Ebook TW 2 2023 4

1 BIOCHAR: ORGANIC SOIL FOR SUSTAINABLE APPROACH TO IMPROVE OIL PALM SEEDLING GROWTH Erwan Syah Tugiman1,2, Mohd Ali Hassan2 , Mohd Yusoff bin Abd Samad3 , Yoshihito Shirai4 1. FGV Agri Services Sdn Bhd, Pusat Penyelidikan Pertanian Tun Razak, 26400 Bandar Tun Razak, Jengka, Pahang, Malaysia. [email protected]. +609-471 8301 2. Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. [email protected]. +603-9769 1181 3. Institute of Plantation Studies, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. [email protected]. + 603-97694856 4. Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808- 0196, Japan. [email protected] +81 93-695-6000 ABSTRACT Statistics shown by Malaysian Palm Oil Board (MPOB), planted mature and immature oil palm trees cover a total of 5.90 million hectares representing 89% of the total area (6.6 million hectares, 2019) designated for agriculture and 18% of overall Malaysian territory. The main problem in the oil palm tree cultivation and its related industries is its substantial amount of biomass wastes. The wastes such as empty fruit bunches (EFB), palm kernel shell (PKS), mesocarp fiber (MF), palm oil mill effluent (POME), oil palm trunks (OPT), oil palm leaves (OPL) and oil palm fronds (OPF) are generated after the oil palm fruits harvesting, palm oil processing or during oil palm trees replantation. Beside over application of chemical fertilizer can result in negative effects such as leaching, pollution of water resources, destruction of microorganisms and friendly insects, crop susceptibility to disease attack, acidification or alkalization of the soil or reduction in soil fertility thus causing irreparable damage to the overall system. The purpose of this trial is to overcome the planting media, soil fertility and fertilizer issues and there is a lot of work that has been done by other institutions and companies in order to solve this puzzle. This trial presents biochar as a solution for the problems raised, biochar is a solid material obtained from thermochemical conversion which is called pyrolysis. Biochar can be used for a range of applications as an agent for soil improvement, improved resource use efficiency, remediation or protection against particular environmental pollution and as an avenue for greenhouse gas (GHG) mitigation. This study is to determine the usage of biochar as an alternative resource of planting media which treatments contained combination of biochar with soil or fully insitu soil. By practicing biochar-soil combination might potentially increase oil palm seedling growth after 8 month of treatments. Oil palm seedling in the main nursery and vegetative growth performance and plant sample was measured and recorded. Keyword: Biochar, planting media, vegetative growth, nursery, biomass management

2 INTRODUCTIONS Oil Palm Waste The oil palm industry has come under increased pressure from Western countries under Renewable Fuel Standard (RFS2). Based on the statistic (April 2014) as shown by Malaysian Palm Oil Board (MPOB), planted mature and immature oil palm trees cover a total of 5.230 million ha representing 79% of the total area (6.6 million ha, 2009) designated for agriculture and 15% of overall Malaysian territory. The main problem in oil palm tree cultivation and its related industries is its substantial amount of biomass waste. The wastes such as empty fruit bunches (EFB), palm kernel shell (PKS), mesocarp fibre (MF), palm oil mill effluent (POME), oil palm trunks (OPT), oil palm leaves (OPL) and oil palm fronds (OPF) are generated after the oil palm fruits harvesting, palm oil processing or during oil palm trees replantation (Mushtaq et al. 2015). In palm oil mills, palm oil consists of only 10% of the total biomass. The rest, 90% biomass, is discarded as waste. On average, five tonnes of fresh fruit bunches are required to produce one-tonne crude palm oil (Abdullah et al. 2012) Biomass Management In Malaysia’s oil palm industries, biomass management is the least projected. There are several biomass wastes that have a huge potential to be converted into biochar. This alternative conversion of oil palm waste into biochar is a promising strategy as part to obtain carbon credit and achieve sustainability in palm oil production. Dried EFB are used for mulching purposes. It retains moisture and improves the fertility of the soil. It also helps to reduce weed growth, especially around young palm trees. EFB has also been found suitable in the production (by pre-carbonization) of self-adhesive carbon grains (SACG) that can be pelletized without the presence of any binding agent for activated carbon pellets production (Abdul Aziz et al. 2008). However, the impact of biochar on crop production, soil physio-chemical properties, and enhanced root development research is still very limited. Nutrient and Soil In the oil palm agro-ecosystem, the components of nutrient demand are plant nutrient uptake for growth and production, nutrient losses through soil processes such as runoff and leaching (environmental losses) and nutrient immobilization (Corley and Tinker 2015). Over-application can result in negative effects such as leaching, pollution of water resources, destruction of microorganisms and friendly insects, crop susceptibility to disease attack, acidification or alkalization of the soil or reduction in soil fertility — thus causing irreparable damage to the overall system. Nutrients are easily lost from soils through fixation, leaching or gas emission and can lead to reduced fertilizer efficiency.

3 Continuous use of these chemical fertilizers depletes essential soil nutrients and minerals that are naturally found in fertile soil. Nursery management Biochar was a renewable resource and due to its economic and environmental benefits, biochar is a promising resource for soil fertility management. Several studies of oil palm seedlings in the nursery stage showed a positive effect of soil amended with organic material on the vegetative growth of oil palm seedlings (Danso et al. 2013; Suryanto et al. 2015). The use of biochar to increase drop productivity has shown good potential in the past decade, as part of the C sequestration strategy this carbonaceous material also potentially increases cation exchange capacity if it were added into the soil (Jien and Wang 2013; Gray et al. 2014) and stimulate soil micro-organisms activities (Ducey et al. 2013). The use of biochar in nursery polybags may contribute to post transplanting benefits in the field, the faster establishment of seedlings and soil C sequestration in oil palm plantations for long term effects. This study aimed to determine the usage of biochar as an alternative resource of planting media whose treatments contained a combination of biochar with soil by in-situ soil polybag trial. Practicing biochar-soil combination might potentially increase oil palm seedling growth after 8 months of treatments. MATERIALS AND METHODS Location and planting material Recent oil palm seedling industries practiced a double stage nursery system, which consists of the pre-nursery stage and the main nursery stage. Pre-nursery stage refers to the plants the germinated seeds are directly sown into a small polybag or hi-plug pot and it either can be under direct sunlight or under shade for selective planting material for 3 to 4 months. These seedlings are then transferred into larger polybags in the main nursery, which are placed in open field conditions for another 8 months. The study was carried out in PPP Tun Razak Pahang with the location coordinate of 3°52'51.5 N 102°31'51.8 E, for the duration of 8 months in the main nursery stage. Seedlings were planted in polybags with the size of 38 cm x 45 cm and filled with treatment media. Site rainfall distribution and temperature were recorded shown in Fig 1 below.

4 Figure 1: Study Site Rainfall Distribution and Temperature Biochar Preparation There are two types of oil palm biomass selected for this study, due to its abundance and practicality in future application. Empty fruit bunch and mesocarp fibre are commonly either used either for mulching purpose or as fuel in mills. Two tonnes of each oil palm raw material was brought from one of the mills from Jengka, Pahang to Universiti Putra Malaysia (UPM) bio-refinery for conversion into carbon compounds which are also known as biochar. The end product will be transferred back to Tun Razak Research Centre in Jerantut, Pahang where the study was conducted. The carbonization process had been used to produce biochar from these two biomass. From this point onward, biochar that was produced from empty fruit bunch (EFB) will be mentioned as biochar empty fruit bunch (BEFB) and mesocarp fibre based will be using biochar mesocarp fibre (BMF). Slow burning with high temperature was taken place to produce this biochar, 4 Days of the burning process were taken inside the combustion chamber to completely convert biomass into biochar as per displayed in Fig 2. (a) (b) Figure 2: Carbonization final product a) Empty fruit bunch b) Mesocarp fibre

5 Experimental design and plot setup This study was conducted to determine the usage of different types of oil palm biochar in nursery media. The experiment was laid out by 7 media treatments with 10 oil palm seedlings and each treatment contained 3 replications. A total of 210 oil palm seedlings had been used in this experiment, which was then arranged in the main nursery trial plot in a parallel arrangement and blocked by replicates. Fertilizer was supplied as per standard practices for all treatments and pre-packaging techniques were applied for each manuring round to ensure each seedling will get the same amount of fertilizer. Fertilizer was applied on top of the media broadcasted around seedlings. The water supply also was close monitored every day to ensure each palm will get sufficient water supply for its growth. In this system, there are a few 2.5L measuring cups placed at the drips for calibration purposes. Media was prepared by premixing soil and biochar by volume, mixing was done by measuring cup and concrete mixer. Topsoil from bungor series soil was selected to be used in this study. Lists of media used as treatments were based on biochar and soil volume as per Table 1 for details of treatments. TABLE 1: TREATMENTS DETAILS Treatments Types of biochar Ratio of Biochar, v/v (%) Ratio of soil, v/v (%) Control 100% Soils 0 100 25BMF Mesocarp fibre 25 75 25BEFB Empty fruit bunch 25 75 50BMF Mesocarp fibre 50 50 50BEFB Empty fruit bunch 50 50 75BMF Mesocarp fibre 75 25 75BEFB Empty fruit bunch 75 25 *v/v= volume to volume Vegetative Growth and destructive measurement Plant growth parameters were measured such as seedling girth size, height, number of leaflets, SPAD leaf measurement, frond production, frond length, frond number, petiole width and thickness. Girth size also may be called as bole diameter was measured using a calliper and palm height was defined as the length from soil surface to the tip of the highest leaf. Frond length taken from the lowest rudimentary leaflet to the tip of fronds and petiole also was taken at the lower rudimentary part. From the transplanting initiated, the first frond was marked as it needed in order to calculate frond production onwards. Petiole cross section (PCS) then was calculated using the following formulation: PCS cm2 = Width (cm) × Thick (cm)

