f
RU GRANT
Project Code :
(for RCMO use only)
UNlVERsm SAlNS MALAYSIA
FINAL REPORT FORM
Please email [email protected]
A PROJI;CTDETAILS Title of Research:
Exploring the mechanism for
in vitroproduction of anthocyanins from a rare wild orchid
Anoectochilusspp.
ii Account Number: 1001/PBIOLOGI/811211
Name of Research Leader:
Associate Professor Dr. Sreeramanan Subramaniam [School of Biological Sciences, USM]
iii 1.
iv Name of Co-Researcher:
1. Prof. Chan Lai Keng [School of Biological Sciences, USM] - retired 2. Dr. Rahmad Zakaria [School of BiologicalSciences, USM]
v Duration of this research:
a) Start Date 15/07/2012 b) Completion Date 14/07/2015 c) Duration : 3 years
d) Revised Date (if any) : 14/4/2016 (additional of 9 months)
B ABSTRACT OF RESEARCH
(An abstract of between 100 and 200 words must be prepared in Bahasa Malaysia and in English.
This abstract will be included in the Report of the Research and Innovation Section at a later date as a means of presenting the project findings of the researcher/s to the University and the community at large)
Appendix 1
C BUDGET & EXPENDITURE
Total Approved Budget
Total Expenditure Balance
: RM 124,000.00
Yearly Budget Distributed Year 1 : RM 42,000.00 Year 2 : RM 42,500.00 Year 3 : RM 39,500.00
: RM 123,379.03 : RM 420.97 Percentage of Amount Spent (%) : 99.50%
# Please attach final account statement (eStatement) to indicate the project expenditure
Appendix 2
ii Equipment Purchased Under Vot 35000
No. Name of Equipment Amount (RM) Location Status
# Please attach the Assetllnventory Return Form (Borang Penyerahan Asetllnventori) - Appendix 1
Project Objectives (as stated/approved in the project proposal)
No. Project Objectives Achievement
To study efficient in vitro culture techniques for the production Done 1 of callus, cell suspension and PLB of Anoectochilus spp
To determine effects of physical and chemical factors for Done 2 maximum production of anthocyan ins in callus/cell
suspension/plantlet culture
To carry out vitrification protocol for cryopreservation of Done 3 selected plant tissues of Anoectochilus spp producing the
highest anthocyanin yield
To determine the antimicrobial and antioxidant activities of the Done 4 anthocyanin extract of Anoectochilus spp
To identify and quantify the major anthocyanins in the Done 5 established plant cell culture of Anoectochilus spp
ii Research Output
a) Publications In lSI Web of Science/Scopus No.
1.
2.
3.
4.
5
6
7
8
9
Publication
(authors, title,journal, year, volume ,pages ,etc.)
Ranjetta, P.; Rahmad, Z.; Vikneswaran, M.; and Sreeramanan, S. (2016). Histological and autofluorescence observations of Anoectochilus sp. and Ludisia discolor. Journal of Microscopy (Submitted)
Ranjetta, P.; Rahmad, Z.; Vikneswaran, M.; and Sreeramanan, S. (2016) The application of thin cell layer on Ludisia discolor.
Plant Cell, Tissue and Organ Culture (Submitted)
Ranjetta Poobathy, Rahmad Zakaria, Syed Mohd. Edzham Syed Hamzah and Sreeramanan Subramaniam (2016). Early Studies on Protoplast Isolation of Ludisia discolor, A Wild Orchid. Tropical Life Sciences Research, 27(Supp. 1), 15-19, 2016.
Raheleh Dehgahi, Latiffah Zakaria, Azhar Mohamad, Alireza Joniyas and Sreeramanan Subramaniam (2016). Effects of fusnric: nc:id trE'!ntmpnt on thp protor.orm-likp hod ips of Dendrobium sonia-28. Protoplasma. 253: 1373-1383 lSI/Scopus. IF 2.343. --Mini project under this research grant Jasim Uddain, Latiffah Zakaria, Chew Bee Lynn and Sreeramanan Subramaniam (2015). Preliminary assessment on Agrobacterium-mediated transformation of Dendrabium Broga Giant orchid's PLBs. Emirates Journal of Food and Agriculture. 27 (9): 669-677. lSI/Scopus IF: 0.31
-Mini project under this research grant
J.J.J. Antony, R.A. Shamsir, R. Poobathy, B.L. Chew and S.
