EXECUTIVE SUMMARY
Sabah hosts a diverse variety of endemic species of bamboos which can be classified into highland and lowland varieties, with highland varieties associated with the cooler climatic regions such as the Crocker range. Bamboos are an important source of raw material to the local craft industry; however, destruction of their natural habitat has led to a decline in the range and diversity of endemic bamboos. Conservation strategies which focus on the identification of native germplasm, the selection of varieties which can be propagated by tissue culture and the establishment of germplasm banks are some of the approaches which can be undertaken in order to conserve this declining species. Micropropagation is an established protocol which can be applied for the multiplication of germplasm using in vitro technologies. However, issues pertaining to genetic stability of germplasm in tissue culture systems represents a major challenge to mass propagation. The proposed study aims to establish a system for the micropropagation of endemic bamboos in vitro. The fundamental component of this study will focus on the monitoring of genetic stability of in vitro culture of bamboo via mutation detection. The outcomes of the study will lead to new knowledge pertaining to the molecular identity of the endemic bamboos of Sabah and provide the basis for the establishment of tissue culture systems for applications to environmental conservation and economic development in rural areas. The discoveries pertaining to genetic stability of bamboo germplasm in vitro will lead to a further understanding of this fundamental phenomenon and its impact on propagation of this important forest species.
RESEARCH BACKGROUND PROBLEM STATEMENT
1. There is no established or reported protocol for in vitro culture of bamboo varieties from Sabah which can facilitate germplasm conservation.
2. The establishment of germplasm in vitro is likely to lead to somaclonal variation and genetic instability of tissue culture can produce undesirable variants.
HYPOTHESIS
1. The bamboo varieties from Sabah can be established in vitro.
2. The bamboo germplasm established in vitro is genetically stable.
RESEARCH QUESTIONS
The research questions for this study are:
1. Can the bamboo varieties from Sabah be established in vitro?
2. Does the bamboo germplasm exhibit genetic instability in vitro?
LITERATURE REVIEWS
Malaysia is a heavily forested country, and forest products including bamboo are important sources of income especially for rural communities. In Sabah, bamboos grow in lowland and highland (mountain) areas with lowland species occur gregariously on isolated patches, along stream and river banks and degraded forest land, while the highland species mainly found in Kinabalu Park, Ranau (Kulip 2015; Mohamad 2000). Sabah has at least nine genera of bamboo with approximately 35 species (Dransfield 1992).Bambusa vulgaris(the green culm) is the commonest village bamboo occurs often on river banks and the species is cultivated widely throughout the state.
Gigantochloa levis is the most widely used species by local people in Sabah and the species grows well in degraded forest lands and was planted long ago and covers a very large area in Tambunan district (Kulip and Matunjau 1994).Schizostachyum brachyladumis often planted as a village bamboo with religious significance to the Kadazan/Dusun people who only use this bamboo for making drinking vessel for wedding ceremonies.
Bamboo is a very important material for rural communities in Sabah due to its many uses arising from its inherent characteristics such as lightness and strength (Kulip 1992). Apart from making handicrafts, bamboo in can also be used for house construction, musical instrument, kitchen utensils and chopsticks (Kulip 2005). Some species like Bambusa blumeana andB. vulgaristhrive well along river banks, thus helping to prevent erosion. Species likeSchizostachyum blumei,S. brachyladumandB. vulgarismay be planted on hill slopes to prevent landslides.
The climbing species, especiallyDinochloaspp., is very abundant in the logged-over forest throughout the state and is good for bamboo-cement wall panels (Kulip 1992).
There is a wide genetic diversity of bamboo in Sabah and this pool of genetic variation may serve as the base for selection as well as for plant improvement. The conventional methods of propagation of bamboos sexual as well as vegetative have lots of problems that restrict their multiplication in large numbers. Natural regeneration and artificial propagation of bamboo take place mainly by seeds with extreme difficulties. Use of seeds is challenging because of plant’s sporadic flowering and long flowering cycles, together with seed recalcitrance and consumption by wild animals (Sandhu et al. 2018). Vegetative propagation conducted by cuttings and air layering are only useful at small-scale because they damage the mother plants, propagation material is bulky and difficult
to be transported and is only available during few months of the year (Nautiyal et al. 2008). In view of constant increase in the demand of the planting material and the limitations of the conventional methods of propagation, research into newer and more rapid methods of propagation for different bamboo species is urgently called for. In such condition, tissue culture offers rapid and reliable methods such as micropropagation to rapidly produce bamboo plants in large numbers within a reasonable timeframe (Goyal and Sen 2016; Waikhom and Louis 2014).
Micropropagation via in vitro shoot proliferation and somatic embryogenesis offers method for mass propagation from elite genotypes (Sandhu et al. 2018; Yuan et al. 2013). However, culture conditions, explant source, ploidy level and in vitro culture age may cause genetic instability and known to induce somaclonal variation in vitro.