6 After 8 months at the main nursery, 5 seedlings from each replicate were harvested for shoot and root dry matter weight measurement and analysed for nutrient uptake. From the polybag, soil was removed carefully by using special netting placement under low pressure flowing water. The palm was chopped off at the bole part and dry weight for both above ground biomass and root were determined after oven-dried at 60 °C until reached to a constant weight. Analysis for plant tissues and soil Pieces of plant tissue retained in the samples compartment were cleaned, no soaking involved because this might cause rapid leaching of plant nutrients. The midribs are removed and rejected. Plant samples were dried in an air-draught oven at least 6 hours at a temperature of 100-105 °C. The time of drying varied depending on the size and nature of the material. The sample was considered dry when it was crisp to the touch, and quite brittle and stored in closed clean polythene. Plant tissues (leaflets and rachis) were ground to < 2 mm and digested for macronutrient and micronutrient analyses. For foliar and rachis analysis, samples were carefully homogenized before the digestion process. Statistical Analysis The parameters measured were subjected to analysis of variance (ANOVA) using SAS version 9.4 (SAS Institute Incorporation). Mean separations for all parameters were carried out using the least significant difference (LSD) test at p<0.05. RESULTS AND DISCUSSION Several parameters were measured at the interval of 90 days from each recording or were measured by month after treatments (MAT). Results for vegetative measurement at 3 MAT, 6 MAT and 8 MAT were displayed in Table 2 girth size from mixed media between biochar and soil showed significantly thicker bole than control. In addition, girth size was significantly better using treatments of 75BMF, 50BMF and 25BMF compared to biochar material made from EFB (75BEFB, 50BEFB, 25BEFB). This result indicated types of plant parts used as biochar play a major role that affected the end result due to its characteristics variable for each material. TABLE 2: VEGETATIVE MEASUREMENT BETWEEN TREATMENTS OVER MONTHS AFTER TREATMENTS Parameter Treatments 3 MAT 6 MAT 8 MAT Girth Size (cm) Control 2.78ab 4.57b 5.15c 25BMF 2.75ab 5.10a 6.10ab 25BEFB 2.50c 4.34b 5.54bc 50BMF 2.90a 5.10a 6.21a 50BEFB 2.60bc 4.35b 5.55bc 75BMF 2.90a 5.32a 6.49a

7 75BEFB 2.57bc 4.58b 5.97ab Height (cm) Control 27.3ab 48.63bcd 68.02b 25BMF 27.6ab 54.85ab 77.47a 25BEFB 24.74b 44.89d 67.37b 50BMF 28.41a 53.20abc 73.23ab 50BEFB 24.38b 47.63cd 66.88b 75BMF 27.41ab 56.81a 78.11a 75BEFB 24.89b 49.11bcd 71.81ab Number of leaflet Control 7.70ab 12.40bc 16.40bc 25BMF 8.50a 13.41b 18.29b 25BEFB 7.49b 11.61c 15.89c 50BMF 8.29ab 13.31c 17.79bc 50BEFB 7.77ab 11.74c 16.47bc 75BMF 8.06ab 14.79a 20.41a 75BEFB 7.87ab 12.52bc 18.09b Chlorophyll (%) Control 51.75ab 49.75a 44.03b 25BMF 50.91ab 52.93a 52.52a 25BEFB 51.30ab 49.87a 50.05a 50BMF 51.26ab 58.11a 48.98ab 50BEFB 50.39ab 49.68a 48.00ab 75BMF 52.31a 53.86a 52.00a 75BEFB 50.26b 50.70a 49.93a Frond Production (No) Control 4.03a 4.84b 3.36c 25BMF 4.23a 5.29ab 3.93ab 25BEFB 3.88a 5.13ab 3.58bc 50BMF 4.13a 5.63a 3.77abc 50BEFB 4.20a 5.32ab 3.75abc 75BMF 4.05a 5.56a 3.91ab 75BEFB 3.80a 5.39a 4.07a Frond Length (cm) Control 15.53ab 26.17bc 43.34bc 25BMF 16.06ab 28.43ab 48.74ab 25BEFB 14.97ab 24.45c 43.49bc 50BMF 15.31ab 28.34ab 46.91abc 50BEFB 14.60ab 24.32c 41.70c 75BMF 16.19a 29.86a 50.44ab 75BEFB 14.05b 24.62c 44.58abc Frond Number (No) Control 8.87a 12.43e 13.49c 25BMF 8.90a 13.83bc 14.18ab 25BEFB 8.20b 13.26cd 14.33abc 50BMF 8.90a 14.09b 15.18a 50BEFB 8.40ab 12.92de 14.57abc 75BMF 8.88a 14.68a 15.00a 75BEFB 8.63ab 13.45bcd 14.89ab 75BEFB 0.37bc 0.61ab 0.81ab Petiole Cross Section (cm2) Control 0.21abc 0.56ab 0.82cd 25BMF 0.21abc 0.59ab 0.82cd 25BEFB 0.18c 0.46b 0.75d 50BMF 0.25a 0.69a 0.92bc

8 50BEFB 0.19bc 0.47b 0.84cd 75BMF 0.22ab 0.68a 1.08a 75BEFB 0.20bc 0.60ab 0.97ab *MF=Mesocarp Fibre, EFB=Empty fruit bunch Means with the same letter within the column are not significantly different at p<0.05 by LSD’s test The height measurement became one of the crucial parameters to differentiate plant growth responses. Besides, results obtained from height measurement also correspond to the seedling's girth size. Similar trends were observed for treatments using 75BMF, 50BMF, and 25BMF which showed significantly different results compared to EFB (75BEFB, 50BEFB, 25BEFB). In addition, in this observation appeared that using biochar from mesocarp fibre showed better palm growth compared to empty fruit bunch. Two initial parameters indicate treatments using 75BMF significantly gave better responses 6 months after treatments were applied, while compared with other treatments as well as control treatment. Number of leaflets showed 75BMF treatments stand as the highest number of leaflets produced by palms from 6 MAT to 8 MAT. Meanwhile, SPAD greenish level was used to represent palm chlorophyll content, from the observation chlorophyll variation was not significantly different between treatments. Oil palm seedling’s chlorophyll level wasn’t affected by media and biochar treatments, which was supported by Seemann et al. 1987 who noted that leaf chlorophyll content is often correlated with leaf nitrogen status and photosynthetic activity and not planting media. Another study using compost as planting material on oil palm seedlings also showed that watering and fertilizing compost did not affect oil palm’s seedlings leave’s chlorophyll content, which stated the content of chlorophyll leaves was more influenced by genetic factors (Mira et al. 2018). PCS was calculated accordingly and a comparison between similar biochar ratios of 75BMF and 75BEFB showed non-significantly differences between both treatments, while other treatments are significantly low. In short, the remark indicates 25BEFB was inferiorly low compared to other treatments. This may indicate the incorporation of biochar and soil in this ratio, unable to break palm potential to increase oil palm frond sizes. Not every treatment mixed with biochar showed significantly better results in vegetative growth, for example as 25BEFB continuously fell below expectations. Furthermore, treatments with 25BEFB showed the lowest vegetation performances compared to other treatments with biochar and control treatment. The highest vegetation growth performance on the other hand was held by 75BMF, as per observed in girth size, height, leaflet count and frond development parameters and this trend was then followed by other treatments such as 50BMF, 75BEFB and 25BMF.

9 Foliar and Rachis Nutrient Content Macro-element The essential element that was required for oil palm growth was considered macro elements such as Nitrogen, Phosphorus, Potassium and Magnesium. Nutrient balanced approaches are very crucial in palm nutrient demand, this foundation specifically attempts to identify nutrient uptake for growth and production, nutrient losses and nutrient immobilization. Nitrogen (N), Phosphorus (P), Potassium (K) and Magnesium (Mg) are considered as major nutrients in oil palm growth, and palm oil trees need NPK in large quantities. Nitrogen losses via topsoil erosion are usually associated with palm deficiency. Biochar, on the other hand, has the capability of soil amendment and retains nutrients in media. Fig 3.0 showed the results of foliar status and rachis nutrient concentration (P and K). Total nitrogen in 75MF and 25MF may be related to the intense growth responses of oil palm seedlings. Total N uptake also was influenced by the type of growing medium used (Ofosu et al. 2018). Correlation analysis indicated that the spectral parameter was more sensitive to foliar nitrogen (Amiratul et al. 2017). 3.29 3.11 3.24 3.38 3.37 3.06 3.25 2.9 3 3.1 3.2 3.3 3.4 3.5 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Total-Nitrogen (% on dry matter) 0.178 0.166 0.171 0.167 0.172 0.168 0.164 0.155 0.16 0.165 0.17 0.175 0.18 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Phosphorus (% on dry matter) 1.523 1.285 1.066 1.281 1.248 1.182 1.323 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Potassium (% on dry matter) 0.253 0.315 0.326 0.338 0.312 0.33 0.309 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Magnesium (% on dry matter)

10 Figure 3: Effect of treatments on the macro element uptake for foliar and rachis after 8 month of treatment application. Potassium has an important role in both plant cells and membrane transport systems. Hanum et al. 2016 states that high Potassium levels in intercrops would affect the absorption and translocation of phosphorus nutrients. In the younger palm, N and P had no effect on micro elements such as Cu and Zn and longer time required for the effect to become significant (Tohiruddin et al. 2010). In this study, the foliar P determined was at level of 0.17% of dry matter. Meanwhile rachis P level was lower than 0.10% as the level of Mg was higher. This level was also reported by Foster and Probowo (2002) where the rachis P concentration of the palms was at a critical P level of 0.10%, which was found in this study. It revealed that even at a lower P level in rachis, the level status was more reflective of the P nutrient status compared to foliar P status. Therefore, lower rachis P level can be used to identify a better biochar mixture that improves nutrient uptake and enhances palm growth, as seedling absorbs nutrient in the production of palm tissue. Microelement Abdul and Abdul (2017) in their study using rice husk biochar and EFB biochar stated that with the addition of biochars, calcium becomes increasingly available in the soil as the negatively charged surface of biochars attracts the positively charged ions such as Ca and make it available to plants. Ca is an activator of several enzymes systems in protein and carbohydrate transfer. Besides, Ca has a major role in the formation of the cell wall membrane in plant cells. 0 0.5 1 1.5 2 2.5 3 0 0.02 0.04 0.06 0.08 0.1 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 P K Rachis P and K (% dry matter) P K

