Effects Of Chitosan As The Growth Stimulator

For Grammatophyllum Speciosum In Vitro

Culture

PRESENT BY;
MALEESHA MADUBHASHINI :00020021
MITHUNI YOMALI : 00020006
CONTENT

1. Background
2. Research problem
3. Objective
4. Literature review
5. Materials and methods
6. Results and discussion
7. Conclusion
8. References
1) BACKGROUND

• Grammatophyllum speciosum, a prized orchid, faces challenges with traditional

propagation and prompting exploration into tissue culture despite its cost and slow pace

• Studies suggest Chitosan, a natural polymer, can act as a plant growth stimulator.

• This research investigated the effectiveness of Chitosan in boosting the growth rate of G.
speciosum during in vitro culture

• Chitosan successfully increased the growth rate of G. speciosum protocorm-like bodies

(PLBs) in liquid culture medium, showing promise as a growth promoter
2) RESEARCH PROBLEMS

• How does the growth rate of G. speciosum vary with different concentrations of Chitosan in
liquid culture?

• What is the difference in growth rate between G. speciosum PLBs cultured in Chitosan-
supplemented medium and those in a control group?

• What are the feasibility and effectiveness of Chitosan as a growth promoter in G. speciosum
tissue culture, based on its impact on growth rate and overall viability?
 3) OBJECTIVES
Main Objective

• To investigate the potential of Chitosan as a growth stimulator for Grammatophyllum speciosum in

vitro culture
Sub Objectives

• To investigate the impact of varying Chitosan concentrations on the growth rate of G. speciosum
using liquid culture
• To compare the growth rate of G. speciosum PLBs in Chitosan-supplemented medium versus a
control group
• To analyze the feasibility and effectiveness of Chitosan as a growth promoter in the context of G.
speciosum tissue culture
 4) LITERATURE REVIEW
• Grammatophyllum speciosum, a rare Thai orchid, is the largest
member of the orchid family, native to Thailand but also found in
Southeast Asia to New Guinea

• It possesses remarkable characteristics, such as prolonged

blooming for up to 2 months and traditional medicinal use in
Thailand against inflammation
Grammatophyllum
• Asexual propagation of orchids is slow, prompting the need for speciosum ( Source by
rapid techniques like tissue culture researchGate article)
LITERATURE REVIEW

• Tissue culture, pioneered by Morel in 1960 for Cymbidium orchids, has since been
developed with various methods, culture media, and explants

• Chitosan, a biodegradable polymer derived from deacetylated chitin, has shown promise as
a plant growth stimulator in various species, including orchids

• The supplementation of chitosan in culture medium enhanced root and shoot biomass of
grapevine plantlets in vitro
5) MATERIALS AND METHODS
Materials :

Plant : G. speciosum shoot tips Equipment : Stereo microscope

Scalpel or sharp blade
Flasks or culture vessels
Medium : Shaking platform
1/2 MS basal liquid medium
and 1/2 MS agar medium with 2%
(w/v) sucrose
MATERIALS AND METHODS
Methods

A. PLBs induction from G. speciosum shoot tips

• Shoot tips were extracted from 8-10 cm long in vitro seedlings using a stereo microscope under
sterile conditions for precision and cleanliness

• The shoot tips were cultured in a liquid medium containing 1/2 MS (Murashige and Skoog) and
2% sucrose, without any additional plant growth regulators.

• The cultures were maintained under standard conditions:

Temperature: 25±2°C
Light: White fluorescent light at an intensity of 37 µmol m² sec for 16 hours per day
Shaking: The cultures were subjected to shaking at approximately 110 rpm for 24 hours
Methods

B. Effects of chitosan on G. speciosum PLBs growth

• 1.0 g of G. speciosum PLBs with diameters ranging from 0.1 to 0.2 cm were selected for the
experiment

• A 1/2 MS liquid medium was prepared and supplemented with 2% (w/v) sucrose

• Various concentrations of chitosan (0, 5, 10, 15, 20, 25, 30, 50, or 100 mg/l) were added to the
medium

• The pH of the medium was adjusted to 5.7 to maintain optimal conditions for PLB growth

• After 1 month of culture, note the fresh weight of PLBs.

