Ebook TW 2 2023 4

­ 11 ­  RD­RMU­REC02 REV05_1/1/2020 (iv) Contract Staff (Bil) None Reviewed by: Reviewed by: ............................................................ ............................................................ ( Norhafizah Ghazali ) (Dr Lee Yang Ping )    Statistician             Head of FGVIC, Precisian Agriculture & Genomics     FGV R&D Sdn Bhd                     FGV R&D Sdn Bhd Approved by: ............................................................. ( Noor Hisham Hamid ) Chief Executive Officer   FGV R&D Sdn Bhd  

Development of Planting Materials with Long Stalk through Molecular Breeding Approach Siti Khadijah Abdullah, Amer Izzat Samsudin, Sharmilah Vetaryan, Muhammad Farid Abdul Rahim and Mohd Azinuddin Ahmad Mokhtar Abstract FGV has various collections of oil palm materials with diverse favourable traits, which can be introgressed into advanced planting materials. However, conventional breeding methods are lacking as the phenotypic measurements and selection of palms with favourable traits require a long wait, due to the approximately 12 years of its breeding cycle. To shorten the breeding duration and accelerate the introgression of favourable traits, the usage of molecular breeding can be explored. Palms with favourable traits can be selected as early as the nursery stage through marker-assisted selection (MAS). Oil palm material with longer stalk lengths has been of interest to the industry since there is a need to move towards the mechanization of oil palm bunch harvesting technique, hence combating the ongoing labour shortages issue. This project aims to identify molecular markers associated with the long stalk trait, and then use the markers to facilitate the introgression of this trait into the advanced planting materials. Population with long stalk traits will be identified and genotyped by sequencing (GBS) for single nucleotide polymorphism marker (SNP) mining. The SNP data will be used for linkage mapping and genome-wide association study (GWAS) analyses to identify molecular markers associated with the long stalk trait. The identified markers will be validated in other populations with long stalk traits, and markers with satisfactory accuracy in identifying palms with long stalk traits will be developed into a panel for MAS breeding trial and field validation. Establishing a panel for the long stalk trait will fasten the introgression of the trait into the advanced planting materials and eventually, encourage harvesting mechanisation as well as reduce the negative impact of labour shortages.

REV05_1/1/2020 Development of planting materials with long stalk through molecular breeding approach Whole genome re­sequencing and functional genomics approach for development of planting material with specific traits High quality planting material through advanced genomic, clonal and breeding programme Genomics Siti Khadijah Abdullah RD­RMU­REC02 FGV R&D SDN BHD PROJECT PROPOSAL A. Project Title A1. Project Code A2. Program A3. Strategic Thrust ( please refer to the roadmap for FGV R&D Upstream) A4. Unit A5. Presenter

­ 2 ­  RD­RMU­REC02 REV05_1/1/2020 B. Introduction FGV has various collections of oil palm materials with diverse favourable and unfavourable traits. Introgression of favourable traits to improve the crop's yield, quality, or commercial value involves repeated crossing and selection processes. The selection process requires measuring and observing the trait of interest which is known as phenotyping. Plant phenotyping requires persistent attention, especially for perennial plants like oil palm, as the breeding cycle of oil palm is approximately 12 years. The usage of molecular breeding can be explored to shorten the breeding duration and accelerate the introgression of favourable traits. Molecular breeding or marker­assisted selection (MAS)is the usage of DNA markers that are tightly linked to specific phenotypic traits to assist the selection process in breeding programs. Oil palm material with longer stalk lengths has been of interest to the industry due to the difficulties in harvesting fresh fruit bunch (FFB) with short stalks. Harvesting FFB is a tedious and time­consuming process that involves identifying the ripe bunch, removing subtending fronds, locating the stalk, and finally cutting the bunch using a chisel attached to a pole. As the process is time­consuming, a huge labour force is required to harvest the FFB before overriping. Unfortunately, the country is still suffering from a blue­collar labour shortage. Besides, the workers require some experience and good skills as FFBs are embedded between the frond axil and trunk, limiting access to the stalk. Thus, the harvesting process needs to be simplified and mechanised. The minimum stalk length recommend for a mechanical cutter is 20 cm and the current planting material has a shorter stalk length of 10­15 cm. This poses difficulty in reaching and cutting the stalk either manually or mechanically (Ahmad Hitam and Solah Deraman, 2001). This project aims to identify molecular markers associated with the long stalk trait and establish a panel of validated markers to perform MAS. MAS will facilitate and fasten the introgression of the long stalk trait into the advanced planting materials. To achieve the aim, populations with long stalk traits will be identified and genotyped by sequencing (GBS) for single nucleotide polymorphism marker (SNP) mining. The SNP data will be used for linkage mapping and genome­wide association study (GWAS) analyses to identify molecular markers associated with the long stalk trait. The identified markers will be validated in other populations from the same background with long stalk traits, and markers with satisfactory accuracy in identifying palms with long stalk traits will be developed into a SNP panel for MAS breeding programs. Establishing a panel for the long stalk trait will fasten the introgression of the trait into the advanced planting materials and eventually, encourage harvesting mechanisation as well as reduce the negative impact of labour shortages.

­ 3 ­  RD­RMU­REC02 REV05_1/1/2020 1. The conventional breeding methods are lacking as the phenotypic measurements and selection of palms with favourable traits require a long wait, due to the approximately 12 years of its breeding cycle. 2. Harvesting FFB requires labour with experience and skills as it is a tedious and time­  consuming process. In the view of labour shortage in Malaysia, the harvesting process should be simplified and mechanised. The stalk length of current materials is short and not suitable for mechanisation. This project will identify molecular markers associated with the long stalk trait and a panel for MAS will be developed. An established panel for the long stalk trait will fasten the introgression of the trait into the advanced planting materials and eventually, encourage harvesting mechanisation as well as reduce the negative impact of labour shortages. Project’s goal: To establish a panel of molecular markers associated with the long stalk trait. Objectives: 1. To identify the long stalk population and search for SNPs. 2. To perform linkage mapping and GWAS to identify molecular markers associated with the long stalk trait. 3. To validate the identified molecular markers associated with the long stalk trait in other populations. 4. To develop a SNP panel from validated markers for MAS breeding programs. C. Problem Statement (please indicate background of problem statement or requisition) D. Project Description (how the propose project can solve the problems) E. Goal & Objective

­ 4 ­  RD­RMU­REC02 REV05_1/1/2020 ((i) Project Status C ü New Improvement from existing project Continued from existing project (ii) Involvement of Research Instituition/Other Organization Malaysian Palm Oil Board 1. Genomics Laboratory, FGV Innovation Centre 2. Breeding Unit, Pusat Penyelidikan Pertanian Tun Razak F. Project Background G. Project Location H. Methodology (detail in material & method of the propose project) 1. Identification of population segregating for long stalk trait Preliminary population will be selected based on the long stalk data collected by the breeding unit and subjected to an in­house CTAB DNA extraction protocol. The population selection will be narrowed down based on the legitimacy analysis. Legitimate progenies and their respective available parents will be subjected to GBS by the service provider. The phenotype data will be collected for all the samples subjected to GBS, approximately in 6 to 8 months. The phenotype data will be subjected to a normality test to ensure the phenotype data are normally distributed and the outliers are removed in downstream analysis 2. SNP mining through GBS The quality of extracted DNA samples will be checked by FGV and the service provider. The DNA samples will be mined for SNPs through the GBS method. The GBS data will be cleaned according to the service provider’s in­house QC requirement of per base sequence quality, per tile sequence quality, per quality sequence scores, per base sequence content, per sequence GC content, etc before preparing the SNP file. 3. GWAS, linkage mapping, and QTL analysis The cleaned SNP data will be imputed and filtered using different softwares and parameters. The relatedness of the samples will be studied through phylogenetics, sample distances, PCA, structure, and admixture analyses.

­ 5 ­  RD­RMU­REC02 REV05_1/1/2020 (i) Project Risk 1. Limited sources of the legitimate population with long stalk traits to select the population for initial screening and validation of molecular markers. 2. Limited number of molecular markers that are associated with the long stalk trait. 3. Laborious phenotype measurement due to tall palms and peculiar requirements for a consistent measurement SOP. 4. Variation in stalk lengths between bunches from the same palm. The imputed and filtered SNP data and the phenotype data of all progenies will be subjected to statistical analysis in R Studio for GWAS. The data for biparental crossing progenies will be subjected to statistical analysis of linkage mapping and QTL analysis. 4. Molecular marker identification SNPs and LD blocks related to the long stalk trait will be identified based on the GWAS, linkage mapping, and QTL analysis. The identified SNPs and LD blocks will be selected and developed for targeted genotyping. 5. Validate identified molecular markers in other population Other populations associated with the Yangambi parent (expected to carry the long stalk trait) and other Angola populations will be identified and phenotyped. This is because progenies from the biparental crosses (Deli Dura x Yangambi and Tanzania x Yangambi) and open­pollinated Angola population in FGV’s collection with long stalk trait were utilised for GBS. Validating the association of the markers with the traits in similar backgrounds is essential before utilising them in populations from different backgrounds. The progenies of the identified population will be subjected to an in­house CTAB DNA extraction protocol and legitimacy analysis. The legitimate progenies’ leaf samples will be subjected to targeted genotyping of the identified molecular markers by the service provider. The data from targeted sequencing will be analysed for markers’ accuracy in predicting the long stalk phenotype. 6. Develop molecular markers into a panel of MAS The molecular markers with satisfactory accuracy will be developed into a SNP panel for MAS breeding programs.