11 Figure 4: Effect of microelement by treatments on the nutrient uptake after 8 month of treatment application. Fig 4 represents the microelement status of oil palm seedling’s foliar, the selected microelement such as calcium, boron, zinc and iron which is most affecting oil palm health. Boron as the major microelement component which influences leaflet size and petiole growth, chlorine affects leaves colouring and palm turgidity, zinc is more prominent in peat area that has been called ‘peat yellow’ which chlorotic with small and limp canopies, and iron usually present as iron oxide and commonly soil was acid soil thus this element frequently available (Corley and Tinker, 2003). Furthermore, some heavy metals are adsorbed by biochar, which reduces their availability in soil (Chen et al. 2011; Trakal et al. 2014). This trend can be observed from a few microelements in Fig 4, whereby Cu and Zn showed slightly lower levels with the presence of the biochar. However, the effect of other microelements is still uncertain and further study may be required to justify the effect of biochar over the elements, for example S, Cl and Fe. Bulk Density Bulk density decreased with more biochar used in each treatment and types of biochar been used. Root development commonly relates to bulk density, since higher bulk density indicates dense medium which inhibits root development. Less penetration of the root gave direct impact to palm growth since nutrient uptake might also be limited. Reduction of bulk density may be attributed to the increase of pore space due to biochar application 0.501 0.649 0.651 0.722 0.662 0.793 0.625 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Calcium (% on dry matter) 19 16 19 19 18 18 19 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Boron (% dry matter) 26.26 28.04 27.06 26.27 26.36 22.23 23.09 0 5 10 15 20 25 30 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Zinc (% dry matter) 76.81 85.13 103.08 69.52 79.39 74.58 64.09 0 20 40 60 80 100 120 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Iron (% dry matter)

12 shown in Fig 5, which facilitated root branching and root penetration depth (Brunn et al. 2014). Figure 5: Effect of treatments on bulk density of soil 8 MAT Apparently, in Fig 5 bulk density decreased with the increase of biochar added into the mixture and revealed mesocarp fibre dense than empty fruit bunch biochar based and soil at the higher peak. However, root fresh and dry weights were not affected by mixture variations. There were no significant differences found in root fresh weight and moisture content (Table 3), implied biochar usage in ratio as per treatments did not affect seedling’s root development. TABLE 3: EFFECT OF TREATMENTS ON THE FRESH WEIGHT, DRY WEIGHT AND MOISTURE CONTENT OF FOLIAR AND ROOTS Treatments Fresh weight (g) Dry weight (g) Moisture Content (%) Foliar Root Foliar Root Foliar Root Control 530.00b 222.00ab 168.67cd 82.17ab 66.56a 60.63a 25BMF 675.00ab 252.00a 214.00abcd 114.30ab 67.14a 56.18a 25BEFB 483.00b 192.00ab 157.50d 68.00b 65.73a 63.88a 50BMF 633.00ab 178.00ab 231.00abc 71.00b 61.09a 59.79a 50BEFB 515.00b 200.00b 177.00bcd 63.67b 65.24a 60.09a 75BMF 767.00ab 192.00ab 266.83a 75.83b 63.81a 59.92a 75BEFB 668.00ab 200.00ab 242.67ab 84.67ab 63.93a 56.68a Means with the same letter within the column are not significantly different at p<0.05 by LSD’s test Conclusion Biomass from palm industries can be used in more sustainable products approach, such as biochar made from mesocarp fibre and empty fruit bunch. Considering the potential of both types of biochar in facilitating seedling growth response, this study could lead to better biomass management direction. This study revealed 75MF was the optimum ratio 1.504 1.214 1.314 1.043 0.989 0.692 0.595 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control MF-25 EFB-25 MF-50 EFB-50 MF-75 EFB-75 Bulk density (g/cm3) Bulk density as affected by treatments

13 to boost seedling growth in the main nursery stage. According to the finding from the trial, the potential of MF is significantly higher than the EFB usage, especially using 25EFB rate is not recommended due to its low vegetative growth rate of palm seedlings than the control treatment. Ratio variables of biochar mixture in this study indicated that the amount of biochar (v/v) used in this study gave no effect on root development and moisture content of foliar and roots. Moving forward, a new direction has been established by Malaysian palm industries' which is to enhance bi-product in downstream marketing strategy and this biochar usage may contribute as one of the business plans moving forward. REFERENCES Abdul, R N F, and Abdul, R N S (2017). The Effect of Biochar Application on Nutrient Availability of Soil Planted with MR219. J Microbial Biochem Technol. 9: 583- 586. Amiratul, D A; Farrah, M M; Tan, N P; Daljit, S K S and Martini, M Y (2017). Nitrogen Effects on Growth and Spectral Characteristics of Immature and Mature Oil Palms. Asian J. Plant Sci. 16: 200-210. Alvarez-Campos, O; Lang, T A; Bhadha,J H; Mccray, M J; Galaz, B; Daroub, S H (2018). Biochar and mill ash improve yields of sugarcane on a sand soil in Florida. Agric. Ecosyst. Environ. 253: 122–130. Bruun, E W; Petersen, C T; Hansen, E; Holm, J K and Hauggaard-Nielsen, H (2014). Biochar amendment to coarse sandy subsoil improves root growth and increases water retention. Soil use manage. Vol 30 (1):109– 118. DOI: 10.1111/sum.12102. Chen, X; Guangcun, C; Chen, L; Chen, Y; Lehmann, J; Mcbride, M B and Hay, A G (2011). Adsorption of copper and zinc by biochars produced from pyrolysis of hardwood and corn straw in aqueous solution. Bioresour. Technol. 102: 8877–8884. Corley, R H V and Tinker, P B (2013). The Oil Palm, Fourth Edition. Oxford: Blackwell Publishing. p. 346-348. Danso, F; Adu C; Opoku, A; Danso, I; Anim Okyere, S and Larbi, E (2013). Raising oil palm seedlings using sole and amended green-gro compost. International Research Journal of Agricultural Science and Soil Science 3: 362-368. Ducey, T F; Ippolito J A; Cantrell K B; Novak J M and Lentz R D (2013). Addition of activated switchgrass biochar to an aridic subsoil increases microbial nitrogen cycling gene abundances. Applied Soil Ecology Vol 65:65–72. DOI: 10.1016/j.apsoil.2013.01.006. Foster, H L and Probowo, N E (2002). Overcoming the limitations of foliar diagnosis in oil palm. Paper presented at International Oil Palm Conference, Indonesian Oil Palm Research Institute, Bali.

14 Gray, M; Johnson, M G; Dragila, M I; and Kleber, M (2014). Water uptake in biochars: The roles of porosity and hydrophobicity. Biomass and Bioenergy. 61:196–205. DOI: 10.1016/j.biombioe.2013.12.010. Hanum, C; Rauf, A; Nasution, I; Fazrin, D A and Habibi, A R (2016). Nitrogen, Phosphorus, and Potassium Level in Soil and Oil Palm Tree at various Composition of plant species mixtures grown. IOP Conference Series: Earth and Environmental Science: 41 (1). iopscience.iop.org/article/10.1088/1755-1315/41/1/012008/pdf. Jeffrey, R S; Thomas, D S; Jinlang, W and Barry, C O (1987). Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant physiology. 84(3). pp. 796-802. http://www.plantphysiol.org/content/84/3/796, accessed on: 24 January 2021. Jien, S H and Wang, C S (2013). Effects of biochar on soil properties and erosion potential in a highly weathered soil. CATENA 110: 225–233. DOI: 10.1016/j.catena.2013.06.021. Mira, A; Santi, R; Intan, R D and Aldo, F (2018). The Growth Response of Oil Palm Seedling at Main Nursery against Watering at Different Volume and Frequency and Against Provision of Compost. International Journal of Sciences: Basic and Applied Research (IJSBAR). ISSN 2307-4531. 37 (3): 226-233. Olmo (2016). Changes in soil nutrient availability explain biochar’s impact on wheat root development. Plant Soil 399 (1-2): 333–343. Peng, Y; Chen, S; Zhenhao, Z; Jvianyu, F; Xin, L; Liping, Z; Lan, Z; Wenyan, H and Lichao, F (2021). Biochar stimulates tea growth by improving nutrients in acidic soil, Scientia Horticulturae. 283 (2021) 110078. DOI: 10.1016/j.scienta.2021.110078. Suryanto, T; Wachjar, A and Supijatn (2015). The growth of oil palm (Elaeis Guineensis Jacq.) seedlings at various media and containers in double stage nursery. Asian J. Appl. Sci. 3(5): 664–671. Tohiruddin, L; Tandiono, J; Abner, J S; Prabowo, N E and Foster, H L (2010). Effect of N, P and K Fertilizer on Leaf Trace Element Levels of Oil Palm Sumatra. J. Oil Palm Res. 22: 869-877. Trakal, L; Sigut, R; Sillerova, S; Faturikova, D and Komarek, M (2014). Copper removal from aqueous solution using biochar: effect of chemical activation. Arab. J. Chem. 7:43–52. Wang, Y; Villamil, M B and Davidson, P C (2019). A quantitative understanding of the role of co-composted biochar in plant growth using meta-analysis. Science of the Total Environment. 685: 741–752. Zhang, X K; Wang, H L; He, L Z; Lu, K P; Sarmah, A; Li, J W; Bolan, N S; Pei, J C and Huang, H G (2013). Using biochar for remediation of soils contaminated with heavy metals and organic pollutants. Environ. Sci. Pollut. Res. 20: 8472–8483.

