IJERD – International Journal of Environmental and Rural Development (2013) 4-2

Research article
erd

Effects of Chitosan and Lotus Extracts as Growth
Promoter in Dendrobium Orchid
PIYAVADEE CHAROENWATTANA*Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand
Email:piyavadeec@yahoo.com

UMNOUI PETPRAPAI
MahidolUniversity, Bangkok, Thailand
Received 14 January 2013

Accepted 10 June 2013(*Corresponding Author)

Abstract The objective of this research was to investigate the effects of chitosan and lotus
extracts to stimulate growth of orchid plantlets in greenhouse conditions. Chitosan was
extracted from prawn shells (Penaeus monodon) with 90% deacetylation (DD) and 8589% purity. The lotus extracts from the leaves of Nelumbo nucifera ‘Roseum Plenum’
were used as the sources of antimicrobial agents. The Dendrobium orchid ‘Sureepeach’plantlets were treated with 10,30,50 and 100 mg L-1 chitosan and 1,3 and 5 g L-1 lotus
extracts at 7 day intervals. The combination of 100 mg L-1chitosan and 5 g L-1 lotus
extracts gave the maximum number of leaves and shoots of the plantlets after 10 weeks oftransplanting.
Keywords chitosan, lotus extracts, orchid, growth promoter

INTRODUCTION
Thailand is the world premier producer and exporter of orchids. The Thai orchid industry is wellknown for both cut orchid flowers and the supplier of in vitro plantlets and pre-blooming plants.
Most micro-propagated Dendrobium hybrids are the majority of Thai cut flowers. Key export
markets are in Asia (China, Korea, and Japan), Europe (Italy and the Netherlands), the US andAustralia. In 2011, the quantity of orchid exports was 23,392 tons or equivalent to Baht 2,111
million (Office of Economic Agriculture, 2012). The government's orchid strategy (2011-2016)envisages that Thailand will become the world’s leading orchid exporter, with a target export value
reaching 10,000 million Baht by 2016 (Than Sethakit. 2012). Increased demand for orchids could
lead to an expansion of cultivated areas, especially for large scale production of orchid plantlets. To
triple orchid production, the strategy aims to improve the quality of orchids and diversify theirvarieties. The orchid industry will benefit from the development of more efficient micropropagation techniques, especially Dendrobium orchids.
Chitosan is wildly used in agriculture due to its positive effects on plants growth anddevelopment. Chitosan (poly [b-(14)-2-amino-2-deoxy-D-glucopyranose]), a biopolymer
derivative of chitin, is mostly found in the exoskeleton of arthropods and crustaceans (Palpandi, etal., 2009). In Thailand, chitin can be extracted from the abundant prawn and crab shells that are
sustainable and readily available as a natural waste product from the seafood industry. For the cut
flower hybrid orchid, Dendrobium ‘Earsakul’, chitosan of 45 kDa molecular weight and a >90%
degree of deacetylation (DD) applied at a concentration of 1–100 mg/L significantly increases bothquantitatively and qualitatively inflorescence yields (Limpanavech et al., 2008). However, theimpacts of chitosan on in vitro orchids and orchid propagation have been limited. Nge et al. (2006)
found that the size and the origin of the chitosan as well as its concentration affected the number ofDendrobium and Phalaenopsis plantlets regenerated from a protocorm-like body (PLB).
Additionally, the research also found that spraying with chitosan significantly reduced the severityof leaf spot diseases in orchids, promoted plant growth of some orchids, and increased the size andlength of the Dendrobium florets and inflorescences, respectively (Uthairatanakij et al., 2007).