­ 6 ­  RD­RMU­REC02 REV05_1/1/2020 (iv) Start Date 1st January 2022 (v) End Date 31st December 2025 (vi) Gantt Chart No Project Activity 2022 2023 2024 2005 Q 1 Q 2 Q 3 Q 4 Q 1 Q 2 Q 3 Q 4 Q 1 Q 2 Q 3 Q 4 Q1­ Q4 1 Population identification & selection 2 Legitimacy analysis & phenotyping of selected progenies 3 Sample preparation & GBS of selected progenies 4 GWAS, linkage mapping, and QTL analysis 5 Marker validation population selection 5 Legitimacy analysis & phenotyping of validation population 6 Sample preparation & targeted sequencing for marker validation 7 Marker accuracy analysis and finalisation of markers for MAS 8 Development of SNP panel for MAS breeding program H. Budget (Cost of Project) Kategori Jumlah (2022) (2023) (2024) (2025) (RM) (RM) (RM) (RM) (RM) Bahan/ Bekalan Penyelidikan 8,000 6,500 5,000 5,000 Perkhidmatan Teknikal 95,000 50,000 50,000  ­  Kos pengangkutan 3,500 3,500 3,500 3,500 Kos penyelenggaraan 5,000 5,000 5,000 5,000 Sewaan peralatan/bangunan ­  ­  ­  ­  Lain­lain ­  ­  ­  ­  Jumlah 111,500 65,000 63,500 13,500 Jumlah kos 253,500

­ 7 ­  RD­RMU­REC02 REV05_1/1/2020 A SNP panel of molecular markers associated with the long stalk trait. The SNP panel will be utilised for MAS breeding program to fasten the introgression of the trait into the advanced planting materials and progeny testing. I. Planned Outcome (Potential Product for Commercialization/Technical Recommendation & etc) The outcome expected from this project is the development of a SNP panel for the MAS breeding program of the long stalk trait. This panel will be able to genetically select progenies that are most likely to exhibit long stalk phenotypes. In the long run, the panel will enhance the commercialisation of materials with long stalk traits. This project was initiated in Q1 2022. The brief progress of the project is as follows: 1. Identification of populations with the long stalk Preliminary populations were selected based on the long stalk data collected by the breeding department and subjected to an in­house CTAB DNA extraction protocol. The population selection was narrowed down based on the legitimacy analysis. Legitimate progenies and their respective available parents were delivered to the vendor. The phenotype data collection were completed for all the samples subjected for GBS. 5 out 6 of populations subjected for GBS has a normal distribution for the stalk length. 2. SNP mining through GBS Every extracted sample’s DNA quality was assessed through the agarose gel electrophoresis method for DNA integrity and Qubit Fluorometer (dsDNA HS Assay Kit) method for DNA quantity and purity. The DNA samples were double digested using the Pstl (6­base cutter) and Mspl (4­base cutter) restriction enzymes. The digested sample was subjected to paired­ends sequencing using the Illumina platform. The sequencing data quality was assessed and ~89% of sequencing data was retained for each sample. The sequencing data were aligned with the reference genome, EG5, and a total of 412,230 SNPs were called. 3. Validate identified molecular markers in other population Identification and preliminary phenotype data collection of other long stalk progenies from similar backgrounds are ongoing. J. Business Opportunity (any opportunity for services / professional development) L. Benefit to Group/Cluster/Company/Unit (Tangible and Intangible)

­ 8 ­  RD­RMU­REC02 REV05_1/1/2020 Establishing a SNP panel for the long stalk trait will fasten the introgression of the trait into the advanced planting materials and its commercialisation. This eventually, encourage FFB harvesting mechanisation as well as reduce the negative impact of labour shortages. Not applicable (ii) Project ‘Intellectual Property Rights’ (Please state the organization). FGV­MPOB Consultancy Agreement (i) State any contract obligation with third party that involve to this project M. Impact of the project (please state also what is the impact if the project is not approve) N. Collaboration Opportunity

­ 9 ­  RD­RMU­REC02 REV05_1/1/2020 O. Research Team (pls specify % contribution) Name Organization (i) Project Leader Siti Khadijah (45%) FGV R&D Sdn Bhd (ii) Team members Amer Izzat Samsudin (15%) Sharmilah Vetaryan (15%) Muhammad Farid Abdul Rahim (5%) Mohd Azinuddin Ahmad Mokhtar (5%) Suradi Mohamad (5%) FGV R&D Sdn Bhd FGV R&D Sdn Bhd FGV R&D Sdn Bhd FGV R&D Sdn Bhd (iii) Supporting Staff (Bil) 1 lab analyst (5%) 1 staff breeding team (5%) FGV R&D Sdn Bhd (iv) Contract Staff (Bil) Reviewed by: Reviewed by: ............................................................ ............................................................ ( Norhafizah Ghazali ) (Dr Lee Yang Ping )      Statistician          Head of FGVIC, Precisian Agriculture & Genomics      FGV R&D Sdn Bhd         FGV R&D Sdn Bhd   Approved by: ............................................................. ( Noor Hisham Hamid ) Chief Executive Officer   FGV R&D Sdn Bhd  

Screening of potential Ganoderma-tolerance marker using multi-approach analysis Anis Farhan Fatimi Ab Wahab, Nur Syafiqah Muhammed, Hooi Wei Yeng and Sharmilah Vetaryan ABSTRACT Oil palm is critical to the economies of several nations, particularly Indonesia and Malaysia, where vast amounts of its products are exported as oil, ready-to-eat food, and other derivatives. However, the production of palm oil has been hampered by the serious disease, known as basal stem rot disease (BSR) caused mainly by Ganoderma boninense followed by Ganoderma zonatum and Ganoderma miniatocinctum. BSR-infected palms have been reported to have lower productive life spans and generate less weight of fresh fruit bunches, resulting in significant economic losses for oil palm industries. The prevalence of BSR disease was on the rise due to several factors; climate change, increased virulence of the aforementioned fungi, and lacking of proper surveillance mechanisms and phytosanitary procedure at the plantation areas. Apart from developing control techniques to restrict the spread of Ganoderma sp. by direct targeting of the disease itself, more efforts are currently being made to seek for potential Ganoderma-tolerance markers (GTM) for planting material using molecular-assisted breeding to facilitate introgression of the resistance genes into advanced breeding material. As a result, this project is being commenced to accelerate the screening for potential GTM using a multiapproach analysis. This includes differential display polymerase chain reaction (dd-PCR), a comprehensive and sensitive analytical tool based on reverse-transcriptase PCR (RT-PCR) to obtain gene expression profiles of different samples (i.e. between different degrees of Ganoderma tolerance of oil palm clones), development of in-vitro screening (IVS) protocol to screen for potential Ganoderma tolerance planting material of which can be further utilized for genome-wide association studies (GWAS) and transcriptomics analysis. Besides that, validation of previously published potential GTM markers will be performed in FGV’s material as well. In conclusion, the findings from this multi-approach research to accelerate the search for prospective GTM will be beneficial in the future, assisting in the screening of potential Ganoderma-tolerance planting material.

REV05_1/1/2020 Screening of potential Ganoderma­tolerance marker using multi­approach analysis High quality planting material through advanced genomics, clonal and breeding programme Genomics Dr. Anis Farhan Fatimi Ab Wahab RD­RMU­REC02 FGV R&D SDN BHD PROJECT PROPOSAL A. Project Title A1. Project Code A2. Program A3. Strategic Thrust ( please refer to the roadmap for FGV R&D Upstream) A4. Unit A5. Presenter

­ 2 ­  RD­RMU­REC02 REV05_1/1/2020 B. Introduction The world's most significant oil crop is the oil palm tree, Elaeis guineensis, which provides around 40% of all traded vegetable oil. Over three billion people, especially in Asia, regularly eat palm oil as essential dietary components. Palm oil also have a variety of significant non­food applications, such as use in cosmetic industries, biofuels, and pharmaceutical industries (Murphy et al. 2021). Compared to crops like sunflower, soybean, and rapeseed, oil palm provides a higher yield at a cheaper cost of production (Nomanbhay et al. 2017). However. future oil palm crop production will confront a number of difficulties, such as new dangers from climate change, pests, and diseases. In addition to battling with climate change, the productivity of oil palm has also been greatly threatened by the fungal infection known as basal stem rot disease (BSR) which can reduce the yield by 50­80% (Paterson et al. 2019). The BSR incidence has been reported to increase over the years, most probably due to improper land sanitation practices, repeated cycles of crop planting on infested sites, and increasing virulence of the fungus due to its faster rate of evolution for adaptation. This disease can kill up to 80% of the oil palm while they are halfway through its 25­year of productive lifespan (Murphy et al. 2021; Hambali et al. 2017). Without proper strategies to reduce BSR incidence, the economic loss will further increase and possess a greater effect on the oil palm industry. Currently, most strategies to combat the spread of the BSR disease mostly focus on the pathogen itself rather than the oil palm. This is presumably owing to the simplicity of testing, as the fungus grows more readily and responds more quickly to the appropriate treatments. At present, limited information is available on the potential Ganoderma­tolerant marker (GTM)that can be used to screen for planting material. It is very important to have GTMs that can facilitate the molecular breeding process to attain oil palm varieties with increased disease resistance so that they can stay productive for a long time and reduce the overall cost of disease management. * Ruangan ini di penuhisecara ringkas, padat dan tepat.

­ 3 ­  RD­RMU­REC02 REV05_1/1/2020 The majority of research being done to combat the BSR­disease brought on by G. boninense, Ganoderma miniatocinctum, and Ganoderma zonatum is currently concentrated on developing products that specifically target the pathogens rather than developing oil palm varieties that are more resilient to infection. However, the majority of the strategies and control products demonstrated varying degrees of efficacy as a result of the high genetic variability of the Ganoderma sp. Therefore, this project has been conducted to identify Ganoderma­tolerant markers that can be used to screen for planting material that can highly tolerate the infection of Ganoderma sp., regardless of the Ganoderma sp. genetic variation. * Ruangan ini di penuhisecara ringkas, padat dan tepat. This project may facilitate the identification of potential GTM that can be used in screening for planting materials in the future. The GTM screening process involved numerous ways to expedite the identification of possible GTMs and was reliable due to multiple levels of assessments using a variety of candidate samples that had been evaluated for their Ganoderma­tolerant trait using the established IVS method. Choosing the correct planting materials with high Ganoderma­tolerant trait may reduce the BSR incidents in the future, of which may mitigate financial loss especially to the oil palm planters. C. Problem Statement (please indicate background of problem statement or requisition) D. Project Description (how the propose project can solve the problems)