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FGV R&D SDN BHD (COMPANY NO. 1012623-V) PROGRESS REPORT Project Title : Jackfruit Tissue Culture Program : Other Crop Cloning Project Leader : Mohamad Farkhan bin Mohamad Ishak Team Members : Dr Siti Habsah Roowi, Nurul Asyikin Mohamad Zim, Norshafiqah binti Khalid, Dr. Then Kek Hoe Unit : Tissue Culture Research Presenter : Mohamad Farkhan bin Mohamad Ishak Abstract On average, Malaysia produces 20,000 metric tonnes of jackfruit produce with an average of over 8,000 metric tonnes for export to countries like China and Thailand. These figures are steadily rising as higher awareness towards environmental and health issues have slowly increase the demands for fresh fruits like jackfruits. This means that there is potential for jackfruit to enter the market further, both locally and overseas. In order to meet consumer demands, there is a need to increase jackfruit production which can be achieved through tissue culture micropropagation. One of the advantages of using tissue culture micropropagation is that the desired traits from the parents can be preserved from parents to offspring. This means that traits like taste and texture can be passed down and produce identical progenies with similar high qualities. Desirable traits in jackfruits include sweet flavour and less juicy texture like those found in the J33 Tekam Yellow variety, a highly favoured variety in comparison to others as verified by consumer preference studies. Therefore, in this study, we aim to establish tissue culture strategies and protocols until field observation for jackfruit, specifically J33 Tekam Yellow. This project has the potential to assist in FGV’s new business plan to diversify our agricultural business portfolio and strengthen our position as an integrated_agri-business_leader.

1.0 INTRODUCTION The jackfruit tree (Artocarpus heterophyllus) is widely cultivated in many tropical and subtropical countries, especially in Asia and South Asia such as Bangladesh, Pakistan, Malaysia, Thailand and the Philippines (Amin, 1992). In general, each tree is capable of producing fruits between 25 - 250 fruits per year, weighing between 45 – 50 kg, with mature trees producing almost 500 fruits in their prime time (El-Zaher, 2008). Apart from being a food source, each part of the jackfruit tree has its own uses (Figure 1), such as making furniture using its wood and making varnish from its latex (Ranasinghe et al., 2019). This makes jackfruit one of the better planting materials to improve the country's economy as an alternative crop. Figure 1. The Potential Uses of Different Parts of the Jackfruit Tree Due to the versatility of the fruit, the overall jackfruit market is expected to grow exponentially within the next few years. Since 2017, jackfruit production in Malaysia has showed a steady yearly increase with the latest figure in 2022 showing a total of 41252 metric tonnes in production value (Table 1). Meanwhile, the jackfruit global market is also expected to reach USD359.1M in 2026 with trade value increasing by 3.3% from 2021 to 2026. If managed properly, Malaysia has the potential to become a major producer of jackfruit for the international market in the foreseeable future. With the rise in global fresh fruits consumption in both the local and international markets, the demands for fresh fruits will slowly increase in the coming years, resulting in need of larger production capacity. Table 1. Data on jackfruit production in Malaysia from 2017 to 2022 (Jabatan Pertanian Malaysia, 2021). Year 2017 2018 2019 2020 2021 2022 Hectarage (Ha) 5097 4879 4657 4677 5074 5089 Production (MT) 28042 31,175 31,281 35624 41047 41252

Application of tissue culture techniques could potentially increase the production capacity of jackfruit planting material at a faster rate compared to conventional techniques like grafting. J33 Tekam Yellow was selected for this project because through consumer preference study, it was discovered that due to its sweeter flavour and less juicy texture, J33 Tekam Yellow is more preferred for consumption over other varieties like Mastura or Mantin (Ismail & Kaur, 2013) 2.0 PROBLEM STATEMENT Growing demand in global fresh fruit consumption in both local and international markets necessitate the need for large production of viable jackfruit planting materials. Though conventional method of propagating jackfruit is an option, the varying degrees of success pose a risk of hindering efficiency in large-scale production of planting material. This can pose problems in later stages when trying to meet consumer demands hence tissue culture method is required to successfully mass propagate J33 Tekam Yellow. 3.0 GOAL & OBJECTIVE Goal: To develop and establish method for the multiplication of jackfruit through tissue culture technique. Objective 1. Developing and optimizing sampling techniques and sterilization procedures to minimize contamination of jackfruit explant before culturing 2. Developing and optimizing media conditions and culturing conditions to promote multiplication, elongation, rooting and acclimatization of jackfruit cultures. 4.0 METHODOLOGY Figure 2. The Overall Experiment Flow of the Project. Explant Collection Explants were collected from two locations; Pusat Penyelidikan Pertanian Tun Razak (PPPTR), Jengka and the mininursery in FGV Innovation Centre (FGVIC), Bandar Enstek. Parental plants in both locations have been certified to be of the J33 Tekam Yellow variety. The type of explants used for the tissue culture project are shoot cuttings, nodal segments and jackfruit seeds.

Sterilization All explants were cleaned using 7X soap solution followed by rinsing with water to remove external debris. The explants were then submerged in 1% w/v Benomyl fungicide solution for one hour and rinsed with sterile water. The remaining sterilization procedure is then conducted under the laminar flow hood. Shoot cuttings and nodal segments were submerged in 30% bleach solution with a few drops of Tween-20 for 30 minutes before being rinsed with sterile water. The process in repeated again with 50% bleach solution with Tween-20 for 30 minutes before being rinsed for 3 rounds with sterile water. Due to the durability of jackfruit seeds, they were instead submerged in 10% bleach solution with a few drops of Tween-20. After being rinsed with sterile water, the process is repeated again before the seeds were rinsed again with sterile water for 3 rounds. Culturing and Multiplication Shoot cuttings and nodal segments were cut to about 1cm while jackfruit seeds were cut to form 1cm x 1cm x 1cm cube around the cotyledon. All explants were placed on modified MS media without hormone and incubated at 28 ± 2oC under LED lighting (9 hrs light/ 15 hrs dark). Presence of contamination were observed for 2 – 4 weeks and only explants that were free of contaminants were subcultured onto FGV proprietary growth media to encourage shoot development and multiplication. The subculture process was conducted every month to ensure enough materials were being produced for the rooting procedure Rooting In vitro root induction of jackfruit ramets were done by cutting the base of the ramets in a slanted manner to encourage root growth. The cut ramets were then cultured onto test tubes containing FGV proprietary rooting media and the success rate of each rooting session were recorded and analysed after 2 months. Acclimatization Jackfruit ramets that have developed roots were cleansed under running water to remove all traces of media. The clean ramets were then packaged and sent to the nursery in PPPTR where each ramet will be placed in planting trays containing a mix of peat and fertilizer as their nutrition source. The planted ramets were then left under black netting for one month to encourage acclimatization. Manual herbicide and fungicide procedures were done to ensure the plants survive their one-month acclimatization period. After a month, the surviving acclimatized ramets were then transplanted onto 6 x 9 inches polybags for trial purposes. Nursery Trial In May 2022, 116 clonal jackfruit trees were planted near the mini-mill area of Pusat Penyelidikan Pertanian Tun Razak (PPPTR), Tekam, Pahang. Out of the 116 clonal jackfruit trees, 54 trees were selected to collect vegetative measurements which include tree height, tree diameter at 15 cm and 4 feet (122cm) from tree base, as well as canopy radius. Vegetative measurements will be conducted every 6 months for three years after planting. Trees bearing fruits were also noted in records and the fruits are left to mature to collect data for future comparison with true-to-type J33 jackfruits. 5.0 GANTT CHART Year 2020 2021 2022 2023 Quarter 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Activity Explant Sampling & Sterilization Shoot Development & Multiplication Rooting Ramet Delivery & Acclimatization Nursery & Field Trial

6.0 OUTCOME Explant Sampling and Sterilization From certified J33 Tekam Yellow jackfruit trees planted in the nursery fields at PPPTR and at the mini-nursery behind the cabin at FGVIC, apical meristems and petioles were sampled for sterilization. Currently, there is success in getting clean initial cultures from apical meristems but not petioles. The difficulty in obtaining clean initial culture is possibly due to endogenous microbes present in the explant (Zamir & Abdur-Rab, 2014). Further efforts of sterilization to reduce contamination only increase chances of wilting and death due to frequent exposure to sterilant. Because of the difficulty in obtaining clean cultures from apical meristems and petioles, seeds from certified J33 Tekam Yellow fruits were also used as starting materials for this project. Sterilization of jackfruit seeds provided a better rate of success compared to sterilization of young shoots and nodal segments, showing a positive response towards the growth media as shoots began to emerge from the seeds (Figure 3). It is important to note that, due to heterozygosity, plants obtained from seeds may share physical traits similar to J33 Tekam Yellow but are not true-to-type to the parental plant. Figure 3. Young jackfruit shoots emerging from sterilized J33 Tekam Yellow seeds. Shoot Development and Multiplication Multiplication of jackfruit tissue culture shoots begins with subculturing germinated seed shoots on FGV proprietary growth media (Figure 4). Since jackfruit seeds are heterozygous in nature, the shoots that developed will not have the same characteristics as the parental plant. Due to the variations in development, each jackfruit seeds that develop shoots were given a clone number, to differentiate it from J33 Tekam Yellow. In both lab and field trials, certain clones were observed to share the same dome-shaped leaves phenotype similar to those found on J33 Tekam Yellow (Figure 5). Due to the variation in development, multiplication rate for each clone varies between clones as well (Figure 6). It is possible that different clones take up nutrients in different rates, thus reacting differently inside the growth media. Clones with high multiplication rates of 60% and above were selected for the rooting process. Among those selected were NJ10 (70.36%), NJ14 (65.96%), and NJ22 (64.79%) to name a few.

Figure 4. Various J33 Tekam Yellow clonal cultures in the multiplication stage. Figure 5. Comparison of leaf shape between jackfruit tissue culture plants and certified J33 Tekam Yellow trees. A) Various jackfruit clonal cultures on FGV proprietary growth media. The characteristic dome-shaped leaf (arrow) can be observed in the middle culture jar. B) The dome-shaped leaf growing on certified J33 Tekam Yellow trees in FGVIC mini nursery. Figure 6. A side-by-side comparison of the multiplication rate of all jackfruit clones obtained through the multiplication process. Each bar represents the multiplication rate for that particular clone.

