Prospective oil palm (Elaeis sp) materials for high tocotrienol content

Crude palm oil (CPO) contains various forms of vitamin E, including tocopherol, tocotrienol and a small amount of tocomonoenol, collectively known as tocols. Among these, tocotrienol is dominant in CPO, constituting approximately 70% of the total tocols; unlike tocopherol, which is known for its anti-ageing properties, tocotrienol exhibits potent antioxidant activities, 60 times stronger than tocopherol. Tocotrienol is recognized for its diverse health benefits as a neuroprotective, nephroprotective, anti-cancer, anti-cholesterol, anti-diabetic, and anti-inflammatory agent. Consequently, tocotrienol holds immense potential for pharmaceutical and nutraceutical applications. The exploration of oil palm materials planted in Kebun Adolina PTPN IV, North Sumatera, has been conducted since 2020, encompassing diverse genetic backgrounds, including commercial palm DxP varieties, an interspecific hybrid of Elaeis oleifera x Elaeis guineensis, wild-type palm materials originated from Angola and Cameroon, and palm clones of commercial DxP varieties. The analysis revealed a wide range of total vitamin E (tocol) content, ranging from 310.78 ppm to 1681.45 ppm. Among the genotypes studied, AD14S311 and AD14S2220 emerged as promising candidates for ortets to develop oil palm varieties with high content, especially tocotrienol. These genotypes demonstrated a high oil extraction rate (OER) and a total vitamin E level above 1000 ppm. These findings remade the potential genotypes as genetic resources for breeding programs in developing oil palm varieties enriched with tocotrienol and fulfilled the commercial value.


Introduction
Oil palm (Elaeis guineensis Jacq.) plays an essential role in Indonesia as a valuable commodity.It supplies more than 50% of the world's demand for vegetable oil, providing employment for more than 5.5 million workers and contributing significantly to regional development (Astuti et al., 2020).The rapid oil palm industry is bolstered by its extensive utilization and diverse range of products.The oil palm fruit produces two types of oil: crude palm oil (CPO) from the mesocarp and palm kernel oil (PKO) from the kernel.
The medical application of tocotrienol encourages appropriate research on tocotrienols in oil palm.Other issues that concern researchers are methods of extracting vitamin E (Mata et al., 2017;Saini et al., 2016;Tai et al., 2019) and methods of selecting oil palm materials with high vitamin E content (Babura et al., 2017;Kong et al., 2019;Kushairi & Amiruddin, 2020).This breeding process involves assessing materials with diverse genetic backgrounds, including African oil palm originating from Angola (Adon et al., 2021), Cameroon (Mathieu et al., (2016)) and American oil palm E. oleifera (Mozzon et al., 2020).To expedite and enhance the selection accuracy, the breeders employ molecular approaches and develop the molecular marker for vitamin E traits (Dou et al., 2021;Karim et al., 2021).These efforts aim to unlock the full potential of tocotrienols and vitamin E from oil palm for various medicinal uses

Material and method
The plant materials used in this study are the gene pool (Gpool) of DxP PPKS 540 (540), PPKS DyP (DyP), PPKS DxP Yangambi (YA), wild E. guineensis from Cameroon (CMR) and Angola (AGO), interspecific backcross of E. guineensis x E. oleifera (OG), and DxP clones of commercial E. guineensis (MK).This study's palms are tenera palms grown in Kebun Adolina, PT Perkebunan Nusantara IV, North Sumatra.We collected four replicate bunches from each palm sample at four to six-month intervals.Vitamin E content was analysed using high-performance chromatography (HPLC) according to Puah et al. (2007).Cluster analysis, according to Blaak et al. (1963).The oil yield formula considered the correction factor (0.855) Nouy et al. (1989) suggested.According to Mattjik and Sumertajaya (2011), data were analyzed using R Studio software through cluster analysis and multivariate Anova.

Result
A scatter plot was used to examine the distribution of observational data for vitamin E measurements and OER values in the tested samples.The total vitamin E content across all samples was 745.3 ppm, while the average OER was 25.03%.We employed K-means clustering analysis to reduce the impact of outliers on clustering results.This approach helps to achieve more robust and accurate clustering outcomes considering the potential influence of extreme data points.

QQ plot
The QQ Plot is a visual tool illustrating the correlation between the analyzed data and the normal distribution.The QQ plot showed that each variable's data distribution appears univariate and normally distributed.However, when subjected to a multivariate normality test using the Shapiro-Wilk test, the p-value was 0.00455, less than the significance level (α) of 5%.This indicates that the data does not meet the assumption of being multivariate normally distributed; despite the lack of multivariate normality, it is still possible to utilize Manova (multivariate analysis of variance) to analyze this data type.Manova is known for its robustness to deviations from the normality assumption, allowing for valid results even when the multivariate assumption is violated.Therefore, Manova can still be applied to draw meaningful insights from the data.