These somaclonal variations may appear due to cell cycle disturbances caused by exogenously applied growth regulators, increased mutation rate, and accumulation of mutations over time, alteration in DNA methylation patterns, DNA damage, and alteration of cell’s ability to repair damaged and mutated DNA (Singh et al. 2013a). It is therefore extremely important to ascertain the clonal uniformity of the in vitro raised plants and the use of reliable DNA based markers such as RAPD, ISSR, SSR, AFLP, etc. is highly recommended to test the genetic fidelity of in vitro raised bamboo plants (Singh et al. 2013b). The use of more than one marker system has also been suggested to target a wider region of the genome as applied in the assessment of the genetic fidelity of Dendrocalamus asperplants raised through axillary shoot proliferation (Singh et al. 2013a). This study focuses on the establishment of an in vitro propagation method for the endemic bamboos of Sabah, particularly the ecologically and commercially important species viz. Racemobambos cockburnii (highland species) and Gigantochloa levis (lowland species), and to determine the genetic variability or stability of tissue-cultured plantlets via mutation detection using Targeting Induced Local Lesions IN Genomes (TILLING) which is a highly sensitive technique for the detection of mutations in the genome.
RESEARCH RELEVANCE
Bamboo species are widely used for construction and reinforcing fibers, paper, textiles and board, food, combustion and other bioenergy applications. According to Forest Research Institute Malaysia (FRIM), only a few bamboo plantations in Malaysia are owned by small and private enterprises but most bamboo resources can be found in natural forest. However, in order to commercialise bamboo products, Malaysia should therefore establish specific and protected bamboo plantations and policies outside its already given natural forest. Aside from the problems posed by changing weather conditions, there are other challenges which affect the bamboo industry in Malaysia. Barriers for investments in the bamboo industry include a lack of raw materials, lack of product variety and lack of properly linked supply chains. To overcome these barriers, tissue culture methods can be used to achieve large-scale production of plants for operational planting, providing material for breeding programs and also for conservation of germplasm. Besides, additional bamboo products, such as bamboo fibre, can be promoted to increase the local demand for new and further innovative products, thereby encouraging domestic and foreign investments. Investments can then be used for the development of necessary value chains and bamboo plantations to address the challenge of raw material supply. This is in line with the Sabah government in looking at promoting bamboo as an alternative to timber in the state and reducing its reliance on depleting timber reserves.
REFERENCES
1. Dransfield S (1992) The bamboos of Sabah. Sabah Forest Record No: 14. Forestry Department Sabah, Malaysia.
2. Goyal AK, Sen A (2016) In vitro regeneration of bamboos, the “Green Gold”: an overview. Indian Journal of Biotechnology 15: 9-16.
3. Kulip J (1992) A note on bamboos in Sabah. Journal of Tropical Forest Science 4: 266-269.
4. Kulip J (2005) The use of bamboos among local communities in Sabah, Malaysia. Sepilok Bulletin 2:
49-61.
5. Kulip J (2015) Bamboos in Tambunan: origin and diversity. Buku Cenderamata Pesta Bambu dan Halia Daerah Tambuhah. Bamboo and Ginger Festival Tambunan 2016.
6. Kulip J, Matunjau CA (1994) The utilization of bamboos in Tambunan, Sabah, East Malaysia. In:
11. Singh SR, Singh R, Kalia S, Dalal S, Dhawan AK, Kalia RK (2013b) Limitations, progress and prospects of application of biotechnological tools in improvement of bamboo – a plant with extraordinary qualities.
Physiology and Molecular Biology of Plants 19: 21-41.
12. Waikhom SD, Louis B (2014) An effective protocol for micropropagation of edible bamboo species (Bambusa tulda andMelocanna baccifera) through nodal culture. The Scientific World Journal 2014:
article 345794.
13. Yeasmin L, Ali MN, Gantait S, Chakraborty S (2015) Bamboo: an overview on its genetic diversity and characterization. 3 Biotech 5: 1-11.
14. Yuan JL, Yue JJ, Wu XL, Gu XP (2013) Protocol for callus induction and somatic embryogenesis in Moso bamboo. PLoS ONE 8: e81954.
15. Chen, L., Huang, L., Min, D., Phillips, A., Wang, S., Madgwick, P. J., ... & Hu, Y. G. (2012).
Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.).PLoS One,7(7), e41570.
OBJECTIVE(S) OF THE RESEARCH The objectives of this study are:
1. to validate the genetic identity of endemic bamboos of Sabah using DNA barcoding;
2. to optimise the tissue culture systems for bamboo varieties from Sabah; and 3. to assess the genetic stability of tissue-cultured bamboos using molecular method.
