203 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Culture of Borneos Endemic Orchid AsPac J. Mol.

Biol. Biotechnol. 2010

Vol. 18 (1) : 203-207
In Vitro Culture of Borneos Endemic Orchid, Vanda dearei
Roslina Jawan
1
, Jualang Azlan Gansau
1
* and Janna Ong Abdullah
2
1
School of Science and Technology, Universiti Malaysia Sabah, Locked Bag 2073, 88999, Kota Kinabalu, Sabah.
2
Faculty of Biotechnology and Biomolecule Science, Universiti Putra Malaysia. 43400 Serdang, Selangor DE.
Proceedings Asia Pacifc Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007
* Author for correspondence:
Jualang Azlan Gansau, School of Science and Technology, Universiti Ma-
laysia Sabah, Locked Bag 2073, 88999, Kota Kinabalu, Sabah, Telephone:
+6088-320000 ext: 5875, Fax: +6088-435324, e mail: azlanajg@ums.edu.
my.
Abstract. Te efect of basal media, complex additives and sugars to the growth and development of V. dearei protocorms were
reported in this study. Protocorms were initiated from in vitro seeds germination from immature capsule. Among the three basal
media, growth of protocorms was superior in MS medium followed by KC and VW with maximum percentage of protocorms
with leaf and root, and mean number of leaf and root produced. Meanwhile, 0.2% (w/v) yeast extract markedly enhanced the
growth of protocorms. In sugar study, results show that protocorms preferred sucrose as compared to fructose and glucose. Te
leaf and root formation was signifcantly enhanced as the sugar concentration was decreased. Medium supplemented with 2%
(w/v) sucrose was the best compared to the other treatments and sugar at a concentration of 4% (w/v) induced the formation of
large size seedlings.
Keywords: Vanda dearei; Protocorms; Propagation; Growth medium.
INTRODUCTION
Vanda dearei (Orchidaceae) is a tropical epiphytic or-
chid and native to the Borneo Island. It can be found in
the Sapulot and Tomani area in Sabah, Kuching in Sarawak,
Sekayan River in West Kalimantan and Kutai in East Kali-
mantan (Chan et al., 1994). A large, pale yellow and strong-
ly scented fowers and fowering throughout the year make
this species one of the beautiful orchid and become favorite
and ecstatic orchid species (Lamb, 1978). It has been culti-
vated for hybrid production as this species is one of the chief
sources of yellow color in Vanda hybrids (Chan et al., 1994;
Motes & Hofman, 1997). Te damaging efect of human
activities on natural habitats has resulted in great loss of the
species. In addition, intensive and illegally collection lead-
ing to alternated of this species (Fay, 1994). Terefore, there
is an urgent need to conserve this ecstatic species. Numer-
ous studies on efcient propagation of orchid plants have
been published (Arditti, 1977; Colli & Kerbauy, 1993), and
suggested that method is species-specifc oriented. Optimi-
zation of medium compositions was an important approach
to fasten the propagation process and improve the quality
of regenerated plantlets (Ichihashi, 1992; Chen et al., 2000;
Park et al., 2002). Among them, diferent carbon sources
such as sucrose, glucose, maltose, fructose and sorbitol also
were proven to have efects on protocorm-like bodies (PLBs)
growth (Islam & Ichihashi, 1999; Tokuhara & Mii, 2003).
Rahman et al. (2004) had demonstrated that the addition
of crops enhanced growth of the Doritaenopsis sp. calli and
promoted the regeneration of PLBs. In the economic im-
portance of view, the need for formulation of simple culture
media is important to reduce the cost of production. Tis
study reports the infuence of basal media, complex addi-
tives and sugars to the growth and development of V. dearei
protocorms.
MATERIALS AND METHODS
Ninety days old protocorms of V. dearei aseptically raised
through in vitro immature seed germination were used as
starting material. Fifteen protocorms at 0.7-1.0mm in size
were cultured on plastic Petri dish containing 25ml of me-
dia. Tree basal media were assessed for growth and develop-
ment efciency: Knudson C (KC) (Knudson, 1946), Mu-
rashige and Skoog (MS) (Murashige & Skoog, 1962), and
Vacin and Went (VW) (Vacin & Went, 1945). In complex
additives study selected basal medium (KC) supplemented
with various types and concentrations of complex additives
such as coconut water (10-20%, v/v), tomato juice (10-
20%, v/v), banana pulp (2.5-12.5%, w/v), peptone (0.2%,
w/v) and yeast extract (0.2%, w/v). For the efect of sugars,
sucrose, glucose and fructose at concentrations of 1%, 2%,
204 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Culture of Borneos Endemic Orchid
Figure 1. Efect of basal media on growth and development
of V. dearei protocorms. (A) Percentage of protocorms with
leaf; (B) Mean number of leaf; (C) Percentage of protocorms
with root; (D) Mean number of root.
