Transformation of CRISPR/Cas9 expression construct to enhance saturated fatty acid synthesis in oil palm for biofuel production

New technology innovations and the utilization of elite varieties are necessary for oil palm intensification to improve productivity. In addition to productivity improvement, oil palm intensification is also directed to improve the quality of crude palm oil (CPO) to be used in biofuel production. The modification of the fatty acid biosynthesis pathway through genetic engineering is one of the most promising approaches. The research aims to apply a genetic transformation to oil palm calli and to improve success regeneration of the transformed explants. A CRISPR/Cas9 expression construct to edit the PTE (palmitoyl-acyl carrier protein thioesterase) gene, which protein is involved in the formation of palmitic acid, was generated in the previous research. In this study, the genetic transformation of the construct into 4month-old oil palm calli was conducted via Agrobacterium tumefaciens. The transformed calli were subsequently subcultured into selection media containing 100 ppm of cefotaxime and 50 ppm of kanamycin antibiotics. After several weeks, the transformed calli were transferred into DF10 media without antibiotics. Non-transformed calli were able to grow on the selection media with antibiotics but still in the globular phase. In contrast, the transformed calli on the selection media were able to grow to the scutellar phase with a compact structure in the embryogenic callus development stages. The next step is to optimize the transformation method as well as the regeneration media composition.


Introduction
Oil palm (Elaeis guineensis L.) is one of the producers of vegetable oils with high productivity and has an important role as a source of income for the Indonesian economy.Together with Malaysia, Indonesia is a big contributor to 90% of CPO (crude palm oil) in the world.Oil palm is a plant with the highest productivity of vegetable oil i.e. 57 million tons/year in 2013 compared with soybean, canola, sunflower, or coconut [1].
Since oil palm has a high productivity, it was considered a good source of biofuel as a sustainable bioenergy that can compete with fossil fuels.Highly productive oil palms produce high-quality crude palm oil (CPO), usually measured by the composition of their fatty acids.Naturally, the composition of the fatty acid in palm oil consisted of 48% saturated fatty acid and 52% of unsaturated fatty acid.Palmitic acid is the highest saturated fatty acid that reaches 85% in palm oil.Whereas, oleic acid is the highest unsaturated fatty acid that can reach 88% in palm oil [2].Besides that, triglyceride is a neutral lipid used as raw material to produce biodiesel/biofuel.Triglyceride consists of three long chain saturated or unsaturated fatty acids bound to a glycerol molecule.
The composition of saturated and unsaturated fatty acids in palm oil can determine the quality of the produced biofuel.Based on this issue, oil palm seedlings with a high yield of saturated fatty acid are needed to produce biofuel with less production budget and more optimum yield.An attempt to improve oil palm traits with the target of providing good raw materials for biofuel production can be conducted by genetic engineering of the fatty acid biosynthetic pathway [3].One of the techniques of genetic engineering that can be used for this purpose is through genome editing (GE) using CRISPR/Cas9.By this method, a new type of variety can be generated without inserting genes from other plants or organisms, but only by editing the target genes.
GE technology has been applied to engineer fatty acid biosynthetic pathways in soybean, [4,5,6], canola [7], Camelina sativa [8], peanut [9], and oil palm [10].Based on the successful stories on those plants it shown that this technique can be applied to oil palm.Several important enzymes in the fatty acid biosynthetic pathway that is responsible for determining the fatty acid composition which are βketoacyl ACP synthase II (KASII), palmitoyl-ACP thioesterase (PTE), stearoyl-ACP desaturase (SAD), dan oleoyl-CoA desaturase [3,11,12].Moreover, Dar et al [13] revealed that plants have enzymes that play a role in the most reaction of glycerolipid desaturase in the tissues.
In previous research in 2021, our cisgenic research team had successfully conducted the initiation of genetic engineering on oil palm on genes involved in fatty acid biosynthesis i.e.PTE, SAD, FAD2, dan MADS21 using CRISPR/Cas9 technology.The results included comparative in silico studies on target genes, prediction of networking analysis, gene target construction plasmid using CRISPR/Cas9 technique, and transformation of CRISPR/Cas9 constructs into Agrobacterium tumefaciens system.Following that, the transformation of the CRISPR/Cas9 construct was also conducted into oil palm calli until validation.The transformed calli need to be maintained in optimum condition until it's regenerated into cisgenic plantlets with high oil quality for biofuel production.This will bring benefits to the oil palm companies as well as to the oil palm farmers that can provide biofuel massively and independently.This research aims to apply a genetic transformation to oil palm calli with CRISPR/Cas9 constructs and to improve success regeneration of the transformed explants carrying gene with the target of oil palm with suitable quality for biofuel production.

Plant material preparation
The plant material that was used in the breeding of oil palm using the genome editing technique is embryogenic calli induced from young leaves of oil palm material from Dami group.Calli was cultured on in vitro solid initiation media (Media 3 and Media 4) supplemented with sucrose, coconut water, active charcoal, auxin, and kinetin hormones following the protocol of Sumaryono et al [14].Calli culture was stored in the dark at 25 o C for about 12 weeks.

Transformation of CRISPR/Cas9 constructs to oil palm
Transformation of CRISPR/Cas9 constructs with sgRNA to the target gene was conducted by Agrobacterium tumefaciens LBA4404 strain to the oil palm calli following the prior protocol that has been published [15].The calli were then incubated in the Agrobacterium recombinant suspension at 27 o C for 15 min in the shaker at 150 rpm in the dark.The resuspended calli were subsequently cultured on DF proliferation media supplemented with 100 ppm acetosyringone in the dark.After 2-3 days of incubation in the acetosyringone containing-media calli were subcultured to another DF proliferation media supplemented with 250 ppm cefotaxime in the dark.One week after incubation in the cefotaxime-containing media, calli were then transferred into selection media which is DF media added with 100 ppm cefotaxime and 50 ppm kanamycin in the dark [15].After 2 weeks, explants were then subcultured to another selection media with 25 ppm kanamycin and 100 ppm cefotaxime for 2 weeks in the dark.The transformed explants were then transferred to proliferation media and embryo induction.