­ 4 ­  RD­RMU­REC02 REV05_1/1/2020 C Objectives: 1. To identify potential GTM via PCR­based analysis using the Ganoderma­infected and non­infected root and leave samples. 2. To enhance the in vitro screening (IVS) technique to facilitate the discovery and selection of ideal Ganoderma­tolerant or ­susceptible oil palm tissue culture varieties that are available in FGV R&D Sdn. Bhd. for further downstream analyses 3. To perform  ­omics analysis for the selected potential candidates attained from thorough IVS screening to further identify potential GTMs (i) Project Status New Improvement from existing project Continued from existing project (ii) Involvement of Research Instituition/Other Organization ­ / FGVIC – Biotechnology (Genomics) 1. Identification of potential GTM via PCR­based analysis i­ DNA profiling via ddRT­PCR analysis For this analysis, Ganoderma­infected tissues (root and leave) at different infection time points will be used. The RNA will be extracted from the tissue samples and converted to cDNA before can be used as a template in PCR analysis. The forward primer for this analysis is known as an arbitrary primer (10­mer) designed to anneal to cDNAs at random distances from the poly(A) tails while the reverse primer is known as anchor primer, dT10XX, where X refers to base A, T, G or C. Anchor primer will anneal at the downstream of the cDNAs. The amplification of the PCR was performed for 25 cycles at 95 °C for 40 seconds, 36 °C for 30 seconds, 72 °C for 1 minute, and 15 cycles at 95 °C for 40 seconds, 42 °C for 30 seconds, E. Goal & Objective F. Project Background G. Project Location H. Methodology (detail in material & method of the propose project)

­ 5 ­  RD­RMU­REC02 REV05_1/1/2020 72 °C for 1 minute. The PCR products will be analysed using 2% 1X TAE agarose gel to observe and compare the DNA profile. Any intriguing DNA band that shows up exclusively once or has a distinctive intensity at particular infection time points will be excised, cloned, sequenced, and analysed. This method has the advantages of being straightforward, cost­effective, and only targeting expressed genes. In addition to this, screening of SNPs or SSR markers which are located in proximity with the identified bands from ddRT­PCR analysis will be conducted in order to identify any DNA sequence variations for screening purposes. ii­ Targeted gene expression profile using qPCR analysis The tolerance to infection is not only due to the presence of the resistance genes (R­genes) toward particular pathogen, but, it has also been contributed by the pre­existing levels of general resistance proteins that have been present in the plants regardless of whether the plant is under attack or not. A higher concentration of pre­existing defense molecules that are toxic to the pathogens will cause the plant to be more disease tolerant as this condition can delay the progression of the infection. Hence, for this reason, the targeted genes that are thought to play a role in plant general resistance will be chosen for the gene expression study. Samples from the IVS analysis will be used for this analysis. The expression of these genes will be compared between before and after infection analyses and may also be compared to the degree of severity score from IVS analysis. 2. Optimizing parameters for IVS method to screen for Ganoderma­tolerant and Ganoderma­susceptible planting material The development of an in vitro screening method is necessary to identify possible candidates for planting material that show traits indicative of potential Ganoderma­  tolerance and Ganoderma­susceptible. This technique enables the infection assay to be carried out in a controlled setting, hence minimizing mistakes and variance during data collection. The symptoms of the infected ramets will be recorded every week for 10 weeks, and the results will be used to score the disease severity index (dsi). The tissue culture clones will be ranked based on how well they can tolerate the infection by G. boninense using the final results of the dsi scores. The selected candidate will be further analysis using ­omics approach.

­ 6 ­  RD­RMU­REC02 REV05_1/1/2020 Experimental design: The ramets were arranged according to Randomized Completely Block design (RCBD) with the number of ramets and the biological replicates as requirement for statistical analysis which has been verified by FGV’s biostatistician from previous IVS preliminary analysis as per the following: Table 1 The total number of tissue culture ramet per clone per phase * If there are sufficient ramets, it may be possible to repeat the analysis to produce more solid data. The disease severity indices (dsi) per week basis will be employed in this study to assess the performance and establish the level of infection tolerance of the tissue culture clones with respect to the response towards infection by G. boninense under the controlled treatments. The data for dsi of the treated clones was assessed based on the disease score (0 to 5 scales) as listed in Table 2. The dsi will be computed according to the formula described by Abdullah et al. (2003). Table 2 The score (0-5) of the disease class on based on the external signs and symptoms of the treated ramets Where, ∑   = sum of the symptomatic leave with their corresponding scale N = total leave K = highest score scale

­ 7 ­  RD­RMU­REC02 REV05_1/1/2020 Analysis of variance (ANOVA) was performed as per RCBD mainly to determine whether there were statistically significant differences among the test clones with respect to their level of resistance for each week. Meanwhile, score data recorded from week 1 to week 10 for each tissue culture clones will be subjected to repeated measures ANOVA to compare group means on a dependent variable across repeated measurements of time (same individuals are measured on the same outcome variable under different time points or conditions). Time is often referred to as the within­subjects factor, whereas a fixed or nonchanging variable is referred to as the between­subjects factor. Where appropriate, mean comparisons then were performed using the Tukeys’s Honest Signifcant Diference (HSD) test at p≤0.05. The mean of the dsi at different week after inoculation for each clone will be evaluated to rank Ganoderma ­tolerant’s order. 3. Performing ­omics analysis on the potential candidates The ­omics (genomic, transcriptomic, and/or proteomic) analysis will be performed on the selected candidate based on the IVS analysis. These approaches are important to screen for more potential GTMs that can be used widely to screen for potential planting materials in the future. 4. Scientific writing Interesting information and findings from this project will be delivered to the public through the publication of scientific writing. (i) Project Risk 1. Limited number and type of tissue culture materials to be used in this project. 2. The overall rating of Ganoderma­tolerance may be impacted by inconsistent profiles of the dsi caused by phenotypic or somaclonal variance. 3. Efficient imaging and scoring method for IVS experiment (iv) Start Date May 2023 (v) End Date Dec 2025

­ 8 ­  RD­RMU­REC02 REV05_1/1/2020 (vi) Gantt Chart 2023 2024 2025 No Activity Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 1 DNA profiling via ddRT­PCR analysis 2 Targeted gene expression profile using qPCR analysis 3 Conversion of targeted GTM into suitable screening method 4 Enhancement of IVS method to screen for Ganoderma­tolerant and Ganoderma­susceptible planting material 5 IVS of tissue culture ramets to obtain reliable PT and PS candidates (may involve other strain of G. boninense to compare the reponse and obtain a more reliable result) 6 GTM validation with PT and PS samples to determine prediction accuracy 7 Performing ­omics analysis on the potential candidates to search and screen for more potential biomarkers 8 Scientific writing and publication I. Budget (Cost of Project) Kategori Jumlah (RM) (2023) (RM) (2024) (RM) (2025) (RM) Bahan/ Bekalan Penyelidikan 272 000 10 000 90 910 102 000 Perkhidmatan Teknikal ­  ­  Kos pengangkutan 4000  ­  2000 2000 Kos penyelenggaraan ­  ­  Sewaan peralatan/bangunan 5000 5000 5000 Lain­lain 13 000  ­  ­  5000 8000 ­  Jumlah 234 910 15 000 102 910 117 000

­ 9 ­  RD­RMU­REC02 REV05_1/1/2020 J. Planned Outcome (Potential Product for Commercialization/Technical Recommendation & etc) The following results are expected once this project is completed: (i) A list of potential GTMs will be successfully obtained and assessed to be used in marker­  assisted breeding and Ganoderma­tolerant planting materials screening analysis (ii) An enhanced and reliable in vitro screening method for identification of potential Ganoderma tolerant material can be established to aid the selection of the desirable planting materials. (iii) Through thorough investigation using the ­omics method, further in­depth GTMs that can support reliable screening analysis and improve comprehension of the complex functions of the Ganoderma­tolerant mechanism will be found. Current progress: i­ The preliminary analysis of DNA profiling via ddRT­PCR has been performed to screen for potential arbitrary and anchor primers to be used in subsequent analysis. The sample used for this preliminary analysis were infected roots from 7 d.p.i and 49 d.p.i (d.p.i stand for day­  post­inoculation) and uninfected root was used as the control. Figure 1.0 shows one example of the outcomes of this analysis. From the analysis, a few bands that showed a distinct profile as compared to the controls will be selected for cloning and sequence analysis. This analysis is still ongoing. O – control oil palm G – control G. boninense 7 – 7 d.p.i infected root 49 – 49 d.p.i infected root Figure 1.0 Profile of the PCR products from ddRT-PCR on 2% of 1X TAE agarose gel. ii­ To establish the IVS method, a few parameters need to be optimized in order to obtain a consistent result. For this preliminary analysis, the ramets were grown on three different growth media; solid media, semi­solid media, and liquid media. The symptoms of the infection for each infected ramet were observed and recorded weekly, for 10 weeks. In order to determine which media was optimal for IVS analysis, the recorded data were then compared.

­ 10 ­  RD­RMU­REC02 REV05_1/1/2020 Collaboration Opportunity Providing potential GTMs to help the company to screen for planting materials that can survive the Ganoderma­infection longer as one of the solutions to reduce the economic loss and controlling the spread of the disease. A more cogent and systematic approach to control and break the chain of BSR spreading is currently urgently needed due to the significant economic impact of the G. boninense­caused BSR disease of oil palm. This study will help in discovering prospective GTMs to employ in screening for planting material that can tolerate the Ganoderma­infection better, therefore can have a longer productive life span and lower the economic loss. K. Business Opportunity (any opportunity for services / professional development) L. Benefit to Group/Cluster/Company/Unit (Tangible and Intangible) M. Impact of the project (please state also what is the impact if the project is not approve) N. From the result analysis, the solid media was the best media to be used in IVS analysis. Most of the BSR symptoms appear almost at the same time for the same clone, whereas, semi­solid and liquid media have caused delays in the appearance of the symptoms, most probably due to the unconducive condition for the growth of G. boninense, which does not favour the high humidity. Therefore, solid media will be used for the next IVS infection analysis. This project will improve the quality of the planting materials of which more tolerant toward the infection by G. boninense. If this project is not approved, it will be more difficult to choose planting materials that are more resistant to the BSR disease and the financial damage caused by the growing spread of the disease would be greater. (i) State any contract obligation with third party that involve to this project (ii) Project ‘Intellectual Property Rights’ (Please state the organization). FGVR&D

