Cocoa is one of the food commodities that is favored by its high in polyphenols. The polyphenol and flavonoid in cocoa beans can prevent arteriosclerosis, diabetes, and their related risk factors. This research aimed to analyze the total polyphenolic contents (TPC) and flavonoid contents (TFC) of three cocoa bean varieties, namely LAM, TSH, and SUL1 collected from Lampung, Indonesia. The study also aimed to understand the effect of adding starter culture on TPC and TFC contents during cocoa beans fermentation. The TPC and TFC were extracted using ethanol from ground-dried beans and measured three times using a spectrophotometric method based on the Folin-Ciocalteu method at 760 nm and aluminum reagents at 510 nm. LAM variety contained the highest TPC and TFC among other samples at 62.8 mg GAE/g and 21.9 mg QE/g, respectively. The TPC and TFC decreased during both fermentation treatments with starter culture and non-starter culture. The TPC of cocoa bean with starter culture and non-starter culture decreased to 58% and 59% after fermentation. The TFC of cocoa beans with starter culture and with non-starter culture decreased after five days of fermentation, up to 58% and 75%, respectively. Remarkably, the TPC and TFC in fermented cocoa beans with starter culture were higher than those with non-starter culture. Therefore, it is concluded that three days of fermentation with starter culture can produce fermented cocoa beans with the preferred flavor/quality and maintain the TPC and TFC. However, the polyphenols and flavonoids content were reduced during the fermentation process.

Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense. Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense. A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense.

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AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract      Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]