Cluster plot
Tabel 1 Status of vitamin E and OER  Cluster 1 has high vitamin E levels with an average OER, cluster 2 has high OER with moderate vitamin E levels, and Cluster 3 has the lowest OER and vitamin E content.

Manova analysis Figure 3. Multivariate analysis of Gpool on observation parameters
The Manova test gave a significant F-count of 2.48 and a p-value of 0.00642 (<5%), indicating a significant difference in the global mean of the combined response variables (total vitamin E and OER) among Gpools.Subsequent univariate Anova comparisons, using a Bonferroni-adjusted α (5%), revealed that only the OER between MK and OG displayed a statistical difference.Apart from the MK and OG, no other significant variations in OER were found among the remaining Gpools.

Discussion
The data obtained from the observations were considered quality and suitable for analysis.Figure 1 indicates the possibility of some data points being outliers.Although the data was not multivariate normally distributed, it showed univariate normal distribution.Outliers representing extreme values were considered noise, usually excluded from analysis due to potential process errors.However, extreme data may sometimes occur naturally due to features or anomalies (Salgado et al., 2016).In this study, they were included in the analysis despite outliers.This decision was made because the data was obtained from multiple bunches of replications and consistently exhibited these extreme values.It was assumed that these indicators do not represent measurement or sampling errors but represent specific inherent traits of the genotype.Therefore, these extreme values were retained for further analysis.
According to the results of a previous study by Setiowati et al. (2021), tocotrienols dominate the vitamin E content in commercial varieties of palm PPKS.In this study, 70% of the total value of vitamin E in CPO is tocotrienols.Therefore, the mention of total vitamin E in this article refers to the value of tocotrienols without neglecting the contribution of α-tocopherol.
The study's findings showed that most vitamin E in CPO is tocotrienol, with a small amount of tocopherol (70-80%), considering that tocotrienol dominates.The total indicator of vitamin E reflects tocotrienol while acknowledging the contribution of α-tocopherol.In this study, the total vitamin E content ranged from 310.78 to 1681.45 ppm, while the oil yield (OER) ranged from 14.46% to 32.69%.Notably, the amount of vitamin E determined in this study closely aligns with Abdullah et al. 2017 report on vitamin E content in the MPOB population, which was 300 ppm-1600 ppm.
Karim et al. ( 2021) defined a high vitamin E content oil palm as having a 1000 ppm or more vitamin E content.According to this definition, none of the groups in this study met the criteria of having vitamin E levels exceeding 1000 ppm while maintaining a high yield.Despite not meeting the combined criteria as a group, individual genotypes showed both high vitamin E content and high yield simultaneously.Specifically, genotype AD14S311 exhibited a vitamin E content of 1505.40 ppm and an OER of 28.84%, while genotype AD14S2220 displayed a vitamin E content of 1277.83 ppm with an OER of 30.39%.These two genotypes stand out as they possess high tocotrienol and high yield.As a result, these palms are considered promising candidates as ortets for future clone variety as they serve as excellent starting material for developing oil palm clones with additional value of high tocotrienol content and high oil yield.
Cluster analysis was performed for vitamin E content and OER traits to identify ideal genotypes with good scores.Cluster analysis considers the lowest within-group (between clusters, within SS) and the highest between-groups (within-cluster, between SS) sum of squares for three groups (Sinaga & Yang, 2020).The three distinct groups represent a genotype with similar vitamin E and OER characteristics.However, creating more than three clusters would lead to challenges in data interpretation.Too many clusters may lead to smaller, less meaningful grouping and make it harder to discern meaningful patterns or trends within the data.Three clusters are enough to pinpoint the stands out genotypes and avoid excessive complexity.
The result of Figure 3 indicates that genotypes belonging to Cluster 1 are promising genotypes with the potential to select high-quality plant material due to their high tocotrienol content.This cluster is high in vitamin E and has a high or moderate OER.Ideally, genotypes or pools with high vitamin E and high OER can be obtained from this cluster since it has a combination of high vitamin E and high or moderately high OER.In other words, this group is ideal for a nutritious variety.Cluster 2 mainly consists of non-commercial materials derived from various Gpools such as OG, AGO, and CMR, along with some genotypes of commercial varieties.Cluster 3, on the other hand, mainly includes palm clones.However, this group displays lower vitamin E content and oil yield than the other clusters.
The AGO and OG generally displayed good OER values (24.4% -32.69%).However, their vitamin E content was not as desired.The AGO's oil yield in this experiment was higher than the Angola x Lame hybrid reported by Adon et al. (2021), which fell from 20.8% to 21.9%.Nevertheless, the vitamin E of AGO was lower when compared to the findings of Karim et al. (2021) (2021) showed that the total level of vitamin E in African oil palms ranged from 200 ppm to 1500 ppm.Moreover, their study highlighted a significant link (P = 0.0062) between the origin of the African palm and the levels of tocopherols and tocotrienols.Oil palms from Côte d'Ivoire tend to be high in tocopherols, while oil palms from LáMe and Yangambi have higher tocotrienol levels.However, in this study, the contribution of tocopherols to the total vitamin E did not exceed 60%.These findings emphasize the genetic diversity in vitamin E composition among different oil palm genotypes and highlight the potential, significant considering the origin of genotypes in tocopherol and tocotrienol analysis.The genotypes AD14S311 and AD14S2220 also stand out as the remarkable combination of high vitamin E and OER.It makes them attractive candidates for further breeding selection.