­ 11 ­  RD­RMU­REC02 REV05_1/1/2020 O. Research Team (pls specify % contribution) Name Organization (i) Project Leader Dr. Anis Farhan Fatimi Ab Wahab (55%) FGV R&D Sdn. Bhd. (ii) Team members Dr. Hooi Wei Yeng (15%) Ms. Nur Syafiqah Mohammed (15%) Ms. Siti Mardhiah Mustafha (10%) Ms. Sharmilah Vetaryan (5%) Ms. Nurul Asyikin Mohd Zim Ms. Rafidah Mohamed Kassim Ms. Raja Bahiyah Nur Raja Hirdan Dr. Lee Yang Ping FGV R&D Sdn. Bhd. (iii) Supporting Staff (Bil) (iv) Contract Staff (Bil) Reviewed by: Reviewed by: ............................................................ ............................................................ ( Norhafizah Ghazali ) (Dr. Lee Yang Ping )    Statistician             Head of FGVIC, Precisian Agriculture & Genomics      FGV R&D Sdn Bhd           FGV R&D Sdn Bhd         Approved by: ............................................................. (Noor Hisham Hamid) Chief Executive Officer FGV R&D Sdn  Bhd  

Establishment of Gene editing system for Ganoderma boninense Anis Farhan Fatimi Ab Wahab; Mohd Azinuddin Ahmad Mokhtar; Wong Mui Yun and Sharmilah Vetaryan ABSTRACT Ganoderma boninense has been a major threat to the productivity of oil palm, especially in Malaysia and Indonesia by causing a destructive vascular disease, known as basal stem rot disease (BSR), resulting in annual losses of up to USD 500 million. Despite the fact that many control methods have been implemented to combat the BSR disease, none of them are effective enough to reach the desired objectives. This could be due to a lack of understanding of the genetic variability and virulent characteristics of G. boninense, which has impeded the development of effective treatments for this disease. Therefore, the purpose of this project is to develop CRISPR/Cas9 gene editing technique to enable functional gene analysis in G. boninense. The targeted gene in this project was orotidine-5-monophosphate (pyrG), a key enzyme in uridine biosynthesis. This gene was chosen due to the ease of screening by the uracil auxotrophic characteristic and the use of 5-fluoroorotic acid (5-FoA) as the selection agent. To select CRISPR-Cas9 target sites and create possible sgRNAs, the CCTop online target prediction tool was employed. Based on the analysis, six potential sgRNAs (sgRNA-1 to sgRNA-6) were chosen and cloned. In vitro transcription of the cloned sgRNAs was carried out using a commercial kit. The generated sgRNAs and vector harboring the Ganoderma lucidum codon-optimized cas9 gene (pGreenGlCas9) were co-transformed into G. boninense using a protoplast PEG-mediated transformation method. The transformation plates were overlaid with 1800 µM/mL 5-fluoroorotic acid (5-FoA) and incubated at 28°C for 7 days. The transformation plates yielded around 43 putative G. boninense pyrG- mutants. However, only two out of the 43 putative mutants can stably grow on 1800 µM/mL 5-FoA at third generation and exhibit similar phenotype and growth rate as the wild type. The sequencing analysis for both putative mutants has confirmed the presence of indels at the targeted region for sgRNA1. No indels were detected for the other sgRNAs. In conclusion, CRISPR/Cas9 gene editing in G. boninense was successful. At present, this is the first effective CRISPR/Cas9 gene editing in G. boninense that has been described. Nonetheless, more work needs to be done to make this strategy a viable and promising technique for Ganoderma-functional genomics research in the future.

FGV R&D SDN BHD (COMPANY NO. 1012623-V) PROJECT CLOSURE Project Title : Establishment of gene editing system for Ganoderma boninense (FR-0604-80-0219) Program : Ganoderma Research Project Leader : Dr. Anis Farhan Fatimi Ab Wahab Team Members : Dr. Mohd Azinuddin Ahmad Mokhtar, Prof. Dr. Wong Mui Yen and Sharmilah Vetaryan Unit : Genomics Presenter : Dr. Anis Farhan Fatimi Ab Wahab Abstract Ganoderma boninense has been a major threat to the productivity of oil palm, especially in Malaysia and Indonesia by causing a destructive vascular disease, known as basal stem rot disease (BSR), resulting in annual losses of up to USD 500 million. Despite the fact that many control methods have been implemented to combat the BSR disease, none of them are effective enough to reach the desired objectives. This could be due to a lack of understanding of the genetic variability and virulent characteristics of G. boninense, which has impeded the development of effective treatments for this disease. Therefore, the purpose of this project is to develop CRISPR/Cas9 gene editing technique to enable functional gene analysis in G. boninense. The targeted gene in this project was orotidine-5-monophosphate (pyrG), a key enzyme in uridine biosynthesis. This gene was chosen due to the ease of screening by the uracil auxotrophic characteristic and the use of 5-fluoroorotic acid (5-FoA) as the selection agent. To select the CRISPR/Cas9 target sites and create possible sgRNAs, the CCTop online target prediction tool was employed. Based on the analysis, six potential sgRNAs (sgRNA-1 to sgRNA-6) were chosen and cloned. In vitro transcription of the cloned sgRNAs was carried out using a commercial kit. The generated sgRNAs and vector harbouring the Ganoderma lucidum codon-optimized cas9 gene (pGreenGlCas9) were co-transformed into G. boninense using a protoplast PEG-mediated transformation method. The transformation analysis produced two of the 43 putative mutants that can stably grow on 1800 M/mL 5-FoA at third generation and had a similar phenotypic and growth rate as the wild type. The sequencing analysis for both putative mutants has confirmed the presence of indels at the targeted region for sgRNA-1. No indels were detected for the other sgRNAs. In conclusion, CRISPR/Cas9 gene editing in G. boninense was successful. At present, this is the first effective CRISPR/Cas9 gene editing in G. boninense that has been described. Nonetheless, more work needs to be done to make this strategy a viable and promising technique for Ganoderma-functional genomics research in the future.

1.0 INTRODUCTION Oil palm is crucial to the economies of several countries, particularly Indonesia and Malaysia, where enormous volumes of its products are exported as oil, meal, and other derivatives (Asyraf et al. 2021). However, the production of palm oil has been hampered by the serious disease, known as basal stem rot disease (BSR) caused mainly by Ganoderma boninense, a white rot basidiomycete fungus, and the disease has shown a rising trend since it was discovered in the 1930s (Mohd Shukri et al. 2020). BSRinfected palms have been shown to have shorter productive life spans and produce less fresh fruit bunch weight, resulting in annual losses of up to USD 500 million to Malaysia and Indonesia. Despite the fact that many strategies have been implemented to combat the BSR disease, none of them are effective enough to achieve the intended outcome (Siddiqui et al. 2021). This could be due to a lack of understanding, particularly of the genetic variability owned by different strains of G. boninense, that governs traits such as adaptation, virulence, and aggressiveness, which has hampered the development of effective treatments to control the spread of this disease. To elucidate the pathogenicity of the fungus, , particularly at the molecular level, an efficient approach for functional analysis of the genes that may be utilized to elucidate the factors contributing to the pathogenicity of this fungus must be developed. However, there is currently no information on the established approach for functional gene analysis in G. boninense (Ab Wahab et al. 2022). As a result, efforts are needed to develop methods for molecular functional analysis in order to speed up the design of more efficient strategies to battle and reduce the occurrence of BSR disease in the near future. There are a few ways that could be utilized to knock out or knock down the targeted gene in order to understand the true function of the genes in a certain organism. Among available methods for functional gene analysis is RNA interference which employs the use of small double-stranded RNA (siRNA) complementary to the mRNA target to forbid the translation process of the targeted gene (Svoboda 2020) and knocking out the targeted gene via homologous recombination process by taking advantage of cell’s own DNA repair machinery to replace the targeted region with homologous genomic sequence (Catlett et al. 2003). However, currently, a more advanced technology known as CRISPR/Cas9 gene editing, which serves the same function as the aforementioned methods, has been introduced to facilitate functional gene study. This method makes use of a straightforward two-component system. The first component is the Cas9 protein, an endonuclease that will cleave the DNA strand, and the second component is single guide RNA (sgRNA), which carries a scaffold sequence to anchor the Cas9 and a 20 base pairs spacer sequence (complementary to the target gene and adjacent to the protospacer adjacent motif, PAM) to guide the Cas9 to its intended genomic location. When DNA double-strand breaks occur at the targeted location, the DNA repairing system frequently initiates a non-homologous end joining (NHEJ) event, which involves random indels at the cutting site and results in a frameshift mutation, which frequently results in a premature stop codon or shifts the way the DNA sequence is read, which may result in a malfunctioning product of the expressed gene (Su et al. 2016). The most recent technology has shown to be a powerful tool because it is less expensive, more accurate, easier to use, and faster than any other approach for biological analysis. As a result, investigating the possible application of the CRISPR/Cas9 gene editing technology for G. boninense could be extremely beneficial in identifying the critical virulence components of G. boninense to be targeted for the development of efficient antifungal compounds to manage the BSR disease in the future. Keywords: basal stem rot, Ganoderma boninense, CRISPR/cas9 gene editing

2.0 PROBLEM STATEMENT There is currently no record of effective gene deletion or editing for G. boninense for functional gene analysis except for gene knock-in analysis (Lim et al. 2021, Ab Wahab et al 2022). Gene deletion using a substitution approach via homologous recombination was reported unsuitable to be used in G. boninense as no stable mutants were obtained due to several reasons as discussed by Ab Wahab et al. 2022. The CRISPR/Cas9 gene editing technique is a new technology that has proven successful in a variety of organisms across species. This technique is also reported to serve faster and more effective editing of the targeted gene. As a result, this project has been initiated to establish a CRISPR/Cas9 gene editing approach for G. boninense, which will facilitate functional analysis on G. boninense genes implicated in disease at the molecular level. The information attained from the preceding analysis will be a foundation to develop more efficient strategies and bio-fungicides to control the spread of the BSR disease in the future. 3.0 GOAL & OBJECTIVE Objectives: 1. To identify and select the best chemical compounds to be used in putative mutant screening media 2. To establish a method for transformation using a protoplast PEG-mediated approach 3. To evaluate the gene editing events via PCR-based and auxotrophic trait analyses. 4.0 METHODOLOGY 1. Identification of the best chemical compound for putative mutant selection A few candidates were chosen for this purpose to examine their suitability as selection markers at various concentrations in the event of screening G. boninense putative mutants. Those candidates are: a. Ampicillin (50 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL) b. Kanamycin (50 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL) c. Streptomycin (50 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL) d. Gentamycin (50 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL) e. Carboxin (10.0 µg/mL, 20.0 µg/mL, 30 µg/mL, 40 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL) f. 5-fluoroorotic acid (5-FoA) (100 µM/mL, 200 µM/mL, 300 µM/mL, 400 µM/mL, 500 µM/mL, 600 µM/mL, 700 µM/mL, 800 µM/mL, 900 µM/mL, 1000 µM/mL, 1100 µM/mL, 1200 µM/mL, 1300 µM/mL, 1400 µM/mL, 1500 µg/mL) g. Hygromycin (50 µg/mL, 60 µg/mL, 70 µg/mL, 80 µg/mL 90 µg/mL, 100 µg/mL) The growth of the G. boninense on the PDA containing the antibiotic, 5-FoA or antifungal (carboxin) was observed after 7-10 days of incubation at 28 °C. 2. Construction of the vectors i- Synthesis of the codon-optimized cas9 gene The cas9 gene was originally isolated from bacteria (Streptococcus pyogenes), a prokaryote. For this project, the cas9 gene was synthesized based on the Ganoderma lucidum codon-bias to assure its functionality in the eukaryote system, particularly in Ganoderma sp., by an external vendor.