C. Relative growth rate measurement

Fresh weight of PLBs was recorded after 1-month culture and expressed as relative growth rate
calculated based on the fresh weight of cultured tissue as follows:

Relative Growth Rate (%) = Wf-Wi x100 %

Wi

Wf final weight
Wi initial weight
6) RESULTS AND DISCUSSION
 A) Effects of chitosan on PLB in
vitro propagation

 Relative Growth Rate was ,

• Highest (756%) – 15 mg/l chitosan
• Signefficiently reduced - Through Concentration
more than 15 mg/l chitosan. And caused PLB
necrosis
• Solid media was lower than those obtained using
liquid media at the same concentration of
chitosan.
Figure 01: G. speciosum pLBs cultured in ½ Figure 02: Some PLBs did not survive when
MS liquid medium supplemented with 100 culture on 1/2 MS solid medium
mg/l chitosan, supplemented with 100 mg/l chitosan.
 B) Effects of chitosan on PLB development
SIGNIFICANT

 Verified that chitosan can act as a plant growth stimulant for orchid production or tissue culture using
G. speciosum micropropagatiion.
concentration needs to be optimized for the specific type of orchid and culture medium.
 Optimal amount of 15 mg/l of chitosan: Highest PLB growth rate
and 5 mg/l and 15 mg/l Concentration best for promoting shoots and leafs Formation.(similar to the
results of NGE et al.
 Through a tryptophan-independent signalling pathway leading to auxin production, chitosan may
contribute to the enhancement of growth and development. (Uthairatanakij, Teixeira da Silva, & Obi
Wan, 2007))Studies suggest chitosan might influence plant hormones like gibberellins and auxins,
which play a crucial role in various growth processes (Román-Doval et al., 2023)
7) CONCLUSION

Chitosan can be used to promote the growth of Grammatophyllum speciosum protocorm-like bodies
(PLBs) in vitro culture. Optimam concentration for liquid medium supplemented is 15 mg/l chitosan
And for 1/2 MS agar medium it should be supplemented with 25 mg/l chitosan .Supplementation with
Chitosan to agar medium promoted shoot formation but not rooting. However, supplementation at too high
a level is not suitable such as 100 mg/l because it can inhibit growth and kill PLBs. Liquid medium is
significantly give better growth than on agar medium.
 8) REFERENCES
1. K.L. Nge, N. Nwe, S. Chandrkrachang and S. Stevens, Chitosan as a growth stimulator in orchid
tissue culture. PlantSci. vol. 170, pp. 1185-1190, 2005.
2. A. Uthairatanakij, J.A. Teixeira da Silva and K. Obi Wan, Chitosan for improving orchid production
and quality. Orchid Sci Biotechnol, vol. 1, pp. 1–5, 2007.
3. R. Román-Doval, Torres-Arellanes, S. P., Tenorio-Barajas, A. Y., A. Gomez‐Sanchez, & Valencia-
Lazcano, A. A. (2023). Chitosan: Properties and Its Application in Agriculture in Context of
Molecular Weight. Polymers, 15(13), 2867–2867. https://doi.org/10.3390/polym15132867
4. Azian E, Zaki ARM, Mohamed MTM, Kamuruzaman S (2004) The use of chitosan on vase life of ait
chrysanthemum (Dendranthema morifolium Ramat). Proceedings of APEC Symposium on quality
management in post- harvest system, August3-5, 2004 at Bangkok, Thailand, p 403
5. Boonlertnirun S, Sarobol E, Sooksathan (2005) Studies on chitosan concentration and frequency of
foliar application on rice yield potential cv Suphun- buri 1. 31 Congress on Science and Technology
of Thailand October 18-20, 2005, Suranaree University of Technology, Thailand, pp 40-44
THANK YOU