205 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Culture of Borneos Endemic Orchid
Figure 2. Efect of complex additives on growth and devel-
opment of V. dearei protocorms on KC medium (Knudson,
1946) added with 0.2% (w/v) yeast extract. (A) Percentage
of protocorms with leaf; (B) Mean number of leaf; (C) Per-
centage of protocorms with root; (D) Mean number of root.
Figure 3. Efect of sugars on growth and development of V.
dearei protocorms on KC medium (Knudson, 1946) added
with 0.2% (w/v) yeast extract. (A) Percentage of protocorms
with leaf; (B) Mean number of leaf; (C) Percentage of pro-
tocorms with root; (D) Mean number of root.
or 4% (w/v) were separately added into the KC medium
supplemented with 0.2% (w/v) yeast extract. Te pH of the
medium was adjusted to pH 5.3 before solidifed with 0.9%
(w/v) agar and autoclaved at 121 oC (15p.s.i) for 20 min.
Te cultures were grown at 252 oC under continuous illu-
mination (20-50molm-2s-1) provided by cool white fuo-
rescent tubes (Philips, Malaysia).
Experiments were performed in a completely randomized
design. Each treatment was conducted in 10 replicates. Te
number of protocorms with leaf and root, and number of
leaf and root produced were observed and recorded after 30
days of culture. Te data collected was analyzed by SPSS
(Statistical Package for Social Science) Ver. 12 and means
were compared with Duncan Multiple Test.
RESULTS AND DISCUSSION
Growth and development. After 14 days of culture (DAC)
V. dearei protocorms appear with the upper site curve surface
and papillae structure. Te shoot emergence was observed
at 21 DAC and leaf develops at 28 DAC. Te development
was continued with the formation of second leaf and root
(56 DAC), third leaf (63 DAC) and matured seedling at
150 DAC.
Efect of basal media. Protocorms responded variably on
three diferent basal media (Figure 1). Among the three basal
media had tested, growth of protocorms was superior in
MS to that in KC and VW medium. Tis basal medium
achieved the highest percentage of protocorms with leaf
(65%) and roots (21.67%) that produced up to 1.890.49
leaves and 0.850.61 roots after 60 DAC. Tis might per-
haps be due to its richness in micro element regime (Kishor
et al., 2006) and organic and inorganic nitrogen sources
206 AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010 In Vitro Culture of Borneos Endemic Orchid
(Majerowicz et al., 2000). After 120 DAC protocorms cul-
tured on KC basal medium show the better response on
development compared to MS (data not shown). Te
nutrient requirement was specifc at diferent growth stages
(Kauth et al., 2006).
Efect of complex additives. Protocorms cultured on basal
medium supplemented with various type and concentra-
tion of complex additives showed a varied growth response
(Figure 2). Treatment with 0.2% (w/v) yeast extract was
the best to promote the fastest response for the induction
of shoot (20DAC) and root (30DAC) followed by coconut
water, peptone, tomato juice and banana pulp. Tis treat-
ment attained maximum percentage of protocorms with
leaf (100%) and root (70%) that produced up to 3.150.5
leaves and 1.10.09 roots after 60 DAC. Similar result also
has been reported by Mathews & Rao (1980) in protocorms
development of Vanda hybrids. Complex additives have
reported containing carbohydrates, protein, fat, vitamins,
phenolic compounds, and a low level of amino and organ-
ic acids which can infuence the growth and development
of protocorm culture (Rahman et al., 2004). Yeast extract
containing about 9.8% total nitrogen comprising prima-
rily 5.1% amino nitrogen as amino acids (Arditti & Ernst,
1993). Supplementation of organic extracts to the orchid
culture medium is simple and practical method to improve
culture media used for commercial production.
Efect of sugars. Protocorms growth and development were
greatly afected by source and concentration of sugars added
in the culture media (Figure 3). Sucrose was the most pref-
erable sugar followed by glucose and fructose. Te leaf and
root formation was signifcantly enhanced as the sugar con-
centration was decreased. Medium supplemented with 2%
(w/v) sucrose was the best compared to the other treatments
with 98.67% protocorms produce leaf and 94.57% produce
root with an average of 2.810.18 leaves and 1.670.12
roots after 60DAC. Beside that sugar at a concentration of
4% (w/v) induced the formation of large size seedlings (data
not shown). Sugar has a great potential efect on the physi-
ology, growth and diferentiation of the cultured cells and
tissues (Nowark et al., 2004).
CONCLUSION
In conclusion MS medium, 0.2% (w/v) yeast extract
and 0.2% (w/v) sucrose was the best factors for the growth
and development of V. dearei protocorms.
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