Rooting, Ramet Delivery and Acclimatization Rooting process is still being refined to boost the rooting efficiency of jackfruit tissue culture plants to 70% and above. Initial rooting strategies uses FGV proprietary rooting media supplemented with various rooting hormones such as 1-Naphthaleneacetic acid (NAA) and 6-Benzyl Amino Purine (BAP) of differing concentrations (Media P71R1-P71R6). Later rooting strategies uses a different media base for rooting from the standard Murashige-Skoog (MS) to Woody Plant Media (WPM). At this stage of the rooting trials, Indole-3-Butyric acid (IBA) was selected as the main hormone for jackfruit rooting, following evidence presented in various literature review stating that woody plants like jackfruit favour IBA as a rooting hormone compared to other hormones like NAA and BAP. Using WPM with IBA hormone, there was a boost of rooting efficiency from a range of 30% - 40% upwards to almost 60%. Recent rooting trials uses B20, another FGV proprietary rooting media that is half in strength compared to the FGV rooting media used in earlier rooting strategies. Rooting efficiency results for B20 is still being tabulated. Ramets that develop roots were sent to PPPTR for acclimatization (Figure 7). After a month of acclimatization, the surviving ramets were then transplanted onto 6 x 9-inch polybags and sent to the trial plots to observe tree development. Figure 7. Ramets that survive acclimatization are transplanted into 6 x 9 inches polybag. Ramet with roots were sent to PPPTR for acclimatization using a peat soil-fertilizer mix with manual pesticide and herbicide control to maintain plant health. Field Trial On 13th April, vegetative measurements were recorded from the clonal jackfruit trees planted near the mini-mill area of PPPTR (Figure 8). Out of the 116 plants planted, 54 were selected out of the total 116 trees planted to represent the average vegetative measurements of the clones planted in Tekam. The vegetative measurements taken are tree height, tree diameter at 15 cm and 4 feet (122cm) from tree base, as well as canopy radius. Based on the vegetative measurement data, there seem to be a few potential candidates for good jackfruit harvest namely clones 14, 37, and 42. Other clones such as 28, 32.2, 48.1 and 55 should also be monitored for potential good harvest (Figure 8).

Figure 8. Clonal jackfruit plants planted in PPPTR. (A) Clonal jackfruit trees planted in PPPTR. 54 trees were selected to represent the average vegetative measurement for each clone planted in the field. (B) A diagram showing the position of each clone in the field.

Figure 9. The bar charts show the average values for each vegetative measurements recorded for each clone present in the field. (A) Average tree height. (B) Average canopy radius. (C) Average diameter 15cm from tree base. (D) Average diameter 4 feet (122 cm) from tree base. In terms of fruit observation and data measurements, two jackfruits belong to a single clone NJ14 has been harvested on 22nd June and 14th July, respectively. Upon inspection, it was noted that both fruits have a very soft texture and a sweet taste, though not as sweet as J33 Tekam Yellow. Another noted physical difference is that the thorns found on NJ14 is much longer than the one from J33 Tekam Yellow (Figure 10). Data on the fruit morphology such as length and width as well as fruit content and brix content were also recorded and provided below (Table 2). As for the NJ14 tree itself, it shares characteristics similar to J33 Tekam Yellow, most noticeably its leaf morphology with the dome-like characteristics common in certified J33 Tekam Yellow trees (Figure 11). Clonal trees planted in PPPTR that display the dome leaf characteristic were also identified (Table 3).

Figure 10. The NJ14 jackfruit harvested from the clonal jackfruit planting area. (A) The jackfruit still attached to the tree. (B) The jackfruit after it has been cut open. The fruitlets are pale in colour, different from J33 which has a more vibrant yellow colour on the inside. The J33 jackfruit also has shorter thorns (C) compared to the thorns of the clonal jackfruit (D). Table 2. Characteristic of clonal fruit harvested from PPPTR (as of July 2023)    # Clone Harvest Date Mass (Kg) Length (cm) Width (cm) Number of Edible Fruitlets Sugar Content (Brix, oBx) Top Mid Bottom Top Mid Bottom 1 NJ14 22/06 11.4 82 72 75 70 190 14.1 12.2 14.1 2 NJ14 14/07 6.5 32 58 55 57 48 14 13 16

Figure 11. Comparison of leaf morphology between NJ14 clonal jackfruit and J33 jackfruit plants. (A) Leaf from clonal jackfruit tree. The middle leaf (black arrow) has the dome-shape at the top as per (B) the leaf morphology of a J33 Tekam Yellow jackfruit tree. Table 3. Sorting the planted jackfruit clonal trees based on the presence of dome-shaped leaves. Characteristic With dome-shaped leaves (Both Sides) With dome-shaped leaves (One side) Without dome shape leaves Jackfruit Clones NJ4, NJ8, NJ10, NJ14, NJ18, NJ20, NJ28, NJ30, NJ31, NJ32, NJ33, NJ35, NJ37, NJ42, NJ48, NJ52, NJ55, NJ59 NJ19 NJ21, NJ41, Total Number of Clones 18 1 2 DISCUSSION The project has progressed to the nursery trial stage but given that true-to-type J33 Tekam Yellow jackfruit clones has not been successfully obtained, this project will still proceed to obtain the J33 Tekam Yellow end product. Based on the success rate of obtaining ramets in the laboratory, the current cloning system is suitable for jackfruit tissue culture, however two areas in the cloning system require further improvement. First, it’s the sterilization stage. Materials obtained in the field and greenhouse are exposed to all forms of contamination hence the sterilization stage needs to be optimized to reduce as much contamination as possible while maintaining plant viability to acquire clean starting materials for J33 Tekam Yellow tissue culture. The second point for improving is the rooting stage. While there is some success in getting jackfruit shoots to develop roots, optimization is required to bring the success rate of jackfruit rooting up to 70% and above. This is to ease the transfer of technology from the research block to the commercial block. As of now, there are 7 bottles of clean starting culture to develop the true-to-type J33 Tekam Yellow Jackfruit planting material (Figure 12). Figure 12. Clean culture of J33 Tekam Yellow Jackfruit. These clean cultures will be multiplied and developed to produce true-to-type J33 Tekam Yellow Jackfruit planting material.

7.0 BUDGET Category Total 2020 2021 2022 2023 (RM) (RM) (RM) (RM) (RM) Research Material - - - - - Laboratory Expenditure 6,000 1,500 1,500 1,500 1,500 Technical Services - - - - - Manpower cost (Salary) - - - - - Transportation cost - - - - - Maintenances 800 200 200 200 200 Equipment/Building Rental - - - - - Administration (Electricity etc.) 800 200 200 200 200 Others - - - - - Total Cost* 7,600 1,900 1,900 1,900 1,900 NOTE: * Excluding Manpower Cost 8.0 BUSINESS OPPORTUNITY Successful development of the jackfruit clonal system can be used to mass-propagate jackfruit trees as well as the development of other woody plant cloning system for commercialization purposes. 9.0 TANGIBLE AND INTANGIBLE BENEFITS TO GROUP/ CLUSTER/ COMPANY/ UNIT OR IMPACT OF THE PROJECT This project can be used to commercialize various jackfruit variety and even give potential to develop new varieties of jackfruit. There is also potential to develop and commercialize other woody plant of financial value which can be a new source of income for FGV. 10.0 REFERENCES 1. Amin, M.N. (1992). In vitro enhanced proliferation of shoots and regeneration of plants from explants of Jackfruit trees. Plant Tissue Culture, 2, 27-30. 2. El-Zaher, M. (2008). Studies on Micropropagation of Jackfruit 1-Behaviour of the Jackfruit Plants through the Micropropagation Stages. World Journal of Agricultural Sciences, 4(2), 263- 279. 3. Ismail, N. & Kaur, B. (2013). Consumer Preference for Jackfruit Varieties in Malaysia. Journal of Agribusiness Marketing, 6, 37-51. http://www.fama.gov.my/documents/20143/0/v6+3.pdf/1727b12d-f077-f5e4- 21d1- 73aa6166470d 4. Jabatan Pertanian Malaysia. (2021). Statistik Tanaman Buah-Buahan 2021. http://www.doa.gov.my/index/resources/aktiviti_sumber/sumber_awam/maklumat_pertanian/perangkaan_tanaman/ statistik_tanaman_buah_2021.pdf 5. Ranasinghe, R., Maduwanthi, S., & Marapana, R. (2019). Nutritional and Health Benefits of Jackfruit (Artocarpus heterophyllus Lam.): A Review. International Journal of Food Science, 2019, 1-12. 6. Zamir, R. & Abdur-Rab. (2014). Effect of exposure time and incubation period of various sterilants and antioxidants on the in vitro morphogenesis of guava explants. Journal of Agriculture and Veterinary Science, 7(5). 81-86

1 FGV'S PLANTING MATERIALS IN SABAH: 7 YEARS PERFORMANCE RESULTS AND ANALYSIS Noramiza Sabturani, 1,*, Mohd Azinuddin Ahmad Mokhtar2 , Nurul Fatiha Farhana Hanafi2 , Muhammad Farid Abdul Rahim2 , Muhammad Azhar Abdul Wahid2 , Nur Adibah Ishak3 , Nurul Syafika Mohammad Fauzi2 , Mohd Hazim Bin Zakaria2 , Siti Habsah Roowi3 and Noor Hisham Hamid4 1Breeding Unit, Department of Planting Material, FGV Research & Development Sdn Bhd, Pos Cenderawasih 02, 91150, Lahad Datu, Sabah, Malaysia. 2Breeding Unit, Department of Planting Material, FGV Research & Development Sdn Bhd, P.P.P. Tun Razak, PO BOX 11, Jerantut, Pahang, Malaysia. 3FGV Research & Development Sdn Bhd, PT 23417 Lengkuk Teknologi, 71760 Bandar Enstek Negeri Sembilan, Malaysia. 4FGV Research & Development Sdn Bhd, PT. 23417, Lengkuk Teknologi, 71760, Bandar Enstek, Negeri Sembilan, Malaysia. Abstract Oil palm, a monoecious plant, is the world's most efficient oil-yielding crop, producing two types of vegetable oil: palm oil and kernel oil, with an extended economic life of up to 25 years. In the past, oil palm plantations are planted material with the Dura fruit type, later, in the 1970s, Dura x Pisifera (DxP) hybrid was introduced with a 30% increase in oil yield. In addition to planting DxP, cloning technology was explored to expedite replicating of high-yielding palms to increase yields and optimising land use. In Malaysia, seed and clonal materials producers have commercialised a range of oil palm varieties to meet the industry's needs. FGV has conducted a study to assess the performance of planting material supplied by well-established oil palm companies. The planting materials used in the research comprised six progenies (DxP Agency 1, Clone Agency 2, DxP Agency 3, DxP Agency 4, FGV Clone FC4628, and FGV DxP TK3873), planted in 2012 in Randomised Complete Block Design (RCBD) with four replications at FGVAS Plantation Sahabat 17, Lahad Datu, Sabah. The data were analysed with SAS (P≤0.05) using Duncan multiple range test. From the analysis, FGV DxP TK3873, a 3-Way cross of Deli-NPM x Yangambi produced the highest cumulative mean for Fresh Fruit Bunches (FFB) for seven years of yield recording and mean Number of Bunches (BNO) at 175.58 tonne/hectare and 19.60 number/palm/year, respectively. Interestingly, the mean BNO of this progeny was significantly different from that of the other materials. While the highest average bunch weight (ABW) is from Clone Agency 2 at 12.12 kg/p/yr. For bunch quality criteria, FGV Clone FC4628, a clonal 3-Way cross led in Oil to Bunch (O/B), Oil Extraction rate percentage, Oil Yield (OY), and Total Economic Product (TEP) at 32.15%, 27.49%, 7.74 t/ha, and 8.31 t/ha, respectively. While the highest Kernel Yield (KY) was achieved by Clone Agency 2 and DxP Agency 3 at 1.02 t/ha. This study concluded that FGV DxP and clonal materials originating from the 3-Way crosses had outperformed other planting materials in bunch yield and quality criteria, which may contribute to higher commercial values in the oil palm industry.