ii- Identification and cloning of the potential single guide RNA (sgRNA) for pyrG gene To identify potential sgRNAs for pyrG gene, CCTop – CRISPR/Cas9 target online predictor tool has been employed to predict the best sgRNAs using G. lucidum as a species reference. Only six from the top rank of the potential predicted sgRNAs will be cloned into pGEM-T Easy Vector and validated via sequencing analysis. 3. Establishment of protoplast PEG-mediated transformation In order to introduce foreign DNA into the fungal cell using the PEG-mediated transformation approach, the fungal cell wall need to be removed carefully, usually by using lytic enzymes. Excessive removal of the cell wall will cause the cell to burst or unable to regenerate. Hence, to obtain viable protoplasts cell, a few parameters; the age of the mycelia, the combination of the lytic enzymes, and incubation time have been taken into consideration for the optimization analysis. Prior to optimization, the working protocol was based on the method described by Ab Wahab et al. (2022). The method used for the PEG-mediated transformation analysis was adopted from Yu et al. (2014). The adopted approach has been slightly modified to improve the efficiency of the transformation. The protoplast cells generated from the optimized method were rinsed with 1 mL of STC solution (0.55 M sorbitol, 10 mM CaCl2, and 10 mM Tris-HCl buffer pH 7.5) and pelleted by centrifuge at 833 g , 4 °C for 5 minutes. After that, the protoplast cells were diluted with STC solution to 1 107 cells/mL. Around 100 µL of 1 107 cells/mL were employed for each transformation analysis. The protoplast cells were then mixed with 50 µL of PTC solution (60% w/v PEG4000, 10 mM Tris-HCl buffer pH 7.5 and 50 mM CaCl2) and DNA (2.5 µg pGreenCas9 and 5 µL each of the two in vitro transcribed sgRNAs), and incubated on ice for 10 minutes before 1 mL of PTC buffer was added. For this analysis, two sgRNAs were simultaneously co-transformed into the protoplast cells. The mixture was then incubated at room temperature for 20 minutes before adding 10 mL of YMSB (0.015 M yeast extract, 0.016 M peptone, 0.044 M malt extract, 0.6 M sucrose). Before the mixture was poured into the Petri dish, a warm 10 mL of 2% YMSB (2% agar, 0.015 M yeast extract, 0.016 M peptone, 0,044 M malt extract, 0.6 M sucrose) was added to the mixture. After incubating the transformation plate overnight at 28 °C, 10 mL of 1400 mM 5-fluoroorotic acid (5-FoA) supplemented potato dextrose agar (PDA) was overlayed to screen for putative G. boninense pyrG mutants. The transformation plate was incubated for 4-5 days at 28 °C in the dark before any growth of putative mutants can be observed. 4. Evaluation of the gene editing events via PCR-based and auxotrophic trait analysis The pyrG gene encodes orotidine-5-monophosphate (OMP) decarboxylase, an enzyme of the pyrimidine biosynthesis pathway that is important for uridine synthesis. The presence of a functioning pyrG gene in the cell converts 5-FoA to fluorodeoxyuridine, a toxic chemical that can destroy the cell by suppressing cell proliferation. Resistance towards 5-FoA can be attained by blocking the pyrimidine pathway through the deactivation of pyrG gene via mutational inactivation. Other than resistance to 5- FoA, deactivation of the pyrG gene results in a uridine auxotrophic phenotype that requires uridine to be supplemented in minimum media (MM) to grow. For the auxotrophic trait study, each putative mutant was cultured on a PDA plate containing 1400 mM 5-FoA for at least three generations to select for stable putative mutant. Only stable putative mutants will proceed to further validation using PCR-based and sequencing analysis. 5. Scientific writing

The findings from this project will be presented as a poster at PIPOC 2023. The full research paper write-up is still ongoing. 5.0 GANTT CHART 2019- 2021 2022 Activity Jan Feb Mac Apr May Jun Jul Aug Sep Oct Nov Dec Optimising method for generation of protoplasts Optimising methods for transformation Developing vectors and synthesizing sgRNAs in vitro for targeted genes Transformation analysis Screening of mutants on the selective media for three generation (auxotrophic traits) DNA extraction and PCR verification of gene-editing on putative mutants via sequencing Current percentage of achievement: 100% Problems and issues: Market discontinuation of a certain type of lytic enzyme. This may cause a delay in project progress because new optimization analysis on the optimal composition of the lytic enzymes utilized to make the protoplast will need to be performed again. 6.0 OUTCOME i. Choosing suitable chemical compound for putative mutant screening Based on the analyses of different antibiotics, 5-FoA, and carboxin, the best three chemical compound that can be used for mutant screening are hygromycin, 5-FoA and carboxin. The minimal inhibitory concentration (MIC) for the hygromycin, 5-FoA and, carboxin was 90 µg/mL, 1400 mM/mL and 50 µg/mL, respectively. The genes that confer resistance toward hygromycin and carboxin were hygromycin B phosphotransferase (hph) and succinate dehydrogenase B with a point mutation (sdhBDpm) at position Preliminary work and resignation of the project PIC

240 to change histidine residue to leucine (His240Leu). To utilize hygromycin or carboxin as a selection agent, the putative mutant must carry the respective resistance genes, which can be introduced into the cell via transformation of the vector containing the abovementioned genes. While for 5-FoA, the resistance toward this reagent has been explained in Method Part 4. ii. Synthesizing the codon-optimized cas9 gene The cassette of G. lucidum codon optimized cas9 gene (Glcas9) was synthesized together with GPD promoter (glyceraldehyde-3-phosphate dehydrogenase) to drive the expression in G. boninense and PDC terminator (pyruvate decarboxylase) to stop the transcription of the Glcas9, and EcoRV and SpeI restriction enzyme sites that flank the cassette for future use. The Glcas9 cassette was then cloned into pGreen0179hph to obtain pGreenGlcas9 vector. Figure 1.0 showed the circular plasmid of pGreenGlcas9 and its elements. iii. Identification of potential sgRNAs for pyrG gene, cloning and validation The prediction of potential sgRNAs for pyrG gene from G. boninense was performed using CCTop – CRISPR/Cas9 target online predictor tool (Stemmer et al. 2015; Labuhn et al. 2017). Based on the analysis, six potential sgRNAs (20 nt) was selected, cloned and validated using sequencing analysis prior to in vitro transcription (IVT) analysis. The cloned sgRNA sequence consist of T7 promoter sequences (17 bp), 20 nt of sgRNA sequence, and scaffold sequence for the binding of the Glcas9 protein (Figure 2.0). iv. Co-transformation of pGreenGlcas9 and IVT sgRNAs into the protoplast via PEG-mediated transformation Figure 1.0 The schematic diagram of pGreenGlcas9 vector and its elements Figure 2.0 The schematic diagram of the cloned sgRNA cassette sequence. It consists of T7 promoter sequences, 20 nt of predicted sgRNA sequence and scaffold tracrRNA for the binding of Glcas9 protein

Fungi have different cell wall component compositions depending on the species and strain. As a result, thorough optimization of protoplast generation and regeneration for Ganoderma boninense PER71 was performed to ensure that the number of viable protoplasts was sufficient to be employed in the transformation analysis. Prior to optimization, the working protocol was based on the method described by Ab Wahab et al (2022). Based on the analysis, the optimum parameter for the generation of the protoplast is more or less similar to the result reported by Ab Wahab et al. (2022). The suitable age for mycelia to produce more protoplast cells are day-4 and -5 when a combination of 1% lysing enzyme from Trichoderma harzianum and 0.08% of driselase in osmotic solution (0.6 mannitol, 100 mM phosphate buffer pH 5.8) were used. The incubation time was 4h at 30 °C, a bit longer as compared to the working protocol, most probably due to the thicker cell wall for the G. boninense PER71 (dikaryotic mycelia) used in this experiment as compared to G. boninense Mg12 (monokaryotic mycelia derived from G. boninense PER71)(Ab Wahab 2022). The total protoplast cells obtained from the optimised method were around 1.75 – 2.15 107 cells/mL. Combining more lytic enzymes, such as 1% T. harzianum lysing enzyme, 0.08% Driselase, 0.03% Aspergillus sp. lysing enzyme, and 0.03% yatalase, resulted in more protoplast cells, up to 7.87 107 cells/mL after only 3 hours of incubation. The regeneration media used in this project was YMSA (Chou & Tzean 2016) as the regeneration rate of the protoplast was already >85%. The co-transformation analysis via protoplast PEG-mediated transformation approach has yielded 43 putative mutants. However, only two out of the 43 putative mutants can stably grow in 1800 µM/mL 5- FoA at third generation. Both putative mutants exhibited similar phenotype and growth rate as the wild type (Figure 3.0). PCR and sequencing studies were used to validate the mutations even further. According to the PCR results, the amplified pyrG gene from 4/5pyrG.col.6 produced two distinct DNA bands, one of which was similar in size to the WT and the other of which had a size increase of about 200 bp when compared to the WT, whereas the amplified pyrG gene from 4/5pyrG.col.4 produced no significant differences (Figure 4.0). Figure 3.0 The putative mutants on 1800 µM/mL 5-FoA. Two colonies, 4/5pyrG.col6 and 4/5pyrG.col.4, were putative pyrG putative mutants with 5-FoA resistance. The wild type (WT) G. boninense PER71 was used as the control. Because WT has a functional pyrG gene, it cannot grow on selection media containing 5-FoA because 5-FoA is converted to a toxic substance that can kill the cell. (Note: 4/5 refers to the co-transformed sgRNA-1 and sgRNA-2 that took place in the same transformation cell)

The pyrG gene sequencing result for both putative mutants was further analysed using ICE Synthego and TIDE prediction tools (Brinkman & van Steensel 2019) to screen for indels at the targeted regions. According to the ICE Synthego analysis (Roginsky, 2018), the indels at the targeted area for sgRNA-1 were successfully detected for 4/5pyrG.col.6 (Figure 5.0), but, this prediction technique failed to detect the indels for 4/5pyrG.col.4. However, TIDE prediction tool was able to detect the indels, also at the targeted region for sgRNA-1 (Figure 6.0). This event may be due to the low copy number of the edited pyrG gene amplicons in the samples, hence, affecting the indels prediction analysis. Figure 4.0 The agarose gel profile for the amplified pyrG gene from both mutants ~1.0 kb ~1.2 kb 6 : 4/5pyrG.col.6 4 : 4/5pyrG.col.4 Figure 5.0 Sequence analysis using ICE Synthego prediction tool for pyrG amplicons amplified from 4/5pyrG.col.6. The result showed various indels have been detected at the targeted region. Figure 6.0 Sequence analysis using TIDE prediction tool for pyrG amplicons amplified from 4/5pyrG.col.4. Most of the amplicons in the sample were similar to the wild type (86.5%), while the remaining amplicons showed various indels at the targeted region.