Subramaniam (2015). Somaclonal variations were not induced by the cryopreservation:Levels of somaclonal variations of in vitro and thawed protocorms of Dendrobium Bobby Messina analysed by SCoT and TRAP DNA markers.
South African Journal of Botany. 100: 148-157.1SI/Scopus IF:
0.978 --Mini project under this research gront
Pavallekoodi Gnasekaran, Maziah Mahmood and Sreeramanan Subramaniam (2016). Ultrastructure study of Vanda Kasem's Delight orchid's protocorm-like body.
Horticultura Brasileira. Vol 34: 333-339. lSI/Scopus. IF: 0.822 --Mini project under this research grant
Jasim Uddain, Pavallekoodi Gnasekaran, Latiffah Zakaria, Chew Bee Lynn and Sreeramanan Subramaniam (2015). The effect of different growth media, carbon source, PGRs on Dendrobium Broga Giant orchid's protocorm-like bodies (PLBs) proliferation supported with SEM and TEM analyses.
Pakistan Journal of Botany. 47 (2): 587-593. lSI/Scopus IF:
0.822 --Mini project under this research grant
Jessica Jeyanthi James Antony, Jeevandran Sundarasekar, Xavier Rathinam, Kasi Marimuthu and Sreeramanan Subramaniam (2014). Microscopical analysis of in vitro Mokara Giant orchid's PLBs. Emirates Journal of Food and Agriculture. 26 (i): 73-81. Scopus Journal. --Mini project under this research grant
Status of Publication (published/accepted/
under reviewl Under review
Under review
Gallery proof
Published
Published
Published
Published
Published
Published
b) Publications in Other Journals
Publication Status of Publication
No. (authors, title,journal, year, volume, pages, etc.) (published/accepted/
under review)
c) Other Publications
(book, chapters in book,monograph,magazine,etc.)
Publication Status of Publication
No. (authors, title,journal, year, volume, pages, etc.) (published/accepted/
under review)
d) Conference Proceeding
No. Conference
Title of Abstract/Article Level
(conference name,date, place) (I nternational/National)
1. The 13th
Symposium Malaysian Ranjetta Poobathy, Rahmad Zakaria,
Society of Applied Biology Syed Mohd Edzham Syed Hamzah and International (2014). 8_lOth June 2014. Pg 124- Sreeramanan Subramaniam (2014). A
125. histological study of Anoectochilus sp.
and Ludisia discolor, the jewel orchid.
2. The 2nd Seminar on Sustainable Ranjetta Poobathy, Chan Lai Keng,
Agriculture and Natural Rahmad Zakaria and Sreeramanan International Resources: Strategies for Food Subramaniam (2013). The effect of
Security through Innovative Different Surface sterilization Development.9th
of April 2013, Methods and media composition on Universiti Sains Malaysia. Pg.49. the survival of nodal segments of
Ludisia discolor orchid.
3. Rajshahi University, Bangladesh Sreeramanan Subramaniam (2014).
on the 4th of March 2014. Guest An oral presentation entitled International
l
invited s~eaker. 'Enrichment and Advancement of Knowledge via Plant Science'4. ih Plant Tissue Culture & Sreeramanan Subramaniam,
Biotechnology Conference. Pavaliekoodi Gnasekaran and Jassim International March 1_3rd 2014. Page 49. Oral Uddain (2014). Agrobacterium-
Presentation. mediated transformation of Yanda Kasem's Delight Tom Boykin Orchid.
5.
6
7
8
Invited as a guest lecturer to the Institute of Science and Technology in Tribhuvan University Katmandu, Nepal to present 3 seminars. 8-lih June 2014.
International Association For Plant Biotechnology Congress 2014 (10_15th August 2014), Melbourne, Australia. Pg 97.