Conclusion
The result showed that this study did not identify any cross or population with perfect characteristics; high oil production and high vitamin E content.Instead, two exceptional individuals, namely AD14S311 and AD14S2220, possess the desired traits of more than 1000 ppm of vitamin E and an OER of more than 25%.Therefore, these outstanding genotypes hold significant potential for producing high-yielding oil palm clones with high vitamin E content.These ortets represent valuable genetic resources to improve oil palm varieties with enhanced nutritional profiles and elevated economic value.

Figure 1
Figure 1 Boxplot of total vitamin E and OER The box plot analysis provides insights into the distribution of possible vitamin E and OER indicators.Additionally, calculating the Mahalanobis distance for each Gpool reveals the presence of multivariate indicators.Specifically, one of the samples in Gpool exhibits a total vitamin E value of 1681.45 ppm and an OER of 21.92%.This data point stands out from the rest based on its Mahalanobis distance, indicating its distinct characteristics compared to other samples in the dataset.

Figure 2
Figure 2 Cluster plot using k-mean clusterBased on the cluster analysis for vitamin E and OER using three clusters, the samples were grouped as follows: Cluster 1: This cluster contains 13 samples.These samples have an average OER content of close to 25.03%, the overall average.However, they stand out for having the highest total vitamin E content, surpassing the average of >745.3 ppm.Cluster 2: This cluster consists of 23 samples.The members of this group exhibit the highest average OER, exceeding 25.03%.Their average total vitamin E content falls from 432 ppm to 745.3 ppm, making it moderately high.Cluster 3: This cluster includes 27 samples, representing the group with the lowest OER and total vitamin E content among all the clusters.The three clusters present distinct OER and total vitamin E content characteristics.Cluster 1 has high vitamin E levels with an average OER, cluster 2 has high OER with moderate vitamin E levels, and Cluster 3 has the lowest OER and vitamin E content.
Chew et al. (2021)16) palm, which ranged from 1257 ppm to 1612 ppm.Similarly, the vitamin E content in OG found in this study was also lower than that reported byChaves et al. (2018).Chaves et al. (2018)reported that vitamin E content in E. oleifera origin Coari was 1006.7 ppm, and in the hybrid of E. oleifera x E. guineensis was 1549.69 ppm.These findings suggested that AGO and OG exhibited good OER, and their vitamin E content was comparatively lower than others.However, it is essential to consider the context of each study, including the specific populations and other parameters that may contribute to the variations.Gpool CMR exhibits a fascinating phenomenon.In this study, CMR showed a wide range of total vitamin E content from 372 ppm to 1681.45 ppm, and the OER ranged from 14.46% to 29.88%.The genotype AD13S2516 from this Gpool had the highest OER but the lowest vitamin E, showcasing an exciting contrast between the two traits.Furthermore, the vitamin E content in CMR displayed a wide range, from 372 ppm to 1681.45 ppm, while OER ranged from 14.46% to 29.88%.This variability in vitamin E and oil yield within the same Gpool indicates a considerable genetic diversity among the genotypes studied.A comparison with the findings ofMathieu et al. (2016)reveals a difference in tocopherol content between the two studies.In this current study, tocopherol was observed as a minor component of vitamin E in oil palms.In our study, the highest value of tocopherol was 187.2 ppm.In contrast,Mathieu et al. (2016)report a value of more than 700 ppm of tocopherol in Cameroon oil palm.Moreover, they noted interesting trends in tocopherol content across different planting cycles of Tenera, Dura, and Pisifera oil palm.Their findings showed that tocopherol content was significantly higher in the first cycle (reaching as high as 1681.45ppm in Pisifera palm); it decreased notably in the second cycle for Dura, Tenera and Pisifera.Understanding the underlying factors influencing this fluctuation could shed light on the genetic and environmental interaction affecting the vitamin E content in oil palm genotypes.It is exciting to continue to observe this phenomenon.In this study, two genotypes, AD14S311 from Gpool YA and AD14S2220 from Gpool DyP, exhibited both high vitamin E and OER values.Genotype AD14S311 had a vitamin E content of 1505.40 ppm and OER of 28.84%, while genotype AD14S311 had a total vitamin E level of 1277.83 ppm with an OER of 30.39%, which makes them particularly competitive among other genotypes, especially when it aligns with the target set byChew et al. (2021)which aimed for 23% OER by 2020.Morcillo et al.