In short, only sgRNA-1 was able to cause the gene editing event in this experiment. This finding implies that in order to improve the effectiveness of gene editing analyses, more research on the criteria of suitable sgRNAs to cause DNA breaks by Cas9 and the repairing mechanism in G. boninense needs to be done in the future. v. Conclusion Based on the results, the CRISPR/Cas9 gene editing establishment in G. boninense has been achieved. This is the first successful report of successful gene disruption or mutation in G. boninense at present. More optimizations must be made in the future to increase the rate of effective gene editing. 7.0 BUDGET Kategori Bahan/ Bekalan Penyelidikan Perkhidmatan Teknikal Kos pengangkutan Kos penyelenggaraan Sewaan peralatan/bangunan Lain-lain (training) Jumlah Jumlah bajet (RM) 64000 2000 - 2000 (2019-2021) (RM) 50542 - - - - - 50542 (2022) (RM) 14421 - - - 1092 15513 Jumlah kos 68 000 50542 66055 8.0 BUSINESS OPPORTUNITY Providing consultation on gene editing technology in G. boninense which can be extensively employed for functional gene analysis for product development. 9.0 TANGIBLE AND INTANGIBLE BENEFITS TO GROUP/ CLUSTER/ COMPANY/ UNIT OR IMPACT OF THE PROJECT According to reports, the BSR disease is on the rise on an annual basis. This issue arises from the lack of effective techniques for containing and halting the spread of the pathogenic G. boninense, which can spread by a few different routes, including root-to-root contact, basidiospores, and secondary inoculum. The development of a more target-specific and efficient drug has been impeded due to the lack of information on the molecular level of this disease. The ability to study gene function using methods like CRISPR/Cas9 gene editing has made it possible to identify the key virulence genes (such as effectors, CWDEs, proteases, etc.), and these discoveries will be used to develop new bio-fungicides or treatment methods for BSR disease that are especially more specific, environmentally friendly and exhibiting longer control effect once employed. As a result, the oil palms will be more productive for a longer period of time and require less frequent sanitation and replanting, which will save cost, time and energy. 10.0 REFERENCES 1. Su, T., Liu, F., Gu, P., Jin, H., Chang, Y., Wang, Q., Liang, Q. and Qi, Q., 2016. A CRISPR-Cas9 assisted non-homologous end-joining strategy for one-step engineering of bacterial genome. Scientific Reports, 6(1), p.37895. 2. Ab Wahab, A.F.F., Zairun, M.A., Daud, K.H.M., Bakar, F.D.A., Bharudin, I. and Murad, A.M.A., 2022. Evaluation and Improvement of Protocols for Ganoderma boninense Protoplast Isolation and Regeneration. Malaysian Applied Biology, 51(5), pp.43-57.

3. Lim FH, Rasid OA, Idris AS, As'wad AWM, Vadamalai G, Parveez GKA, Wong MY. Lim, F.H., Rasid, O.A., Idris, A.S., As’ wad, A.W.M., Vadamalai, G., Parveez, G.K.A. and Wong, M.Y., 2021. Enhanced polyethylene glycol (PEG)–mediated protoplast transformation system for the phytopathogenic fungus, Ganoderma boninense. Folia Microbiologica, 66(4), pp.677-688 4. Siddiqui, Y., Surendran, A., Paterson, R.R.M., Ali, A. and Ahmad, K., 2021. Current strategies and perspectives in detection and control of basal stem rot of oil palm. Saudi Journal of Biological Sciences, 28(5), pp.2840-2849. 5. Shukri, I.M., Izzuddin, M.A., Hefni, R.M. and Idris, A.S., 2020, July. Geostatistics of oil palm trees affected by Ganoderma disease in low and high planting density. In IOP Conference Series: Earth and Environmental Science, 540(1), p.012065 6. Svoboda, P., 2020. Key mechanistic principles and considerations concerning RNA interference. Frontiers in Plant Science, 11, p.1237, pp.114-117. 7. Catlett, N.L., Lee, B.N., Yoder, O.C. and Turgeon, B.G., 2003. Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genetics Reports, 50(1), pp.9-11. 8. Yu, X., Ji, S.L., He, Y.L., Ren, M.F. and Xu, J.W., 2014. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher basidiomycetes). International Journal of Medicinal Mushrooms, 16(2), pp.161-168. 9. Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J. and Mateo, J.L. 2015. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PloS one, 10(4), p.e0124633. 10. Labuhn, M., Adams, F.F., Ng, M., Knoess, S., Schambach, A., Charpentier, E.M., Schwarzer, A., Mateo, J.L., Klusmann, J.H. and Heckl, D. 2018. Refined sgRNA efficacy prediction improves large-and smallscale CRISPR–Cas9 applications. Nucleic Acids Research, 46(3), pp.1375-1385. 11. Roginsky, J., 2018. Analyzing CRISPR Editing Results: Synthego Developed a Tool Called ICE to Be More Efficient Than Other Methods. Genetic Engineering & Biotechnology News, 38(11), pp.S24-S26. 12. Brinkman, E.K. and van Steensel, B., 2019. Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER. CRISPR Gene Editing: Methods and Protocols, pp.29-44.

Development of SSR markers for identification of coconut planting material Hooi Wei Yeng, Norshafiqah Khalid, Siti Mardhiah Mustafha and Sharmilah Vetaryan Abstract Coconut is known as ‘Tree of Life’. All parts of this plant have beneficial uses, and hence is a good source of food and income for farmers. In recent years, there is an increase in the worldwide production of coconut. This is due to the renewed interest in coconut for its unique nutritional and medicinal properties. In Malaysia, coconut is the fourth most important agricultural commodity. Coconut is traditionally propagated by seeds, and it takes three to six years to reach reproductive maturity, depends on the variety. Other features such as towering stature, hard seeds, slow reproduction rate and long generation time limit the breeding efforts. Therefore, molecular markers were introduced in coconut breeding programs since 1990s. Among these, simple sequence repeat (SSR) marker is the most widely used. The aim of this project is to screen and develop SSR panel marker suitable for the identification of our coconut planting material. The selected polymorphic markers will be developed into a multiplex panel for genetic profiling application. This marker panel will be used to profile the parental coconut varieties, as well as identifying the legitimate progenies from breeding crosses. With the application of coconut legitimacy test, the reliability of breeding program outcome could be enhanced by ensuring the right material is used in trials. This marker application could also contribute to cost saving by detecting and filtering contaminants at early stages.

RD­RMU­REC02 REV05_1/1/2020        FGV R&D SDN BHD PROJECT PROPOSAL A. Project Title Development of SSR markers for identification of coconut planting material A1. Project Code A2. Program DNA marker development for high purity planting material development and production A3. Strategic Thrust ( please refer to the roadmap for FGV R&D Upstream) High quality planting material through advanced genomic, clonal and breeding programme A4. Unit Genomics A5.   Presenter Hooi Wei Yeng

­ 2 ­ RD­RMU­REC02 REV05_1/1/2020 B. Introduction C. Problem Statement (please indicate background of problem statement or requisition) Coconut is known as ‘tree of life’. All parts of this plant have beneficial uses, and hence is a good source of food and income for farmers. In recent years, there is an increase in the world­wide production of coconut, due to the renewed interestin it for its unique nutritional and medicinal properties. In Malaysia, coconut is the fourth most important agricultural commodity, after oil palm, rubber and rice.   Coconut palms are generally classified into talls and dwarfs, based on their stature.   It is traditionally propagated by seeds, and it takes three to six years to reach reproductive maturity, depending on the variety. As an improvement, hybrids which carry the preferable criteria are being created by crossing different varieties. The hybrid MATAG, which is among the famous high quality varieties in Malaysia, is produced by crossing Malayan Yellow Dwarf (MYD) or Malayan Red Dwarf (MRD) variety with Tagnanan Tall  (TAG) variety. However, the supply of MATAG seeedlings to the planters is far below demand, due to various issues and challenges. Among these is the identification of true­to­type MATAG seedlings from contaminants. Molecular markers were introduced in coconut breeding programs since 1990s. Among these, simple sequence repeat (SSR) marker is the most widely used. The aim of this project is to screen and develop SSR marker panel suitable for the identification of our coconut planting material. The selected polymorphic markers will be developed into a multiplex panel for genetic profiling application. This marker panel could be used to profile the parental coconut varieties, as well as identifying the legitimate hybrid progenies. With the application of coconut legitimacy test, the reliability of breeding program outcome could be enhanced by ensuring the right material is used in trials. This marker application could also contribute to cost saving by detecting and filtering contaminants at early stages. For current practice, the variety of the coconut palms is checked and verified by authorized DOA experts, based on specific list of phenotypic characteristics. However, coconut is a perennial crop which starts to bear fruit after three to five years, depending on the variety. This traditional approach of phenotypic identification of coconut variety is not only laborious and time consuming, but also proneto be affected by environmental factors. This may lead to misidentification. It is more challenging and even impossible to identify and track individuals within a variety which display similar characteristics by this conventional approach. In a breeding program, it is crucial to ensure that the selected individual of parental palms are being used in the crossing process, and the legitimate progenies are planted for evaluation. However, illegitimate progenies might appear due to mishandling, or contamination by foreign or own pollen source. The contamination might only be revealed in later stage if differential phenotypic characters becomes visible. However, this is not for most of the case.  For case of mislabelling of collected pollen, it is impossible to identify the original parent source by phenotypic characteristics. These contaminations will cause misleading results of the breeding trial.