1 FGV'S PLANTING MATERIALS IN SABAH: 7 YEARS PERFORMANCE RESULTS AND ANALYSIS Noramiza Sabturani, 1,*, Mohd Azinuddin Ahmad Mokhtar2 , Nurul Fatiha Farhana Hanafi2 , Muhammad Farid Abdul Rahim2 , Muhammad Azhar Abdul Wahid2 , Nur Adibah Ishak3 , Nurul Syafika Mohammad Fauzi2 , Mohd Hazim Bin Zakaria2 , and Siti Habsah Roowi3 1Breeding Unit, Department of Planting Material, FGV Research & Development Sdn Bhd, Pos Cenderawasih 02, 91150, Lahad Datu, Sabah, Malaysia. 2Breeding Unit, Department of Planting Material, FGV Research & Development Sdn Bhd, P.P.P. Tun Razak, PO BOX 11, Jerantut, Pahang, Malaysia. 3FGV Research & Development Sdn Bhd, PT 23417 Lengkuk Teknologi, 71760 Bandar Enstek Negeri Sembilan, Malaysia. Introduction Oil palm (Elais guineensis) is an important commercial crop, supplying over 40% of the world's vegetable oil demand since it has various non-food uses in industrial applications and downstream products (Denis et al., 2021). In 2019, global oil palm production reached approximately 74.58 million tonnes (mt), with fresh fruit bunch production of 415.90 mt and nearly 84% concentrated in Asia (FAOSTAT, 2022). It is a perennial palm with an economic life span of up to 25 years, widely grown in the Southeast Asia region with the world's major producers are Indonesia and Malaysia at 42.87 mt and 19.86 mt, respectively (FAOSTAT, 2022). The demand for palm oil is increased as the world population is growing. Thus, there is a need for introducing higher-performance planting material for newly expanding plantation areas or during replanting programs. Generally, there are three types of oil palm seedlings available commercially: (1) the advanced Dura x Pisifera (DxP) seeds, (2) semi-cloned seeds, and (3) E. oleifera x E. guineensis (OxG) hybrid seeds. There are nine major oil palm seed producers in the world: (1) ASD Costa Rica, (2) Palmeite-CIRAD®, (3) Sime Darby Plantation, (4) AAR, Malaysia, (5) FGV-FELDA, (6) Benih Sawit Kinabalu, (7) IOI, Malaysia, (8) Asian Agri, Indonesia, and (9) PT Dami Mas Sejahtera, Indonesia (Rethinam & Murugesan, 2018). Apart from the most advanced DxP seedlings, the palm oil industry exploits high-yielding palm clones. The Malaysia Agricommodity Policy 2030 (DKAN 2030) has made it mandatory to include 10% replanting with clonal planting material. With a wide range of varieties available, a trial setup to benchmark and assessed the yield performance of various DxP and clones from five different seed producers in Malaysia.

2 Materials and Methods A trial (Trial code: T265) was laid at PR1, located at FGV Agri Services Sdn Bhd (FGVAS), Sahabat 17, Lahad Datu, Sabah (N 5° 8' 32.381"E 119° 0' 0"), an area of 2.82 hectares (ha), Sahabat series soil-type, and planted in 2012. The trial consists of six progenies with four replications (Table 1) in a Completely Randomised Block Design (RCBD). The yield performance and bunch analysis data were collected from 2016 to 2022. Data were analysed using ANOVA and mean comparisons were run on the data collected using Statistical Analysis System (SAS). The Mean was tested using Duncan Multiple Range Test at p≤0.05 significance level. Table 1: Progenies planted in the T265 trial. No Progeny Parental palms 1 DxP Agency 1 Not available 2 Clone Agency 2 Not available 3 DxP Agency 3 Not available 4 DxP Agency 4 Not available 5 FGV Clone FC4628 Deli-NPM x Yangambi 6 FGV DxP TK3873 Deli-NPM x Yangambi Results and Discussions Bunch Yield Component For the Fresh Fruit Bunches (FFB), the highest yield production during the first and second years was produced by Clone Agency 2 and FGV DxP TK3873 at 19.25 tonne/hectare (t/ha) and 27.97 t/ha, respectively (Figure 1). The yield for FGV DxP has increased by 86% compared to Clone Agency 2 by 31% during the second year. In the third year (2018), the highest FFB was produced by DxP Agency 3 at 29.93 t/ha, followed by FGV DxP TK3873, with a minor difference of 0.08 t/ha. While for 2019, FGV DxP TK3873 produced the highest FFB at 26.23 t/ha. Meanwhile, DxP Agency 1 produced the lowest FFB yield, less than 23 t/ha throughout the five years of data recording. In 2020, the FFB yield for all planting materials showed a downward trend with production between 2.86 - 5.76 t/ha, with the highest FFB produced by DxP Agency 4 due to Movement Control Order 1.0 during the Covid-19 pandemic that

3 impacted the data collection activities. Over the 7-year harvesting record period, the highest FFB value is 175.58 t/ha was produced by FGV DxP TK3873. For the number of bunches (BNO), FGV DxP TK3873 has produced the highest BNO for seven consecutive years at 15.15 - 25.48 BNO/palm/year (Figure 2). The highest Average Bunch Weight (ABW) for the first to the fourth year is from Clone Agency 2, ranging from 8.29 to 11.83 kg/palm/year. While for 2020 to 2022, the highest ABW generated by DxP Agency 4 ranged at 12.04 - 16.20 kg/palm/year. However, for FGV DxP TK3873, the ABW is low for 7 consecutive years (Figure 3). Figure 1: Seven-year period of FFB record for T265

4 Figure 2: Seven-year period of BNO record for T265

5 Figure 3: Seven-year period of ABW record for T265 According to Kushairi et al. (2013), the FFB profile is estimated based on palm age and yield production, which habitually increases from 4 years to 11 years and can be categorised as high yielding at 15.5 t/ha/yr, 22.0 t/ha/yr, and 26.0 t/ha/yr and above for young palms aged 4, 5 and 6 years, respectively. Yield performance shows all materials in these trials are high yielding at four years after planting (Figure 1), with the highest being FGV Clone FC4628 and FGV DxP TK3873. However, FGV DxP TK3873 is categorised as medium FFB yield for 7-year-old palms. While the other planting materials are slightly above the low FFB yield category (20.4 t/ha/yr) particularly DxP Agency 1 (21.10 t/ha/yr) and Clone Agency 2 (21.02 t/ha/yr) . There are 3-phases of dry period phenomenon prevailing in the southern region of Sabah that affected the yield production: early February-April 2019, August-September 2019, and February-April 2020 (Balamurugam & Roslan, 2020). The impact started with flower abortion for 9 - 11 months, bunch failure for 4 - 6 months, and other immediate effects such as bunch weight reduction over 1 - 2 months (Balamurugam & Roslan, 2020). These environmental factors have significantly impacted the FFB yield production across the region. FGV DxP TK3873 is the highest bunch producer for the six consecutive years during this study period. There are several factors led to the optimum number of ripe bunches production: 1) the number of flowers depending on frond production, 2) female ratio, the ratio of female flowers to the total number of flowers, 3) bunch failure, abortion of bunches before full maturity occurs 2-4 months after anthesis (Lotte et al., 2017). The bunch failure is caused by insufficient pollination, lack of water supply, or sunlight (Lotte et al., 2017). Meanwhile, the highest ABW producer is Clone Agency 2 for four consecutive years except 2020-2021 (the highest ABW in 2020-2021 is DxP Agency 4). Several factors responsible for determining bunch weight include the number of spikelets, fruit set, fruit weight, and components other than fruit (Lotte et al., 2017). Besides that, the fresh bunches weight usually increases with the age of the palm, starting from 3-5 kg at 24 months after planting and over 30 kg at 25 years after planting, and the fruit set on the bunch is also determined based on the pollination efficiency (Rajanaidu et al., 1996). However, bunch weight and oil content are less responsive to environmental stress but significantly affect yield (Lotte et al., 2017).