Oral Presentation.
The 9tn RegionallMT-GT UNINET Conference 2014, Penang. 3_5th November 2014. Pg 97. Oral Presentation.
Natural Product 2016 (ICNP 2016), Kuala Terengganu. Pg 60.
Oral Presentation - Best Presenter Award.
Seminarl: Agrobacterium-mediated transformation of orchids
Seminar 2: Enrichment and Advancement of Knowledge via Plant Biotechnology
Seminar 3: Transgenic Orchids for Fungal Disease Resistance
Subramaniam Sreeramanan, Poobathy Ranjetta and Maziah Mahmood (2014). The development of a vitrification protocol for protocorm-like bodies (PLBs) of Dendrobium sonia-28.
Ranjetta Poobathy, Rahmad Zakaria, Syed Mohd. Edzham Syed Hamzah and Sreeramanan Subramaniam (2014). Early studies on protoplast isolation of Ludisia disc%r, a wild orchid.
Ranjetta Poobathy, Rahmad Zakaria, Syed Mohd. Edzham Syed Hamzah, Sreeramanan Subramaniam (2016).
Histology and transmission electron microscopy (TEM) of Anoectochilus sp. and Ludisia disc%r, the wild jewel orchids. International Conference On
# Please attach a full copy of the publication/proceeding listed above
iii Other Research Ouputllmpact From This Project
(patent, products, awards, copyright, external grant, networking, etc.)
International
International
National
National
Establishment of an efficient in vitro culture techniques for the production of callus, cell suspension and PLBs of Anoectochilus spp and Ludisia discolour orchids with the identification and quantification of the major anthocyanins compounds in plant cell cultures. Some fundamental research work also has been carried out in tissue culture in selected orchids.
,'! ,
"';'i~
E HUMAN CAPITAL DEVELOPMENT
'0 ,','" : ,', :,:"
a) Graduated Human Capital
Student Nationality (No.)
Name National International
1 1. Ranjetta Poobathy
PhD
2.
MSc 1.
2.
Undergraduatei 1.
2.
b) On-going Human Capital
Student Nationality (No.)
Name National International
1 1.Hazirah Burkhan
PhD
2.
MSc 1.
2.
Undergraduate 1.
2.
c) Others Human Capital
Student Nationality (No.)
Name National International
Post Doctoral Fellow 1.
2.
Research Officer 1.
2.
Research Assistant 1.
2.
Others (. ... ) 1.
2.
COMPREHENSIVE TECHNICAL REPORT
; ' ': ".}
" ,"s
:.:;~;F
" :i'. ' , , : /.
Applicants are required to prepare a comprehensive technical report explaining the project. The following format should be used (this report must be attached separately):
Refer to Appendix 3 for FULL COMPREHENSIVE REPORT
•
Introduction•
Objectives•
Methods•
Results•
Discussion•
Conclusion and Suggestion•
Acknowledgements- -
, 0 "
P~OBI;;EM~/CONSTRAINTS/CHALLENGES IF ANY
(Please provide issues arising from the project and how they were resolved)
Problems experienced in this research include difficulties in obtaining wild plants, causing the lack of explants, and slow growth of plantlets. Problems experienced in this research include difficulties in obtaining wild plants, causing the lack of explants, and slow growth of plantlets. Callus growth is erratic, not uniform across the treatments, and can only be observed after a period of more than three months, while shoot production can only be observed after two months of culture. Another problem faced is heavy contamination of the explants and media used in this research. These problems have delayed the achievement of the first and second milestones of this research project. Transmission electron microscopy were determined on cross-sections of leaf, stem, root and callus of both orchids to elucidate the location of anthocyanin production within the tissues.
,\};\
"'H:~~~~~~~Nt"'~NDATIOff;'
(Please provide recommendations that can be used to improve the delivery of information, grant management, guidelines and policy, etc.)