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Moideen et al. (2011) found that the medicinal plant of Nelumbo nucifera (lotus) has a wide
range of medicinal properties. Various techniques using different chemical solutions such as
ethanol, methanol and water were applied to the leaves of Nelumbo nucifera to obtain someextracts for testing using preliminary phytochemical analysis. The antimicrobial susceptibility
studies were conducted against gram (-) bacteria such as E. coli, P. aeruginosa, K. pneumonia andgram (+) bacteria such as Staphylococcus aureus. The results show that ethanol extract is antimicrobial.
Li and Xu (2008) tested the anti-microbial of the lotus leaf extracts against fivemicroorganisms. The most active antimicrobial extract was subjected to spectroscopic analysis.
The result shows that the minimum inhibitory concentrations of the most active extract were 0.625,
1.25, 1.25, 0.625 and 2.5 mg/mL for Actinobacillus actinomycetemcomitans Y4, Actinomyces
viscosus 19246, Porphyromonas gingivalis 33277, Fusobacterium nucleatum 25586, andActinomyces naeslundii wvl45. Quercetin from lotus leaves was found to have the greatest
antimicrobial activity; this suggests it could be a potential antibacterial agent for periodontitis.
The use of chitosan and lotus leave extracts to stimulate growth and increase disease
resistance is expected to increase orchid production and thus further develop the orchid exportindustry. They will replace chemical fertilizer and pesticide and are environmental friendly.
OBJECTIVE
This research aims to demonstrate that chitosan and lotus extracts can stimulate the growth ofDendrobium orchid plantlets.
METHODOLOGY
Extraction of chitin and chitosan
Chitin and chitosan were produced from prawn shells through demineralization, deproteinizationand deacetylation (Burrows et al., 2007). Shells of prawns (Penaeus monodon) were washed, dried
and then ground into small pieces. Approximately 25 g of prawn shells were demineralized with
100 ml of 1% (v/v) hydrochloric acid (HCl) for 24 h to remove the minerals (mainly calciumcarbonate). To obtain the chitin, the washed and cleaned shells were transferred to 50 ml of 2%(w/v) sodium hydroxide (NaOH) and soaked for 1 hour in order to dissolve proteins and sugars.
Subsequently, the chitin was cleaned and oven dried, resulting in a chitin product that wassubsequently de-acetylated in 100 ml 50% (w/v) sodium hydroxide (NaOH) and boiled at 100°C
for 2 h on a hot plate to obtain chitosan. The 1% (w/v) chitosan solution was obtained from
dissolving 5 g of chitosan in 500 ml of 0.1M acetic acid and stirred continuously at 55°C for 12 h.
The filtrated chitosan solution had the pH of 3.99, 90% deacetylation (DD), and 85-89% purity.
Concentrated chitosan of 10, 30, 50 and 100 mg L-1was obtained from diluted chitosan in 1% aceticacid.
Lotus extracts
The leaves of Nelumbo nucifera ‘Roseum Plenum’ were air dried for 2-3 days before cutting into
small pieces (2-3 cm.) and ground up. The leaves were coldly extracted using ethanol at roomtemperature. 200-gram leaves were filled in a cloth bag and soaked in 3 L of ethanol three times
during one week until no extracts came out. Subsequently, the ethanol was discarded using a rotary
evaporator to get the brown and concentrated crude extracts that were kept in a cold dry place.
Crude extracts were fractioned using the Column Chromatography technique to derive hexane
crude extracts, which were then diluted into 1, 3 and 5 g L-1 concentrations.
Plantlets preparation© ISERD
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IJERD – International Journal of Environmental and Rural Development (2013) 4-2

The study also used Dendrobium ‘Sureepeach’ plantlets from Mana orchid farm, Nakornpathom
province. 2 to 4 leave plantlets with well-developed roots were removed from medium culture andwashed thoroughly under running tap water to remove traces of agar. Plantlets were acclimatized inpots for 4 weeks, then transferred to a greenhouse and watered daily with 400 g L-1 of 21-21- 21
fertilizer every week for 10 weeks. The Dendrobium orchid ‘Sureepeach’ plantlets were treatedwith 10, 30, 50 and 100 mg L-1 chitosan and 1, 3 and 5 g L-1 lotus extracts at 7 day intervals.
Data analysis
All experiments were carried out in a completely randomized design with three replications underdaily visual observations to record the number of leaves and shoots over the course of 10 weeks.
Subsequently, an analysis of variance (ANOVA) and means were calculated and compared using
Duncan’s new multiple rang test (DMRT).