6 Bunch Quality Component FGV Clone FC4628 (Figures 4a & 4c) produced the highest Oil to Bunch percentage (%O/B) (32.16%) and Oil Extraction Rate percentage (%OER) (27.49%) and was followed by FGV DxP TK3873 and Clone Agency 2 (Table 2). While FGV Clone FC4628 (Figures 4b & 4d) exhibited the highest Oil Yield (OY) at 7.74 t/ha, followed by FGV DxP TK3873 (6.71 t/ha) with a significant difference. DxP Agency 1 was the lowest OY producer compared to all other commercial planting materials. On the other hand, Clone Agency 2 and DxP Agency 3 were comparable in Kernel Yield (KY) at 1.02 t/ha each. Interestingly, FGV Clone FC4628 has the highest Total Economic Product (TEP) producer at 8.31 t/ha over all the commercial planting materials in this trial. Table 2: Average bunch quality component data (2017-2022) No Progeny Parental palms Mean 2017-2022 O/B (%) OER (%) OY (t/ha) KY (t/ha) TEP (t/ha) 1 DxP Agency 1 Not available 24.84c 21.24c 5.39d 0.93a 5.95d 2 Clone Agency 2 Not available 26.54b 22.69b 6.04c 1.02a 6.65c 3 DxP Agency 3 Not available 24.89c 21.28c 6.00c 1.02a 6.61c 4 DxP Agency 4 Not available 25.53bc 21.83bc 6.24bc 0.74b 6.69c 5 FGV Clone FC4628 Deli-NPM x Yangambi 32.15a 27.49a 7.74a 0.95a 8.31a

7 6 FGV DxP TK3873 Deli-NPM x Yangambi 26.75b 22.87b 6.71b 0.96a 7.28b *Mean with the same letters in the same column are not statistically significant using Duncan Multiple Range Test P≤0.05: Significant difference. O/B: Oil to bunch, OER: Oil extraction rate, OY: Oil yield, KY: Kernel yield, TEP: Total economic product a b Figure 4: Phenotypic characteristics of FGV planting material (a) FGV Clone FC4628 (11 years after planting) (b) FGV DxP TK3873 (11 years after planting) (c) FGV Clone FC4628 fruit cross-section (d) FGV DxP TK3873 fruit cross-section. The differences in oil content are expected since different backgrounds of the planting material are involved in this study. Differences in fruit type, Tenera vs Dura has led to OER levels ranging from 22 to 30% (Rajanaidu & Kushairi, 2006). A trend study of OER and Kernel Extraction Rate (KER) by Hoong and Donough (1998) has shown that OER and KER data collected from 21 refineries in Sabah from January 1993 to July 1997, concluded that biological factors and management factors affect both rates significantly. Biological factors include climatic changes that affect the oil palm physiology m, palm age, and pollination. Two factors that are essential for oil palm production in Sabah are rainfall and sunlight. The study also highlighted that management factors, which focus on labour constraints that affect harvesting and processing efficiency, contribute to the palm oil industry's income. c d

8 This study indicates that FGV Clone FC4628 is capable of producing more than 7 t/ha oil yield over the DxP's. The study of Sharma et al. (2006) has also reported that the oil yield of United Plantation Berhad (UPB) semi-clonal and bi-clonal DxP also reached oil yields of 7 t/ha and above. MPOB also featured seven clonal planting materials, including P164, P203, and P162 with high oil yield (Kushairi et al., 2010). Conclusion FGV DxP TK3873, the 3-Way planting material is the highest for FFB and BNO yield in this study. While the Clone Agency produced high in ABW. FGV Clone FC4628, also known as the 3-Way clone planting material, has surpassed the performance of other planting materials in %OB, %OER, OY, and TEP. While Clone Agency 2 and DxP Agency 3 exhibited the highest KY values. These findings prove that FGV planting material has potential and is among the best commercial planting material for the palm oil industry. References Balamurugam, P. and Roslan, M.R. 2020. Yield Analysis Report 4th Quarter 2020: FGVP(M) Sahabat and Kalabakan Region. Agronomy Advisory Unit, FGV AS Sdn. Bhd. Denis, J.M., Kristie, G. and Paterson, R.R.M. 2021. Oil palm in the 2020s and beyond: challenges and solutions. CABI Agriculture and Bioscience 2:39. National Agricommodity Policy 2021-2030. http://online.anyflip.com/kive/jqkm/mobile/ FAOSTAT. 2022. Date accessed: 23rd August 2022. http://www.fao.org/faostat/en/#compare Hoong, H.W. and Donough, C.R. 1998. Recent trends in oil extraction rate (OER) and kernel extraction rate (KER) in Sabah. The Planter, 74 (805): 181-202.

9 Kushairi, A., Tarmizi, A. H., Zamzuri, I., Ong-Abdullah, Mohd. Samsul, K. R., Ooi, S. E. and Rajanaidu, N. 2010. Production, performance, and advances in oil palm tissue culture. International Seminar on Advances in Oil Palm Tissue Culture. International Society for Oil Palm Breeders (ISOPB), Yogyakarta, Indonesia. Pp: 1-23. Khushairi, A., Siti Nurul Hidayah. A., Nur Maisarah, J., Nur Zuhaili, H. A. Z. A., Syahanim, S. and Ainul Mardziah, M. 2013. Oil Palm Biology Facts & Figures. Malaysian Palm Oil Board, Selangor. Pp: 67-68. Lotte, S.W., Mark, T.V.W., Maja, S. and Meine, V.N. 2017. Yield gaps in oil palm: A quantitative review of contributing factors. European Journal of Agronomy 83: 57-77. Rajanaidu, N., Henson, I.E. and Jalani, B.S. 1996. Bunch component studies over the past two decades. International Conference on Oil and Kernel Production in Oil Palm – A Global Perspective. Palm Oil Research Institute Malaysia, Kuala Lumpur. Pp: 133-150. Rajanaidu, N. and Kushairi, A. 2006. Oil palm planting materials and their yield potential. In: International Seminar on Yield Potential in Oil Palm II. Phuket, Thailand. Pp: 11. Rethinam, P. and Murugesan, P. 2018. Global perspective of germplasm and breeding for seed production in oil palm. International Journal of Oil Palm 10 (1&2): 17-34. Sharma, M. 2006. Challenges facing the Malaysian palm oil industry-Multi pronged strategies for raising oil yield, productivity and profitability. International Society for Oil Palm Breeders (ISOPB). Yogyakarta, Indonesia. Pp: 7.

Development of Shorter Rachis Length Planting Material through Molecular Breeding Approach Abstract Tenera planting material based on the Yangambi Pisifera of ML161 family has been the main product of FGV Agri Services Sdn. Bhd. for several years. At maturity, the average rachis length of this material is approximately 7m. The long rachis length is one of the limiting factors influencing the oil palm planting density, which is currently standardised at 148 palms per hectare. To further maximise the usage of our limited land area, planting material with shorter rachis length is favourable to planters. This project aims to develop planting material with shorter rachis length through a molecular breeding approach. Oil palm materials segregating for short (< 5m) and long (>7m) rachis length will be identified from the breeding collection. Their genomes will be sequenced to identify single nucleotide polymorphism (SNP) markers widely distributed across the oil palm genome. Genome-wide association study (GWAS), linkage mapping and quantitative trait locus (QTL) analyses will be performed to identify SNP markers associated with the rachis length trait. To validate its accuracy, the markers will be tested on other populations segregating for the rachis length trait before being utilised in a marker-assisted selection (MAS) breeding trial. The MAS approach has the potential to reduce the cost and time needed for breeding trials, as it bypasses the phenotyping step which typically takes up to 10 years per generation. Moreover, the land area needed for trials will also be reduced, as only the pre-selected seedlings are planted in field. Ultimately, the development of shorter rachis length (<5m) planting material will enable higher oil palm planting density and hence, improves the overall yield per hectare.

REV05_1/1/2020 Development of shorter rachis length planting material 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 Amer Izzat Samsudin 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 Oil palm is one of the most important edible oil crops in the world. In 2022, palm oil made up approximately 32% of the total global vegetable oil production, far exceeding others such as soybean and rapeseed (MPOB, 2022). However, with the ever­increasing demand for vegetable oil in the world, improving oil palm yield has long been a goal of the industry players. One limiting factor hindering oil palm yield is the planting density, which currently stands at 148 palms per hectare. To surpass this standard practice and achieve higher planting density, traits such as rachis length and canopy size will need to be improved. In FGV, the Yangambi ML161 Tenera planting material (a cross between Deli Dura and Yangambi Pisifera) has been the primary planting material product for several years. At maturity, this material possesses an average rachis length of approximately 7m. To further increase planting density per hectare of land, the average rachis length will need to be brought down to <5m without compromising yield. This way, the yield per hectare of land will increase proportionately with the increasing planting density. In plant breeding, there are 2 main methods suitable to be used for the development of a new planting material variety; conventional breeding and molecular breeding. Molecular breeding, in particular, is an approach that involves the utilisation of DNA markers that are strongly linked with specific phenotypic traits, to improve those traits in a breeding program. The general approach in molecular breeding involves the identification of DNA markers associated with specific traits, markers accuracy validation and application of the markers in a marker­  assisted selection (MAS) breeding scheme. The molecular breeding approach can bring desirable advantages to the overall breeding scheme by improving selection efficiency and reducing the land area needed for commercial trials, as only pre­selected seedlings identified using DNA markers will be planted for field trials. The cost and time needed for commercialisation will also be reduced since MAS bypasses the oil palm phenotyping step which typically takes up to 10 years per generation. This project aims to develop a new planting material with a shorter rachis length through a molecular breeding approach. Initially, oil palm materials segregating for short (< 5 m) and long (> 7 m) rachis lengths will be identified from the breeding collection. Their genomes will be sequenced to identify single nucleotide polymorphism (SNP) markers widely distributed across the oil palm genome. Genome­wide association study (GWAS), linkage mapping and quantitative trait locus (QTL) analyses will be performed to identify SNP markers associated with the rachis length trait. To validate its accuracy, the markers will be tested on other populations segregating for the rachis length trait. The markers will then be utilised in a marker assisted selection breeding trial to develop suitable parental palms for commercialisation. Ultimately, planting material developed from this project will enable higher oil palm planting density and hence, improves the overall yield per hectare.