METHODOLOGY
1. Germplasm collection: Bamboo Germplasm will be collected from highland and lowlands of Sabah, Malaysia. Approval for collection of Samples from Tambunan will be obtained from The Sabah Biodiversity Centre. No germplasm will be exported beyond the state boundaries of Sabah. The collection strategy will be developed in consultation with Mr. Julius Kulip, who is an authority in plant taxonomy, distributions and utilizations in Sabah.. Bamboo culms and branches containing internodes will be selected from both highland and lowland bamboos, the geographic location will be recorded using Global Positioning System (GPS), documented using photography, and the samples will be transported to the climate controlled greenhouse located at the Biotechnology Research Institute. The culms will be established in pots for acclimatization to the greenhouse. Leaf samples will be stored at - 80 C until DNA extraction.
2. Germplasm identification:Samples will be identified using the taxonomic identification guide produced by Mr. Julius Kulip, a plant taxonomist..
3. DNA extraction: DNA will be extracted from fully expanded leaves using the DNeasy plant DNA extraction kit (Qiagen) according to the manufacturer's instructions. The purity and concentration of the DNA will be measured using a DNA spectrophotometer.
4. Tissue sterilization:Internode sections which contain actively dividing tissue will be selected. They will be washed thoroughly in tap water to remove any extraneous soil. The washed samples will be section into cuttings with a size of one to two inches, following which they will be immersed in a solution containing 1.0% Commercial bleach and 0.1% v/v Triton X-100 in sterile distilled water for 20 minutes.
Following this, they will be rinsed four times using sterile distilled water and air dried in a clean work station. All of these procedures will be carried out under aseptic conditions.
5. Establishment of in vitro culture:The sterilized tissue will be established in vitro on sterile Murashige and Skoog medium containing the following combinations of Auxins and Cytokinins for induction of callus (Table 1.0), somatic embryos (Table 2.0) or direct regeneration of meri-clones (Table 3.0). Culture conditions will be 25 C with a photoperiod comprising 16 hours of light at 3000 lux provided by white LED fixtures. All experiments will be carried out in triplicates and the statistical significance of the data will be computed using open source software package R (https://www.r-project.org/). The tissue culture plantlets developed in vitro will be hardened in a climate controlled greenhouse prior to establishment in the field.
Table 1.0: Combinations of Phytohormones for the induction of callus in Bamboo. (2,4-D: 2, 4 - Dichlorophenoxyacetic acid); (6-BAP: 6 - Benzyl Amino Purine); (NAA: Naphthalene Acetic Acid); (IBA:
Indole-3-Butyric Acid); (Kinetin: 6 - Furfuryl amino purine). All concentration in milligrams per liter unless indicated otherwise.
HORMONE CONCENTRATION (mg/l)
2,4 D Control 0.1 0.5 1.0 2.0
6-BAP Control 0.1 1.0 2.0 3.0
NAA Control 0.1 1.0 2.0 3.0
KINETIN / IBA Control 0.1 / 1.0 0.5/1.0 1.0/1.0 2.0/1.0
Table 2.0: Combinations of Phytohormones for the induction of somatic embryos in Bamboo. Murashige and Skoog Medium containing 10% Coconut Milk.
6-BAP Control 0.1 1.0 2.0 3.0
NAA Control 0.1 1.0 2.0 3.0
KINETIN / IBA Control 0.1 / 1.0 0.5/1.0 1.0/1.0 2.0/1.0
Table 3.0: Combinations of Phytohormones for the induction of roots and shoots in Bamboo. Murashige and Skoog Medium.
Sucrose Control 1 gm /litre 5 gm /litre 10 gm / litre 15 gm / litre
IBA Control 0.1 1.0 2.0 3.0
KINETIN Control 0.1 0.5 1.0 2.0
6. Assessment of genetic stability:Assessment of genetic stability will be carried out using the Surveyor Nuclease Assay (https://www.future-science.com/doi/10.2144/04364PF01) based on specific PCR amplicons. Ten genes will be selected using the bamboo gene database (http://server.ncgr.ac.cn/bamboo/) , gene specific primers will be designed using the online primer design software (http://bioinfo.ut.ee/primer3-0.4.0/), the length of each amplicon will be adjusted to the range of 1000 - 2000 bases in order to ensure sufficient representation. PCR primers will be applied to amplify these 10 sets of genes from the wild plants as well as their micropropagated versions. The PCR amplicons will be hybridized in vitro, following which they will be digested using the CEL nuclease
according to the manufacturer’s instructions.
(http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/surveyor-kit-for-ge l-electrophoresis-user-guide.pdf?sfvrsn=a9123407_6)
FLOW CHART OF RESEARCH ACTIVITIES
GANTT CHART OF ACTIVITIES
ACTIVITY 2020 2021 2022
Permissions from Sabah Biodiversity Center
Sample Collection and Identification Development of protocol for explant sterilization
Establishment of explants in vitro Development of protocol for callus induction
Development of protocol for regeneration of explants
Establishment in the field and testing Monitoring of Genetic Stability:
TILLING