adbcefFig. 1 Chitin (b) and chitosan (c) are produced from prawn shells (a) throughdemineralization, deproteinization and deacetylation, ethanol extracts comefrom lotus leaves (d), the crude extracts were fractioned by rotary evaporator(e) to obtain brown crude extracts (f)ADabcdFig. 2 Flowers of Dendrobium ‘Sureepeach’ (a), plantlets were transplanted to pots filled withsmall pieces of coconut husk (b), chitosan and lotus leave extracts (c, d)© ISERD135

IJERD – International Journal of Environmental and Rural Development (2013) 4-2

RESULTS AND DISCUSSION
The study found that the number of leaves after 2 weeks of transplanting was not statisticallydifferent. However, the number of leaves after 4, 8 and 10 weeks of transplanting was significantlydifferent. The orchid plantlets treated with 100 mg L-1 chitosan in combination with 5g L-1 lotus
extracts gave the maximum number of 8.5 leaves. The control plantlets without the treatment ofchitosan and lotus extracts gave an average number of 5.1 leaves. The new shoots and leaves thatemerged after 8 weeks were strong compared to those without the chitosan and lotus extract
treatment after 10 weeks. The application of 100 mg L-1 chitosan combined with 5g L-1lotus
extracts resulted in the maximum number of leaves on average. The concentrated chitosan used inthis study was higher than in Dendrobium orchid ‘Earsakul’ that was applied to 1-100 mg L-1
chitosan.
Table 1 The effects of chitosan and lotus extracts on the number of Dendrobium ‘Sureepeach’leaves within 10 weeks after transplanting in the greenhouse
Treatment
Number of leaves per pot
ChitosanLotus extracts
Week(mg L-1)(g L-1)246800
3.0
3.1 a-e
3.2 a
3.9 a-d1
3.6
3.3 a-d
2.2 b-d
2.8 de3
3.3
4.0 a
2.8 ab
4.9 ab53.8
2.7 c-e
2.7 ab
2.8 de100
3.4
3.6 a-c
2.8 ab
3.4 c-e1
3.4
3.7 ab
2.8 ab
4.4 a-c3
3.0
3.0 b-e
2.7 ab2.5 e
5
3.2
2.8 b-e
2.2 b-d
4.1 a-d300
2.8
2.8 b-e
2.2 b-d
4.1 a-d1
3.2
2.7 c-e
2.1 b-d
3.3 c-e3
3.4
3.4 a-d
2.3 a-c
4.4 a-c5
3.3
4.0 a
2.9 ab
3.5 b-e500
3.1
2.9 b-e
2.8 ab
4.5 a-c1
2.7
2.2 e
2.3 a-c
3.6 b-e3
3.4
3.2 a-d
2.6 a-c
3.6 b-e5
2.7
2.6 de
1.4 d
3.7 a-e1000
3.2
2.8 b-e
1.7 cd
3.4 c-e1
2.7
2.8 b-e
2.6 a-c
4.4 a-c3
3.4
2.7 c-e
2.6 a-c
3.8 a-e5
3.3
3.1 a-e
2.5 a-c
5.0 aF-test
Ns
**
*
**
CV (%)
15.7
15.9
18.9
18.6
Means followed by the same letter in the same column had no significant difference by DMRT*, ** indicatessignificance at 5% and 1% levels of probability, respectively.ns = not significant

a105.1 b-f
3.7 ef
6.4 a-d
2.8 f
4.9 b-f
6.3 a-d
3.7 ef
5.6 b-e
4.2 c-f
4.5 c-f
6.6 a-c
4.2 c-f
5.7 b-e
4.9 b-f
4.7 c-f
5.6 b-e
4.0 d-f
6.1 a-e
7.4 ab
8.5 a**
4.2bFig. 3 Comparison of the effects of chitosan and lotus extracts on plantlet growth
controlled plantlets (a) and plantlets treated with chitosan and lotus extracts after10 weeks (b),Photograph is representative of three such independent replications.

