Production of Quality Kelampayan Planting Materials Using Plant Tissue Culture Technique for Industrial Tree Plantation - A Case Study in Sarawak

The main aim of this publication is to bridge the research gap in tissue culture and in vitro mutagenesis of Kelampayan. Significant efforts have been made to produce high-quality planting material of Kelampayan for industrial tree plantations through the "Quality Kelampayan Research Project" (STA-UNIMAS). An efficient micropropagation and acclimatisation protocol has been developed for Kelampayan candidate plus trees by using nodal explants to achieve direct organogenesis. The genetic fidelity of the tissue culture-derived regenerants has been confirmed using DNA-based and target gene-specific markers, thereby validating the true-to-type nature of the produced Kelampayan plantlets. Additionally, polyploid Kelampayan clones have been established through in vitro mutagenesis and micropropagated for field trials, exhibiting improved photosynthetic capacity and growth performance compared to other Kelampayan trees planted in various locations. Although the underlying molecular mechanisms for this improved performance are not yet fully understood, polyploid plants may demonstrate greater tolerance towards environmental stresses. Therefore, further research is essential to gain insight into the molecular mechanisms and poly-tolerance involved in the improved performance of polyploid Kelampayan clones. This publication is the result of years of extensive research and collaboration under the STA-UNIMAS project (2019-2023), and we are delighted to share it with the scientific community. We hope this work will inspire and encourage researchers to continue exploring the frontiers of scientific knowledge in Kelampayan research

Rapid in vitro Propagation and Efficient Acclimatisation Protocols of Neolamarckia cadamba

Background and Objective: Neolamarckia cadamba is a fast-growing commercial timber tree species with considerable economic returns to the growers on a rotation period of 4-10 years. The present study was aimed to establish an efficient micropropagation protocol for N. cadamba through direct organogenesis by using nodal explants. Materials and Methods: The nodal explants from in vitro-germinated seedlings were cultured on B5 medium supplemented with various BAP concentrations. The in vitro shoots were then rooted in 1/2 B5 medium added with growth regulators such as IBA, NAA and PBZ. Three types of potting media were tested for transplantation efficiency. Results: The B5 medium supplemented with 1.0 mg LG1 BAP provided the most suitable medium for shoot induction from nodal explants with a mean of 5.4 shoots per explant. The subculture interval could be shortened by proliferating the regenerated axillary shoots on B5 medium supplemented with 0.8 mg LG1 BAP. Half-strength B5 medium enriched with 0.1 mg LG1 PBZ was able to induce root growth with 100% of root formation and resulted in more than 95% survival during acclimatisation stage. Conclusion: This micropropagation protocol could pave the way for mass production of quality N. cadamba seedlings for industrial tree plantation development in order to assure the local timber industries to meet the global demand for wood. Therefore, this could reduce the reliance on natural forests for wood production

Inter Simple Sequence Repeat (ISSR) Analysis of Neolamarckia cadamba in Sarawak

Sarawak state government’s aspiration to establish one million hectares of planted forests by the year 2020 has only achieved 25 % thus far. It is estimated that at least 30 million of seedlings are needed for annual planting and reforestation. One of the problems causing the slow rate in the establishment of planted forest, despite of the huge number of seedlings needed, is the availability of quality planting materials. To overcome this problem, a tree improvement programme focusing on Neolamarckia cadamba or locally known as Kelampayan was initiated in July 2007. The Kelampayan tree improvement programme is a long-term investment to increase the productivity of forest plantations by providing a source of improved planting stock that will improve the tree growth, better form and wood quality as well as pest and disease resistance. To date, two Kelampayan provenance trials have been established since 2008 in Landeh and Niah, Sarawak. This study is directed at the understanding of the genetic diversity and relatedness of the Kelampayan planted in Block 4 at the Landeh provenance trial using inter simple sequence repeat (ISSR) markers. A total of 34 ISSR markers commonly used in plant genotyping were screened using Kelampayan DNA. Out of these markers, 11 were found to be polymorphic and reproducible. PCR optimizations were carried out on these selected 11 ISSR markers. Genotyping of the Kelampayan using the selected ISSR markers is performed at optimal temperatures (Ta) varying from 44 to 61C. The optimum conditions for these ISSR are as follow: 2ng of DNA template, 2mM MgCl2, 0.5 U Taq DNA polymerase, 0.2mM dNTPs and 10 M primer. The genetic information obtained from this study will be used as one of the parameters for the selection of genetically diverse and nonrelated plus trees from the Landeh provenance trial for future tree improvement programme

GC-MS ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS IN LEAF EXTRACTS OF NEOLAMARCKIA CADAMBA (RUBIACEAE) FROM MALAYSIA