­ 3 ­ RD­RMU­REC02 REV05_1/1/2020 D. Project Description (how the propose project can solve the problems) This project will develop a molecular method for identification and differentiation of individual coconut palms at the DNA level based on SSR markers. Individual identification at DNA level is more stable and reliable compared to phenotypic characters as it is not affected by environmental factors. The SSR markers could be applied on various tissues at different developmental stages,    with only minor modifications in the sample processing methodology. This enable the usage of the SSR markers at different stages as check points to ensure the right materials are being used in the planting material production process, when necessary. Wastage could be minimized if the right materials are being used and the process is well monitored. E. Goal & Objective Goal: To establish a legitimacy test panel for FGV’s collection of coconut parental palms and the hybrid planting materials.   Objectives: 1. To screen and identify suitable SSR markers for differentiation of individual coconut palms.   2. To establish multiplex PCR protocol for coconut legitimacy test. 3. To build SSR profile database for FGV’s collection of coconut palms. F. Project Background ((i)  Project Status    New          Improvement     C             Continued                                   from existing                                       from existing                                                     project                                                   project (ii)  Involvement of Research Instituition/Other Organization Nil Similary, the use of illegitimate parents or occurrence of contamination in large scale hybrid production of planting material will have impact on returns. Although early detection and identification of contaminated individuals might help to reduce losses,  in practice, this is hard to achieve if we are solely dependant on phenotypic characterization.   ü

­ 4 ­ RD­RMU­REC02 REV05_1/1/2020 G. Project Location Genomics Unit, FGV Innovation Centre H. Methodology (detail in material & method of the propose project) 1. Selection of potential polymorphic SSR marker for coconut palms   The tall varieties of coconut are diverse genetically due to their outcrossing nature, while the dwarf varieties are generally inbred, and hence more homogenous. An initial literature search will be conducted to gather information of reported SSR markers for coconut. Among these, the markers which have been tested on the varieties of our interest will be priotized for testing. The second batch of SSR markers to be screened consist of those with reported high polymorphism. Those with reported moderate polymorphism, as well as gene­based SSRs will be selected to top up to a total of 100 SSRs. 2. Coconut palm materials   Leaflet samples of varieities TAG, MYD, MRD and MATAG will be obtained from Department of Agriculture (DOA) stations in Lekir and Teluk Bharu, Perak. Samples in PPPTR which have been verified by DOA will also be tested. Samples of other varieties which are available in PPPTR station, such as Pandan, MYLAG and MARLECA, will be included, to ensure high discrimination power of the selected SSR markers. DNA will be extracted from the leaflets using an in­house CTAB DNA extraction protocol. 3. Screening and evaluation of SSR markers The optimum and sub­optimum annealing temperatures for the primers will be screened. Whenever possible, the common annealing temperature which could be applied for all markers will be used for subsequent analyses. The SSR markers will be tested on all varieties collected. The criteria that will be evaluated include amplifiable in all samples,    stabily in producing clear result, specific or multilocus amplification, and its polymorphism. The SSR markers will be ranked and the top ten will be used for development of FGV’s coconutmarker panel. 4. Redesign primers for multiplex PCR The primers of selected SSRs will be redesigned so that their amplicons span across the analysis range of capillary electrophoresis system. The specificity of the new primers will be confirmed by PCR and sequencing.   The suitability of the primers under multiplex condition will be evaluated. 5. Multiplex PCR optimization   The PCR conditions will be optimized to get clear and reproducible results across all the targets. 6. Pilot analysis with FGV’s coconut panel The optimized protocol will be applied to a larger set of known samples.

­ 5 ­ RD­RMU­REC02 REV05_1/1/2020 7. Coconut SSR profile database The SSR profiles of all the samples will be gathered in a database.   (i) Project Risk The molecular markers might not be able to differentiate each individuals of dwarf varieties. (iv)   Start Date 10th Jan 2023 (v)    End Date 30th June 2024 (vi) Gantt Chart Activities 2023 2024 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Selection of potential polymorphic SSR marker for the coconut palms                         Coconut palms sample collection and DNA extraction                         Screening and evaluation of SSR markers                          Redesign primers for multiplex PCR                           Multiplex PCR optimization                          Pilot analysis with FGV’s coconut panel             Establish coconut SSR profile database                         Project report and launching of routine Marker Services for coconut planting material                          H. Budget (Cost of Project)     Kategori Bahan/ Bekalan Penyelidikan Perkhidmatan Teknikal Kos pengangkutan Kos penyelenggaraan Sewaan peralatan/bangunan Lain­lain Jumlah Jumlah (RM) (2023) (RM) 75,000 ­  500 5,000 ­  ­  80,500 (2024) (RM) 12,000 ­  500 5,000 ­  ­  17,500 Jumlah kos 98,000

­ 6 ­ RD­RMU­REC02 REV05_1/1/2020 I. Planned Outcome (Potential Product for Commercialization/Technical Recommendation & etc) A set of polymorphic SSR markers which are able to differentiate our coconut palm collection could be identified. These markers will be further converted into a genetic profiling test panel, and a proposed workflow for service will be prepared. J. Business Opportunity (any opportunity for services / professional development) A genetic profiling service for parental coconut palms and legitimacy test service for hybrid coconut planting materials. K. Benefit to Group/Cluster/Company/Unit (Tangible and Intangible) The markers could be used to improve efficiency and reliability of coconut breeding programs by ensuring the right materials are being used, and the legitimate progenies are being evaluated. The markers could also be used to assist in the production of hybrid coconut planting material by identification and filtering at early stage. M. Impact of the project (please state also what is the impact if the project is not approve) The SSR panel marker could serve as a quality control step for efficient and reliable breeding programs and hybrids production. Without this maker, contaminations might be undetected until the later stages, causing a waste of resources. Wastage could also happen when true­totype samples be unintentionally filtered due to uncertaincy based on the phenotypic characters alone. N. Collaboration Opportunity (i) State any contract obligation with third party that involve to this project   Not applicable   (ii) Project ‘Intellectual Property Rights’ (Please state the organization).

­ 7 ­ RD­RMU­REC02 REV05_1/1/2020 Not applicable        O. Research Team (pls specify % contribution) Name Organization (i) Project Leader Hooi Wei Yeng (60%) FGV R&D Sdn Bhd   (ii) Team members Sharmilah Vetaryan (20%) Siti Mardhiah Mustafha (10%) Norshafiqah Khalid (5%) FGV R&D Sdn Bhd (iii) Supporting Staff (Bil) 1 lab analyst (10%) FGV R&D Sdn Bhd (iv) Contract Staff (Bil) ­  Reviewed by:           Reviewed by:     N/A .......................................................        ............................................................    ( Norhafizah Ghazali ) (name Head of Department ) Endorsed by:          Approved by:       .............................................................        ........................................................ (   name CEO FGVRD )          (  name of Benefactor ) References

The Oil Palm Variety with The Thinner Tenera Shell Trait Muhamad Azhar Abd Wahid1 , Nurul Fatiha Farhana Hanafi2 , Mohd Azinuddin Ahmad Mokhtar2 , Siti Habsah Roowi3 , and Noor Hisham Hamid3 1Plant Breeding Unit, Planting Material Department, FGV R&D Sdn Bhd, Ulu Belitong Research Station, Felda Ulu Belitong, 86007 Kluang, Johor, Malaysia. 2Department of Planting Material Research, FGV R&D Sdn Bhd, PPP Tun Razak, 27000, Jerantut, Pahang, Malaysia. 3FGV R&D Sdn Bhd, PT. 23417, Lengkuk Teknologi, 71760, Bandar Enstek, Negeri Sembilan, Malaysia. Abstract Palm oil is one of the world's major commodities, with the highest production in 2022 at 73.83 million metric tons, and has a wide range of applications from edible to non-edible products. The demand for edible oil is expected to increase in line with the growth of the world's population to reach 8.5 billion in 2030 and 9.7 billion in 2050. Therefore, planters must continue finding a way to increase fresh fruit bunch production either by increasing the number of palms planted or implementing good agricultural practices. The improvement not only relies on field approaches but through a plant breeding scheme for producing high-yield planting materials, a more sustainable and practical way to boost future oil palm production. Evaluating an oil palm shell thickness has been the breeders' objective previously to increase oil to bunch since 1940. The tenera, a thin-shelled oil palm trait, that is widely used for commercial planting is a hybrid of the thick-shelled dura and the shell-less pisifera. The tenera bunch typically produces 30% more oil than the dura bunch since its thin shell allows more mesocarp to develop. The tenera palms which have a homogenous and thinner shell trait for the entire population have higher oil yield production compared to the existing commercial tenera varieties. Thus, it has high potential as future planting material to meet the world's increasing vegetable oil demand. 1.0 Introduction Palm oil is a major global agricultural commodity, used in a variety of food, non-food items and renewable energy sources (Abideen et al., 2023). There are several crop that can produce vegetable oil and the oil palm is the highest production of oil seed crops among them, representing more than 40% of the total production of edible oils (Reuters, 2022). Therefore,