Growth recovery of transformant calli
The selected cisgenic calli recovered from their growth before regenerating via the somatic embryogenesis phase.Optimization of the calli regeneration of genome editing was conducted by media modification (mineral, vitamin, polyamine, and hormone regulation).The transformed callus was expected to become a nodular phase after several times subcultures.The embryogenic calli that were produced were then induced for the somatic embryo cells on DF media followed by proliferation and maturation phases until performing shoots.

Induction of somatic embryo
The induction of somatic embryo callus was done on DF media and the proliferation was conducted using Temporary Immersion System (TIS) culture.The growth and maturation of somatic embryos were also conducted on DF media containing kinetin and ABA in TIS with subculture every 4-6 weeks.

Germination and maturation of the somatic embryo
The somatic embryo that was produced in the previous stage can be used as induction material for cisgenic oil palm germination.Partly will change into the next growth phases such as cotyledon and shoots.The optimization of germination media was done to increase the germination percentage on cisgenic oil palm.The shoots that emerged were then maturated until become plantlets.The plantlets were transferred into rooting media in the culture bottle/tube.It requires about 29 months to have oil palm plantlets from tissue culture clonal propagation ready for acclimatization to growth in the soil media in the greenhouse.

Confirmation of inserted DNA and identification of target sequence mutations on oil palm cisgenic calli
The template DNA was isolated from oil palm cisgenic calli using the method of [16].The NPTII and actin primers were used for confirmation of the DNA target presence in the oil palm.The amplification process of the target region was conducted with PCR in 35 cycles and with the condition: predenaturation (95 °C; 1 min), denaturation (95 °C; 15 sec), annealing (55 °C; 15 sec), extension (72 °C; 10 sec), and post-extension (72 °C; 10 min).The PCR product was visualized using 1% agarose gel electrophoresis at 80 volts for 35 minutes and documented by UV-Gel Doc.Based on the confirmation results, the number of positive transformants was used for mutation profile analysis on the target sequence using specific primers to target genes.The PCR products were used for DNA sequencing.Mutation profile analyses are conducted using Geneious Prime ® 2020.0.4 (Biomatters Ltd, Selandia Baru) software.

Oil palm callus induction
The plant material for transformation explants was prepared using callus from oil palm of Dami group ortet from Ciomas, Bogor.The callus was inducted on media 3 and media 4. The results showed that about 6.6% callusing explants of Dami group occurred on media 3 and 15.5% callusing explants were initiated on media 4 (Table 1.).Figure 1 showed the representative of callus morphology on induction media.Callus on media 3 revealed a morphology that was rounded, moist, transparent and raised from midrib region.However, the initiated callus on media 4 has a morphology of rounded, dry and on the leaf midrib produced organ that like roots.

Genetic Transformation on oil palm
Genetic transformation of the PTE gene construct was conducted into 4 weeks-old oil palm calli on DF10 proliferation media (Table 1).The morphology of the growth of calli that have been transformed with the PTE gene construct was shown in Table 2.After the co-cultivation step, callus was cultured on selection media with antibiotic cefotaxime 100 ppm and kanamycin 50 ppm.In the post transformation phase at the moment, the DF media is used without antibiotics.It showed in the table that the control plants (wild type) still can grow on the media with antibiotics.However, it showed that they only can be developed to the globular phase.On the other hand, the transformed callus that grew c b a in the selection media with antibiotics revealed a compact structure with yellowish colour and performed a scutellar phase in the embryogenic development.The next step is to initiate the subculture of transformed calli and the control on the regeneration media.These results are in accordance with the report from Budiani et al [15] that the development of transformed somatic embryos was detected after 3-4 weeks culturing on modified DF media.Based on the previous research, the transformed and control callus were grouped into several types.Each type was treated to optimize the regeneration process by adding amino acids, polyamine, and antioxidant compounds.The addition of glutamine, L-cysteine, putrescine, and or ascorbic acid at the concentrations of 0, 10, and 25 ppm on proliferation media showed different conditions for each type of callus.However, most results showed that the callus biomass increased until reached the 2 nd cycle but then became stagnant.In this research, to optimize the regeneration of cisgenic callus, treatments will be applied by increasing the concentration of amino acid, polyamine, and ascorbic acid added to the media.

Conclusion
The transformation of a CRISPR/Cas9 construct to oil palm calli to edit the PTE (palmitoyl-acyl carrier protein thioesterase) gene has been successfully done using Agrobacterium tumefaciens.The transformed calli were subcultured into antibiotic selection media followed by DF10 and DF1 media without antibiotics afterward.The transformed calli on the selection media were able to grow to the scutellar phase with a compact structure in the embryogenic callus development stages.Attempts still have to be done to optimize the cisgenic callus regeneration and to confirm the presence of the target DNA in the next future.

Table 2 .Figure 3 .
Figure 3. Morphology of the development of transformed callus.A) control callus in DF10 media, B) PTE transformed callus on DF10 media, C) Control callus on DF1 media, D) PTE transformed callus on DF1 media, and E) Control callus on DF1 media (after 1 month), F) PTE transformed callus on DF1 media (after 1 month).Bar = 0.5 cm.

Table 1 .
Percentage of the callusing explants of Dami group response at 20 weeks cultured on induction media.