Orchid conservation may prove to be a boon in the preservation of other species as well, as conservation efforts directed towards orchids may help to protect the surrounding species from threats such as deforestation and invasions from alien species. Future research on the jewel orchids Anoectochilus sp. and Ludisia discolor can be improved by expanding the ways of conserving and improving the numbers of the orchids in the wild. Anoectochilus sp. will especially benefit from such efforts, as the number of the orchids available in the wild and the low success in propagating the plants through plant tissue culture techniques were major limiting factors in this research. Quick propagation methods for both the orchids may also be developed for economic and horticultural purposes. Additional studies could be performed on the leaves of both Anoectochilus sp. and Ludisia discolor in order to decipher the role and mechanism of action of anthocyanin-containing cells. The ability of the plants to adapt to shade conditions by localising anthocyanins in strategic cells is indeed an interesting concept to be studied and elucidated, and extrapolated to other studies if the possibility arises. The antioxidative potential of both the jewel orchids could be better explored and enhanced through the use of different extraction solvents and methods.
Microbial and cytotoxicity assays involving a number of cancer cell lines can be conducted to fully assess the potential of both orchids as cure for various diseases. An anthocyanin and ultimately a biochemical profile of both the orchids can be developed through HPLC or other analytical methods using a variety of standards, as there may be other compounds from which the potency of the orchids are derived, as described in folk medicine.
Proje;} Lead£e~'s Signature:
.... L.~~
ASSOC PROFESSOR DR. S, SREERAMANAN Programme Manager
(Agrobio!ogy. Entomology &. Parasitology) School of BiOlogical SCienCeS
IJniversiti Sains Malaysia
Name : Associate Professor Dr. Sreeramanan Subramaniam Date : 25/10/2016
Appendix 1
Abstrak
Anoectochilus sp. dan Ludisia discolor, orkid permata liar daratan, tumbuh di kawasan hutan yang teduh. Orkid tersebut adalah komponen perubatan tradisional di Asia. Mikropropagasi adalah penting untuk memelihara danmembiak tumbuhan yang diingini. Dalam kajian ini, protocol-protokol pensterilan permukaan dan mikropropagasi berjaya dibangunkan untuk Anoectochilus sp. Dan Ludisia discolor melalui penggunaan
OA%(w/v) HgCh. Kadar pencemaran paling rendah (7.7%) dan kadar tertinggi kelangsungan hidup dan pertumbuhan diperoleh pada 63.1% dan 22.5% masing-masing untuk Ludisia discolor. Medium separa pepejal separuh kekuatan Murashige dan Skoog (MS), ditambah dengan 0.2% (w/v) serbuk arang, 8% (w/v) homogenat pisang Mas, 3% (w/v) sukrosa, 3.5g.L-
1Gelrite, 1.0mg.L-
1NAA, dan 0.1mg.L-
1TDZ menghasilkan pertumbuhan tertinggi (19.6%) untuk keratan nod orkid tersebut. Medium Hyponex diubahsuai yang terdiri daripada 0.3% (w/v) Hyponex dan tryptone, 2% (w/v) sukrosa, 2.75g.L-
1Gelrite, 0.05% (w/v) serbuk arang dan 0.00Ig.L-
1TDZ menghasilkan pertumbuhan Anoectochilus sp. yang terbaik. Pembangunan kalus terbaik (50.0%) diperhatikan dalam lapisan sel nipis melintang (tTCL) keratan batang Ludisia discolor dikultur dalam keadaan gelap dalam medium separa pepejal separuh kekuatan MS yang diperkaya dengan 0.33mg.L-
1thidiazuron. Pengasingan protoplas menggunakan daun in vitro Ludisia discolor menunjukkan bahawa tuaian terbaik protoplast hidup (5.2
x10
6sel setiap mL medium) dicapai dengan menggunakan medium K3, langkah pra-osmosis dalam
OAMsukrosa dan penetapan berat daun, kelajuan pengemparan dan tempoh penghadaman enzim masing-masing pada Ig, 600 rpm dan 15 jam. Pemerhatian histologi keratan tumbuhan ex vitro kedua-dua orkid yang diwarnai dengan Safranine O/Fast Green FCF dan Safranine O/Orange G, digabungkan dengan mikroskop elektron aliran menunjukkan bahawa daun-daun memaparkan pembezaan tisu mesofil yang lazimnya didapati dalam tumbuhan dikot. Daun daun terse but memaparkan pengilauan melalui penempatan antosianin dalam kawasan terpilih tisu daun. Ujian antioksidan untuk ekstrak metanol keratan daun, batang dan akar Ludisia discolor berbeza sumber yang tertakluk kepada pelbagai kaedah pengeringan menunjukkan bahawa daun memiliki potensi yang lebih tinggi berbanding bahagian tumbuhan yang lain.