the extensive usage of the palm oil on daily usage such as in food products, detergents, cosmetics and to a small extent, biofuel, its production become constant increase in the last two decades reaching 77 million metric tons (mt) (IndexMundi, 2023). At current growth rates, trade watchers predict the world’s vegetable oil demand to grow to 280 million mt by 2030 (Indonesian Palm Oil Association, 2023). Demand for Malaysia’s palm oil is forecast to notch higher to 19.85 million mt this year (2023) from 19.01 million mt in 2022 (MPOC, 2023). Because of the strong demand for palm oil, cultivation of oil palms has expanded more in the past ten years than cultivation of any other crop. In Indonesia the area of oil palm cultivation more than trebled from 2.5 Mha to over 8 Mha between 2000 and 2014. Murphy et al. (2021). Due to many pressure on non-govermant organization especially on forest conservation organization and europian organization, new land for oil palm plantation was prohibited. Therefore the oil palm industries was looking on research and development approach, especially in agronomic practices and breeding of new planting materials. The higher oil to bunch planting material has become priority material in order to have higher palm oil without additional land required to grow oil palm in case of Malaysia. The O/B is highly contributed by higher mesocarp contains in the fruit, by reducing the shell thickness which is the current of shell to fruit was recorded at 10%. 2.0 The exploitation of shell thickness to increase oil to bunch (O/B) The oil palm is native to west Africa and grow as a tropical forest. The main belt runs through the southern latitudes of Cameroon, Côte d’Ivoire, Ghana, Liberia, Nigeria, Sierra Leone, Togo and into the equatorial region of Angola and the Congo. Processing oil palm fruits for edible oil has been practiced in Africa for thousands of years until the oil palm was distributed around the wold and recognised its potential to become commercial crops. As a wild material, oil palm consists of thick shell fruit and very low oil to bunch content. The important scientific relationship between the thick‐shelled dura and the more desirable tenera type of oil palms was first discovered in the Congo. In 1930, the commercial oil palm was first come from crossing of T ×T palms. This cross contributed to 25% of sterile pisifera palm which reduce the fresh fruit bunch and oil yield production per hectarage plantation. Examination of the progeny of D × T crosses showed no pisifera, and for the majority of these crosses, the segregation was close to 50:50 dura:tenera. Beirnaert (1940) showed clearly the inheritance of the shell thickness character. He also stated that D × T and T × D seed should replace T × T seed for a short period, after which full tenera production should

be assured by the issue of D × P seed. The full information obtained from the Yangambi F1 plantings was published in 1941, after Beirnaert’s death (Corley and Tinker, 2016). The first large‐scale confirmation of tenera production from D × P crosses came when Pichel (1956) reported that several hundred hectares of D × P crosses in the Congo were 98% tenera. In this recent years, dura palm which originated from Deli become as female palms and several pisifera for farther palms which originated in Africa been developed by several reseach institute known as CIRAD, Yangambi, AVROS, IRHO, LaMe and others. According to Kushairi and Rajanaidu (2000), who outlined the origins of material in the major programmes in Malaysia, there were 13 commercial seed producers in 1997. Nine of these produced Deli × AVROS material, and all used Deli duras exclusively as female parents. The FGV programme also produced Deli × Yangambi material, as did Guthrie, IOI and United Plantations. Pisiferas of Dumpy × AVROS ancestry were used by some seed producers (Corley and Tinker, 2016). 3.0 Several attempts from industries to reduce S/F As shell to fruit is the interest trait to increase oil to bunch, the beginning history of breeding program was started using this trait as baseline by oil palm breeder until first D × P program establish, and now the average of the s/f was recorded at 10% (Kushairi and Mohd Din, 2018). Then, the strategy of breeding programs have been shift to the introgression within and between linage to find the best combination which have higher fresh fruit bunch and oil to bunch. However, this ancestor breeder approaches is still significantly relevant to find higher oil to bunch with thinner shell to fruit. In record, there are several attempts have been made by African breeder which has given tenera progenies containing palms only 4–9% shell to fruit using material from Nigeria bred in the Ivory Coast (Corley and Tinker, 2016). Kushairi et al., (2003) found only one palm from Tanzania germplasm population showed 2.8% S/F by which described as ‘paper‐thin’ shell Tanzanian tenera or PS5. The only attempt to ultimately reduce shell thickness was done by Chin and Tang (1960) which they had done trial on the development of pure fertile pisifera planting material which firstly started in Malaysia. They using several fertile pisiferas like S112P, S29/36P, 514P, 418P, 458P and AV476P were self crossed and outcrossed to produce P × P progeny. However, in 1982, Chin was found contamination of thick shell palms on this trial which supposedly P×P produce 100% pisiferas progeny (which is shell less palm). Then in 1983. The project has been stop due to expect contamination was occur in the trial. Other factors like the germination of pisifera seed is difficult and make the seed commercialization

in future become problem to sell the good quantity number of seedling. The factory extraction equipment not suitable for fertile pisifera due to mixing of PKO and CPO. 4.0 Periliminary finding and conclusion Comparison of shell type fertile PxP cross progeny, PS5 (MPOB) and commercial tenera: Fertile pisifera S/F = less than 2.8% Tanzania germplasm PS5, MPOB (2003) S/F = 2.8%

Normal tenera D × P Ml161 S/F = 5% As per shell to fruit (S/F), the P × P material shows the thinnest shell type compared to others material and this could be contributed to higher mesocarp to fruit which directly increases oil to bunch content. However, further study needs to be done in the future to reveal more informations on the fertile pisifera material in FGV. References 1. Abideen AZ, Sundram VPK, Sorooshian S. Scope for Sustainable Development of Small Holder Farmers in the Palm Oil Supply Chain—A Systematic Literature Review and Thematic Scientific Mapping. Logistics. 2023; 7(1):6. https://doi.org/10.3390/logistics7010006 2. Chin C.W. & Shuhaimi S. (1999) Felda oil palm planting materials. In: Proc. 1996 Seminar Sourcing of oil palm planting materials for local and overseas joint ventures (Ed. by N. Rajanaidu & B.S. Jalani), pp. 71‐90, Palm Oil Research Institute of Malaysia, Kuala Lumpur. 3. Corley, R. H. V., & Tinker, P. B. (2016). The oil palm. Fifth Edition. John Wiley & Sons. 4. Indexmundi, 2023. https://www.indexmundi.com/agriculture/?commodity=palmoil&graph=production. 5. Kushairi A. & Rajanaidu N. (2000) Breeding populations, seed production and nursery management. In: Advances in oil palm research, Vol. I (Ed. by Y. Basiron, B.S. Jalani & K.W. Chan), pp. 39‐98, Malaysian Palm Oil Board, Kuala Lumpur. 6. Kushairi, A., Rajanaidu, N., & Mohd Din, A. (2003, October). Mining the germplasm. In ISOPB seminar on the progress of oil palm breeding and selection, Medan, Sumatra, Indonesia (pp. 6-9). 7. MPOC, 2023. Palm oil demand to rise in 2023 to 20 million tonnes. https://mpoc.org.my/palm-oil-demand-to-rise-in-2023-to-20-million-tonnes/ 8. Murphy, D. J., Goggin, K., & Paterson, R. R. M. (2021). Oil palm in the 2020s and beyond: challenges and solutions. CABI Agriculture and Bioscience, 2(1), 1-22. 9. Reuters, 25 April 2022, Factbox: Global edible oil markets simmer after shock Indonesia ban. https://www.reuters.com/business/energy/global-edible-oil-marketssimmer-after-shock-indonesia-ban-2022-04-22/

Yield Evaluation of Chimaera Palms: A Comparative Study Muhamad Azhar Abd Wahid1 Mohd Azinuddin Ahmad Mokhtar1 ; Nurul Fatiha Farhana Hanafi1 and Siti Habsah Roowi1 ABSTRACT The oil palm (Elaeis guineensis Jacq.) is a tropical forest palm that originated from Africa and now become the most economically important crop in Malaysia. Seedling selection is one of the crucial tasks in eliminating any form of abnormality to ensure their performance in fresh fruit bunch (FFB) and oil yield at the field is not affected. One of the seedling abnormalities often occur is a chimaera, the presence of white or bright yellow leaf strips parallel to the stem. However, the effect of chimaera toward yield in field trials was still lacking. In this study, 46 chimaera palms, and 48 normal palms from similar backgrounds were planted at FGV AS plantation in 2007 to evaluate their yield components. The four years yield recording from 2010 until 2014 shows that the FFB mean of the normal palms was slightly higher than chimaera palms at 142.5 kg/p/yr (21.1 t/ha) and 140.4 kg/p/yr (20.7 t/ha), respectively. The oil to bunch result indicates that the normal palms also show slightly higher oil formation than chimaera palms at 28.9% and 28.3%, respectively. Interestingly, all the results including FFB, average bunch weight, average bunch number, mean fruit weight, mean pericarp weight, mean kernel weight, oil to dry pericarp, and oil to bunch did not show statistically significant differences between chimaera and normal palms. This study provides information about the effect of chimaerism on the oil palm yields at plantations. __________________ 1Department of Planting Material Research, FGV R&D Sdn Bhd, PPP Tun Razak, 27000, Jerantut, Pahang, Malaysia.

Recovery Program: PalmaGro Product Elya Masya Mohd Fishal, Nor Hidayah Bohari & Ili Bazilah Abd Razak Abstract PalmaGro is a plant enhancer product containing arbuscular mycorrhiza fungi (AMF) as an active ingredient. The product, developed through research and development by FGV R&D, was commercialised and manufactured by FGV Agri Services in 2012. However, in 2018, there were concerns with the production of PalmaGro product, where the AMF spores did not meet the necessary criteria of 150 spores per 10g of product. As a result, the percentage of offspecification PalmaGro products increased, which increased the losses incurred by FGVAS if no action was taken. In order to prevent this problem from getting worse, FGV R&D has been tasked with looking into its root cause as well as coming up with a suitable solution. This paper discusses the several potential causes of this issue, and we were able to make several improvements in the manufacturing process. Notably, the recovery program was completed by the end of 2020, and quality monitoring has continued since 2021. The PalmaGro product production manual has also been prepared as a guideline.

Extraction of Nitrocellulose using Oil Palm Biomass Presenter: Muhamad Nurfikri Bin Azmi (Downstream Technology) Abstract Generally, every plant and fibrous material cell is made up of cellulose, hemicellulose, and lignin as the main substance. Due to the production of crude palm oil from fresh fruit bunch, by-products such as mesocarp fiber, palm kernel shell, empty fruit bunch, and palm kernel cake are abundantly generated. The by-products are mainly used as fuel sources for the mill’s operation but with a proper extraction process, it can have wider application and generate more profits. One of its applications is the conversion of oil palm biomass into cellulose. Cellulose is a very versatile component and is highly sought after in the industry due to its high mechanical strength, lower density, biodegradability, and bio-compatibility From the cellulose, it can be further developed into nitrocellulose through the nitration process and this gives extra kinetic energy which resulted in explosive properties and a flammable effect of the substance. This nitrocellulose has the potential to be utilized as a nation’s defense mechanism which can contribute to lowering import cost and dependency on imported technology. This project will work on the extraction process of cellulose from oil palm biomass and also the development of nitrocellulose as a propellant.