­ 3 ­  RD­RMU­REC02 REV05_1/1/2020 The national average of oil palm fresh fruit bunch and crude palm oil yield have stagnated at 15­19 t/ha/yr and 3­5 t/ha/yr, respectively, for a very long time (MPOB, 2023). While there are many operational and environmental factors contributing to the numbers, non arguably, oil palm planting density is one of the limiting factors contributing to the stagnating yield. One of the ways to quickly improve yield potential is by developing a new planting material variety with enhanced specific traits. This project aims to develop a new oil palm planting material variety with enhanced specific traits of shorter rachis length (< 5 m). This will be done by utilising molecular breeding and marker­assisted breeding technology. The new variety created will allow for a higher planting density per hectare of land and hence, proportionately improve oil palm’s FFB and oil yield. Project’s goal: To develop oil palm planting material with an enhanced specific trait of shorter rachis length. Objectives: 1. To identify breeding materials segregating for short and long rachis length. 2. To conduct whole genome re­sequencing on breeding materials and identify single nucleotide polymorphism (SNP) DNA markers. 3. To conduct marker­trait association studies and identify DNA markers associated with the rachis length trait. 4. To validate the accuracy of the DNA markers associated with the rachis length trait. 5. To establish a genotyping panel capable of screening future breeding materials with the developed DNA markers. 6. To establish a marker­assisted breeding scheme to produce parental palms for commercialisation. 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 New Improvement C from existing project Continued from existing project (ii) Involvement of Research Institution/Other Organization Advance Biotechnology and Breeding Centre, Malaysian Palm Oil Board (MPOB). X 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 breeding materials segregating for rachis length trait Vegetative measurement data from previous breeding trials will be mined to identify breeding progeny and palms having short (< 5 m) and long (> 7 m) rachis length traits, with good yield data. DNA extraction and legitimacy analysis will be conducted to verify crossing fidelity. Only legitimate palms will proceed for sequencing. 2. Whole genome re­sequencing and SNP mining The DNA of palms with shorter rachis lengths will be pooled together, and similarly for palms with long rachis lengths. The DNA will be sequenced using the next­  generation sequencing technology. SNP markers differentiating the groups of short and long rachis lengths will be generated from the sequencing data. Bioinformatically, the following parameters will be applied to the data; (i) Align and map sequencing reads to the oil palm reference genome (EG5) using bwa mem (ii) Generate raw mapping data and then filter it using bcftools for calls with a minimum read depth of > 5 and a minimum read quality of > Q20. 3. Targeted genotyping and marker­trait association study High­quality SNPs that are well distributed across the oil palm genome will be selected for targeted genotyping. Among the important SNP markers selection criteria include; the coverage of SNP markers across all 16 oil palm chromosomes, the presence in a unique genomic region, and avoiding genomic regions with repetitive sequences.

­ 5 ­  RD­RMU­REC02 REV05_1/1/2020 (i) Project Risk 1) Incomplete vegetative measurement data for all replicates from previous trials may bring unwanted bias in breeding materials selection for the initial work. 2) Poor sequencing/genotyping result. The palms to be used for targeted genotyping will include a few advanced bi­parental crosses, with their progenies compulsory to have segregation for the rachis length trait in the normal distribution. Marker trait association study will be done using 2 methods; (i) genome­wide association study (GWAS) (ii) genetic mapping and quantitative trait loci (QTL) analysis. These will done using molecular breeding package in R Studio, and softwares such as JoinMap and MapQTL. Boxplots of genotype­phenotype distribution will be plotted to determine specific alleles associated with the rachis length trait. 4. Marker validation The genomic region corresponding to the associated SNP markers will be determined. SNP markers within and from nearby linkage blocks will be selected for genotyping. Palms with the same background and related to the ones genotyped in step (3) will be selected for marker validation. The re­occurrence of the same alleles will be assessed to determine the accuracy of SNP markers in predicting short rachis length traits. If there’s no suitable palm available for validation, new crosses will need to be made. 5. SNP genotyping panel The best and most accurate SNP markers will be developed into SNP genotyping panel for routine usage. 6. Marker­assisted selection (MAS) trial and field validation Best SNP markers will be used in a modified reciprocal recurrent selection breeding scheme to introgress shorter rachis length traits into FGV’s advanced parental lines. Breeding crosses will be made and seedlings to be tested for legitimacy. Seedlings will be genotyped using the SNP markers panel and only ones with favourable alleles will be planted for field trial. For each progeny with individuals carrying the favourable alleles, field trial will be conducted using Randomised Complete Block Design (RCBD) with four replications. Each replication will consist of 16 palms, therefore, the total number of palms per progeny is 64. Yield record per palm will be made starting from the 3rd year after planting over a 7 years period. Bunch analysis measurement will be made on 3 bunches per palm, over the 7 years data collection period. Vegetative measurement will be made on the 8th year after planting. The suitability of palms to be declared as parental palms for producing progenies with specific trait (short rachis) will be assessed using the field trial data.

­ 6 ­  RD­RMU­REC02 REV05_1/1/2020 3) Rachis length is most likely a complex trait controlled by multiple genes. Potentially, there will be more than one marker associated with the trait. Selecting and combining the markers to accurately predict the trait presents a significant challenge to molecular breeders. (iv) Start Date July 2020 (v) End Date December 2037

­ 7 ­  RD­RMU­REC02 REV05_1/1/2020 (vi) Gantt Chart

­ 8 ­  RD­RMU­REC02 REV05_1/1/2020 The outcomes expected from this project are SNP markers associated with the rachis length trait. The best markers will be combined into a SNP genotyping panel and will be used in a marker­assisted breeding scheme to produce a new oil palm planting material variety with a shorter rachis length. The new materials will be more suitable to be planted at higher planting densities. This project was initiated in 2020. The project progress is summarised as follows: 1. Identification of breeding materials segregating for rachis length trait Previous progeny testing breeding trial data involving advanced lines were mined for palms with short and long rachis lengths. Tenera palms with complete breeding data were sampled, DNA extracted and tested for legitimacy. In total, the DNA of 48 legitimate palms with short rachis lengths (< 5 m) were pooled together. Vice versa, the same was done on 56 legitimate palms with long rachis lengths (> 7 m). 2. Whole genome re­sequencing and SNP mining The pooled DNAs and 20 other individuals were fragmented, library prepped and sequenced using the Illumina platform (Novaseq6000). Subsamples of the sequencing data were checked for quality by mapping them to the oil palm reference genome. All samples yield a mapping accuracy of > 98%. H. Budget (Cost of Project) Category Marker development Breeding trial 2020 2021 2022 2023 2024 2025 2026­ 2037 (RM) (RM) (RM) (RM) (RM) (RM) (RM) Lab chemicals/r eagents 2,000 10,000 11,000 11,000 8,000 25,000 ­ Sequencing/ Genotyping 63,000 50,000 200,000 50,000 50,000 ­ ­ Transport 1,500 3,000 3,000 3,000 3,000 3,000 ­ Field trial ­ ­ ­ ­ ­ ­ 430,000 Asset ­ ­ 24,000 ­ ­ ­ ­ Total by year 66,500 63,000 238,000 64,000 61,000 28,000 430,000 Total by category 520,500 430,000 Total project cost 950,500 I. Planned Outcome (Potential Product for Commercialization/Technical Recommendation & etc)

­ 9 ­  RD­RMU­REC02 REV05_1/1/2020 In the short term, SNP genotyping panel capable of predicting the short rachis length trait will be developed and applied in a marker assisted breeding program. In the long term, marker assisted breeding program will allow faster introgression of shorter rachis length trait into the advance Dura lines, hence capable for producing oil palm planting material with short (< 5 m) rachis length trait. Benefit to RD division: Faster introgression of shorter rachis length trait into advance planting materials and hence, quicker commercialisation of a new planting material variety. Improve efficiency of future breeding trials. Benefit to FGV’s plantation and oil palm industry: Anewplanting material varietywith shorter rachis length, capable to be planted at higher density than current standardised 148 palms/ha. Increase oil palm planting density, hence improving oil palm FFB and oil yield (t/ha/yr). J. Business Opportunity (any opportunity for services / professional development) L. Benefit to Group/Cluster/Company/Unit (Tangible and Intangible) Reads from each sample were aligned to the EG5 oil palm reference genome. SNP markers differentiating the pooled DNAs were generated using QC criteria of variant call quality of Q20, and minimum read depth of > 5. A total of 180,092 SNP markers were mined from the re­sequencing data. To check the accuracy of SNP markers called, in­silico validation was done on the SNP data of the other 20 samples sent for sequencing. The presence/absence of SNP on 20 other individuals was checked to determine SNP calling accuracy. 3. Targeted genotyping 1st round of targeted genotyping was conducted using 192 SNPs and 200 samples (small­scaled & for training purposes). An association study was conducted, and a few SNPs associated with the rachis length trait was identified. However, the SNP and sample numbers used were not enough to generate a satisfactory correlation. The data will need to be further validated with 2nd round of targeted genotyping. 2nd round of targeted genotyping with more robust SNP and sample selection criteria were conducted using FlexSeq genotyping technology (LGC Genomics, Germany). A total of 500 samples from various advanced breeding lines with backgrounds such as Nigerian, Deli Dura and Yangambi, were genotyped with 5000 pre­selected SNPs. Atthe moment, marker­trait association analysis for the genotyped population is in progress.

­ 10 ­  RD­RMU­REC02 REV05_1/1/2020 Establishment of the SNP genotyping panel and marker assisted selection will save a lot of cost on the future number of progenies to be planted in breeding trial, and reduce the trial size itself. This is because only pre­selected seedlings carrying the favourable alleles will be planted. In turn, manpower requirement to carry out trials will also be more manageable. Financially, FGV Agri Services will have a new source of revenue from the commercialisation of a new planting material variety. The yield for FFB and CPO will also increase which will directly contribute to more revenue per the same area of land. 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 O. Research Team (pls specify % contribution) Name Organization (i) Project Leader Amer Izzat Samsudin (60%) FGV R&D Sdn Bhd (ii) Team members Sharmilah Vetaryan (20%) Dr Mohd Azinuddin Ahmad Mokhtar (10%) Muhammad Farid Abdul Rahim (10%) Suradi Mohamad Dr Suhaila Sulaiman Dr Lee Yang Ping FGV R&D Sdn Bhd FGV R&D Sdn Bhd 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 Genomics lab analyst FGV R&D Sdn Bhd 5 Breeding general workers (for field data collection) FGV R&D Sdn Bhd