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It increased both the yield and quality of the inflorescence (Limpanavech et al., 2008). The plant
responded differently to various type of chitosan molecule. The lower molecular weight of chitosan
was more effective in inducing and disease resisting than the higher molecular weight of chitosan(Lin et al., 2005).
CONCLUSION
The appropriate concentrations of chitosan and lotus extracts were investigated for their effects on
the growth of Dendrobium orchid plantlets in greenhouse conditions. According to the result
obtained, prawn chitosan has the ability to enhance the survival rate and the growth of the plantlets.
Chitosan of 100 mg L-1 are the most effective. The results indicates that plantlets treated with 100
mg L-1 chitosan and 5 g L-1 lotus extracts yielded the maximum number of leaves and shoots after
10 weeks of transplanting. However, further work is required to evaluate the effects of chitosan andlotus extracts interaction or alone for various stages of plant growth.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the Faculty of Agricultural Technology,
Rajamangala University of Technology Thanyaburi, which provided a research grant from theUniversity’s extra-budgetary revenue to finance this study.
REFERENCES
Burrows, F., Louime, C., Abazinge, M. and Onokpise, O. 2007. Extraction and evaluation of chitosan fromcrab exoskeleton as a seed fungicide and plant growth enhancer. American-Eurasian J. Agric. andEnviron. Sci., 2(2), 103-111.
Li, M. and Xu, Z. 2008. Quercetin in lotus leaves extract may be responsible forantibacterial activity. Arch.
Pharm. Res., 31(5), 640-644.
Limpanavech, P., Chaiyasuta, S., Vongpromek, R., Pichyangkura, R., Khunwasi, C. Chadchawan S.,
Lotrakul, P., Bunjongrat, R., chaidee, A., and Bangyeekhun, T. 2008. Chitosan effects on floral
production, gene expression, and anatomical changes in the Dendrobiumorchid. J. Scientia
Horticulturae, 116, 65-72.
Lin, W., Hu, X., Zhang, W., John Rogers, W. and Cai, W. 2005. Hydrogen peroxide mediates defence
responses induced by chitosans of different molecular weights in rice. Journal of Plant Physiology, 162,937-944.
Moideen, M.M.J., Raffick.M., Kumar, S. and Kishore, N. 2011. Antimicrobial activity and phytochemical
analysis of Nelumbonucifera leaves. Journal of Global Trends inPharmaceutical Sciences, 2, 404-410.
Nge, K.L., New, N., Chandrkrachang, S. and Stevens, W.F. 2006. Chitosan as a growth stimulator in orchid
tissue culture. Plant Science, 170, 1185-1190.
Office of Economic Agriculture, 2012. The quantity of orchid exports.
(Available: http://www.oae.go.th/download/bapp/Q3-2555.pdf, accessed: November 15, 2012.)
Palpandi, C., Shanmugam, V. and Shanmugam, A. 2009. Extraction of chitin and chitosan from shell andoperculum of mangrove gastropod Nerita (Dostia)crepidularia Lamarck. International Journal ofMedicine and Medical Sciences, 1(5), 198-205.
Than Sethakit. 2012. Orchid production. (Available: http://www.thannews.th.com, accessed: November 3,2012.)
Uthairatanakij, A., Teixeira da Silva, J.A. and Obsuwan, K. 2007. Chitosan for improving orchidproduction
and quality. Orchid Science and Biotechnology, 1, 1-5.

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