than that of
differential developmental mortality.
After 12 weeks of D. formosum germination only the first three
developmental stages of protocorms were found, but with no
evidence that chitosan could enhance protocorm development.
When D. formosum seeds were germinated on modified VW
medium for 6 weeks, a significant difference in the developmental
stages attained in each medium was found (Fig. 6). More than 77%
of germinating seeds were at stage 3, and only 3.7% were at stage 1
in the medium without chitosan. In contrast, in the media
containing the different chitosans, stage 3 germinating seeds
were found at levels of more than 77%, except for in the media
containing 80 mg/L of P70. The addition of 20 mg/L of P80,
increased the proportion of stage 3 protocorms up to 91%, and
again in the absence of significant mortality this, therefore,
represents increased development of protocorms rather than the
effects of differential mortality. A similar trend, including the
Fig. 7. Seedling diameters of D. bigibbum var. compactum (A) and D. formosum (B) after 8 weeks of germination. Data are shown as the mean  1 S.E. and are derived from four
independent replications. Columns marked with different letters are significantly different (P < 0.05; DMRT).
245
absence of differential mortality, in the enhanced protocorm
development in each medium was found up to 12 weeks after
germination. Pearson’s chi-squared test, performed to distinguish
the significant difference in the proportions of each developmental
stage present in each medium, revealed a significant difference
between the developmental stages in all media containing
chitosan, which were more advanced, than in the medium without
chitosan addition, with the exception of the medium containing
80 mg/L of O80 (Fig. 6).
These data indicate that both the chitosan form and concentra-
tion can have a significant effect on the protocorm development of
some orchids, or at least on D. formosum and D. bigibbum var.
compactum.
The significant effects of chitosan concentration were first
detected from 6 weeks after germination of D. bigibbum var.
compactum. During the first 2 weeks of germination, the seed
diameters were too small to be accurately measured for their
diameter. Therefore, the diameter measurement was performed
after 4 weeks of germination by which time the chitosan had begun
to show apparent effects on protocorm growth. The chitosan types
and concentrations independently affected protocorm growth
after 8 weeks of seed germination, but by 12 weeks no effects on
seedling growth due to different chitosan types was evident.
The growth of D. bigibbum var. compactum germinating seeds on
modified VW medium with and without chitosan supplementation
after 8 weeks is shown in Fig. 7A. A highly significant difference in
the mean seed germination number was detected with factorial
analysis and O70 at 10 mg/L and O80 at 20 mg/L significantly
enhanced embryo development. In contrast, some chitosan types,
and especially P70, showed negative growth effects at higher
concentrations (20, 40 and 80 mg/L) (Fig. 7A).
246 N. Kananont et al. / Scientia Horticulturae 124 (2010) 239–247
In contrast to that the results seen with D. bigibbum var.
compactum, chitosan enhanced D. formosum growth at 6 weeks after
germination. After 8 weeks of germination a clear chitosan dose-
dependence for the enhanced level of embryo growth was also found.
The addition of P70 at 10 or 20 mg/L showed the highest
enhancement for D. formosum seedling development, attaining an
average diameter of about 11% larger than that of the seedlings
grown on the medium without chitosan. However, chitosans at most
tested doses showed either no significant changes (P80, P90 and
O90), or at some concentration of some types of chitosan, such as
40 mg/L of O80 or 80 mg/L of P90, displayed some inhibition such
that the embryos were up to 8–9% smaller in diameter compared to
those germinated and cultured without chitosan (Fig. 7B). After 12
weeks, the same treatments that showed the growth enhancement
effect, 10 or 20 mg/L P70, stimulated seedling development attained
up to 14% larger in diameter seedlings compared to the control (data
not shown). According to the factorial analysis, both chitosan types
and concentrations, independently affected D. formosum growth up
to 12 weeks.
3.4. Positive effects of chitosan on the germination and growth of
seeds shows a species—molecular type and concentration dependent
pattern
Based on these results, it was shown that five (P70, P80, P90, O70
and O80) out of six of the chitosan types tested at 10 mg/L enhanced
the germinating seed growth of D. bigibbum var. compactum. In
contrast, for D. formosum only P70 at 20 mg/L showed a significant
positive effect upon the growth of germinating seeds, although a
weaker positive response was seen with P70 at 10 mg/L and O70 at
20 mg/L. Moreover, all chitosans showed either negative or no
growth effects at 80 mg/L on both orchid species and this was most
evident with O80 on D. formosum. In the case of P70 and P80, the
trends seen for seed growth largely mirror that seen for germination
ratios (Fig. 4) in both orchid species, but the other chitosans show
trends that differ between the two parameters in terms of orchid
species, chitosan formulae and effective dose. Taken together, these
indicate that the appropriate chitosan formulae and concentration
may vary from species to species, but with the appropriate use,
chitosan can significantly enhance plant growth. Most of the previous
studies on the positive effects of chitosan on plant growth (Ohta et al.,
1999, 2004) were carried out with only one type of chitosan molecule.
However, Nge et al. (2006) showed that 1 kDa shrimp chitosan was
more effective than 10 and 100 kDa chitosans in improving D.
phalaenopsis protocorm-liked body culture in vitro, whilst O80 was
shown to be the best of the tested chitosan molecules in improving
floral production in Dendrobium ‘Eiskul’ (Limpanavech et al., 2008).
Based on our studies, we propose that orchid seed germination could
be enhanced by chitosan, but that the appropriate chitosan type and
concentration may need to be studied on a case by case basis, or at
least until sufficient cases have been characterised as to allow any
clear predictive patterns and generalisations, if any, to emerge.
In conclusion, all six chitosan types tested (O70, O80, O90, P70,
P80 and P90) at all concentrations tested (10, 20, 40 and 80 mg/L),
significantly enhanced seed germination in D. formosum, but no
significant improvement of seed germination in D. bigibbum var.
compactum was found. However, for those seeds of D. bigibbum var.
compactum that did germinate, 10 mg/L of all tested chitosan types,
except O90, was a more effective concentration to improve
protocorm growth, while 10 or 20 mg/L of P70 was better for D.
formosum.
Acknowledgements
This research was supported by Agricultural Research Devel-
opment Agency (Public Organization). We would like to thank the
support from Research Funds from the Faculty of Science,
Chulalongkorn University. D. formosum capsules were provided
by Mr. Sittichai Prikdang, the Head of Nam Tok Ngao National Park,
Ranong, Thailand. The authors would like to express our
appreciation to Prof. Dr. Richard A. Criley, Dept. of Tropical Plant
and Soil Science, University of Hawaii at Manoa, and two
anonymous referees for their valuable comments, and Dr. Robert
Butcher for his comments and English language proof reading.
Finally, we would like to thank all members of the research team
for all their contributions to this project.
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