Neolamarckia cadamba is one of the medicinal plants used in the treatment of various diseases traditionally. This study was conducted to identify the phytochemical constituents of N. cadamba leaf extracts using gas chromatography mass spectrometry (GC-MS). Solvents with increasing polarities viz. hexane, petroleum ether, chloroform, ethyl acetate and methanol were used in this study. The solvent extracts were analyzed using GC-MS and the mass spectra of the compounds found in the respective extract were matched with the National Institute of Standards and Technology (NIST) library. A total of 26 compounds were identified and the major chemical constituents were n-hexadecanoic acid (44.88%), hexadecanoic acid ethyl ester (17.96%) and octadecanoic acid ethyl ester (11.71%). Some of the identified compounds have been reported to possess various biological activities such as antioxidant, antimicrobial, anesthetic, antiseptic, antidiabetic, hypocholesterolemic and etc. The results thus concluded that N. cadamba leaves possess various potent bioactive compounds and is recommended as a plant of phytopharmaceutical importance

Development and polymorphism of simple sequence repeats (SSRs) in Kelampayan (Neolamarckia cadamba–Rubiaceae) using ISSR suppression PCR method

Simple sequence repeat (SSR) marker is a polymerase chain reaction (PCR)-based marker system which has become a marker of choice for understanding plant genetic diversity and a powerful tool in addressing genetic resources questions. However, the availability of SSR markers especially for forest tree species is limited thus far due to the high development cost, labour-intensive and time-consuming. The present study aimed to develop an array of SSR markers for Neolamarckia cadamba using inter-simple sequence repeat (ISSR) suppression PCR method and further assessed the polymorphisms and transferability of the markers to other species.In total, 15 out of 31 SSR markers specific for N.cadamba were successfully developed and further characterized and validated by using 30 individuals of N. cadamba. The markers exhibited a considerable high level of polymorphism across the tested N. cadamba genotypes whereby 66 alleles were detected with an average of four alleles per locus. Most of the SSRloci analyzed showed high polymorphism as indicated by their PIC value which was above 0.5. The most polymorphic lociwere: NCAC11 (PIC=0.849), NCAC12 (PIC=0.722) and NCAG01 (PIC=0.712).The transferability rate was ranging from 26.7% to 73.3% among the four cross-genera species tested. The present study is the first report on the development of SSR markers in N. cadamba. These markers provide valuable genomic resources that could pave the way for exploiting SSR genotype data for effective selection of plus trees, provenance trials and establishment of forest seed production areas (SPAs) of N. cadamba in the selected forest areas dedicated to planted forest development, and molecular breeding of N. cadamba and other indigenous tropical tree species in future

Development and polymorphism of simple sequence repeats (SSRs) in Kelampayan(Neolamarckia cadamba–Rubiaceae) using ISSR suppression PCR method

Simple sequence repeat (SSR) marker is a polymerase chain reaction (PCR)-based marker system which has become a marker of choice for understanding plant genetic diversity and a powerful tool in addressing genetic resources questions. However, the availability of SSR markers especially for forest tree species is limited thus far due to the high development cost, labour-intensive and time-consuming. The present study aimed to develop an array of SSR markers for Neolamarckia cadamba using inter-simple sequence repeat (ISSR) suppression PCR method and further assessed the polymorphisms and transferability of the markers to other species.In total, 15 out of 31 SSR markers specific for N.cadamba were successfully developed and further characterized and validated by using 30 individuals of N. cadamba. The markers exhibited a considerable high level of polymorphism across the tested N. cadamba genotypes whereby 66 alleles were detected with an average of four alleles per locus. Most of the SSRloci analyzed showed high polymorphism as indicated by their PIC value which was above 0.5. The most polymorphic lociwere: NCAC11 (PIC=0.849), NCAC12 (PIC=0.722) and NCAG01 (PIC=0.712).The transferability rate was ranging from 26.7% to 73.3% among the four cross-genera species tested. The present study is the first report on the development of SSR markers in N. cadamba. These markers provide valuable genomic resources that could pave the way for exploiting SSR genotype data for effective selection of plus trees, provenance trials and establishment of forest seed production areas (SPAs) of N. cadamba in the selected forest areas dedicated to planted forest development, and molecular breeding of N. cadamba and other indigenous tropical tree species in future

Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests

Applications of Genomics to Plantation Forestry with Kelampayan in Sarawak

Wood-based industries in Sarawak are increasingly encouraged to adapt to " new wood " from planted forests composed of fast-growing species such as Kelampayan with short rotation cycle (6-8 years). The rationale is that natural forests at the most produce about 3m 3 /ha/yr of commercial timber, whereas plantations can produce annually from 10m 3 /ha to 30m 3 /ha of commercial timber. It is estimated at least 30 million seedlings are required for annual planting or reforestation programmes to meet the increasing global demand for raw materials. To date, several molecular genetics studies have been completed for Kelampayan. These include genetic diversity of Kelampayan, genetic marker (SSRs) development, 'Touch-incubate-PCR' approach for preparing plant tissues for high throughput genotyping, and transcriptomics and bioinformatics on wood formation of Kelampayan. This information provides a useful resource for genomic selection of Kelampayan aiming at the production of high value forests for maximum returns