Ujian sitotoksik ekstrak metanol daun-daun in vitro Ludisia discolor yang melalui
pengeringan udara dan ketuhar, serta daun-daun ex vitro yang dikering-beku menunjukkan
bahawa ekstrak-ekstrak tersebut selamat untuk kedua-dua titisan sel kanser rnanusia
kolorektal (HCT -116) dan sel normal endothelial (EA.hy296). Analisis kromatografi cecair
berprestasi tinggi (HPLC) untuk ekstrak metanol Ludisia discolor menunjukkan bahawa
jumlah tertinggi sianidin klorida ditemui dalam daun in vitro yang melalui pengeringan udara
(3.796mg.g-
1ekstrak kering).
Abstract
Anoectochilus sp. and Ludisia discolor, the terrestrial wild jewel orchids, grow in shaded forest areas. The orchids are components of traditional medicine in Asia. Micropropagation is integral for conserving and multiplying desired plants. In this study, surface sterilisation and micropropagation protocols were successfully developed for Anoectochilus sp. and Ludisia discolor through the use of 0.4% (w/v) HgCh. The lowest contamination rate (7.7%) and the highest survival and growth rates were obtained at 63.1 % and 22.5% respectively for Ludisia discolor. Semi-solid half-strength Murashige and Skoog (MS) medium, supplemented with 0.2% (w/v) activated charcoal, 8% (w/v) Mas banana cultivar homogenate, 3% (w/v) sucrose, 3.5g.L-' Gelrite, 1.0mg.L-' NAA, and O.lmg.L-' TDZ produced the highest growth (19.6%) for nodal segments of the orchid. A modified Hyponex medium consisting of 0.3% (w/v) Hyponex and tryptone, 2% (w/v) sucrose, 2.75g.L-' Gelrite, 0.05% (w/v) activated charcoal and O.OOlg.L-' TDZ produced the best growth for Anoectochilus sp. The best callus development (50.0%) was observed in transverse thin cell layers (tTCL) of stem segments of Ludisia discolor cultured under dark conditions in semi-solid half-strength MS medium supplemented with 0.33mg.L-' thidiazuron. Protoplast isolation using in vitro leaves of Ludisia discolor indicated that the best harvest of viable protoplasts (5.2 xl
()6cells per mL medium) was achieved by implementing K3 medium, a pre-osmotic step in O.4M sucrose and the setting of the weight of the leaves, the centrifugation speed and enzymatic digestion period to 19, 600 rpm and 15 hours respectively. Histological observations of ex vitro plant segments of both the orchids stained with Safranine O/Fast Green FCF and Safranine O/Orahge G, combined with transmission electron microscopy indicated that the leaves presented with mesophyll tissue differentiation usually found in dicotyledonous plants. The leaves displayed autofluorescence through the localisation of anthocyanins in select regions of the leaf tissue.
Antioxidant assays of methanolic extracts of differently-sourced leaf, stem and root segments
of Ludisia discolor subjected to various drying methods indicated that the leaves possessed a
higher potency compared to other plant parts. Cytotoxicity assays of methanolic extracts of
air- and oven-dried in vitro leaves, and freeze-dried ex vitro leaves of Ludisia discolor
indicated that the extracts were safe on both human colorectal cancer cell line (HCT-116) and
endothelial normal cell line (EA.hy296). High performance liquid chromatography (HPLC)
analyses of the methanolic extracts of Ludisia discolor indicated that the highest amount of
cyanidin chloride was found in air-dried in vitro leaves (3.796mg.g-' dry extract).
