Variations on The Concentration and Purity of Oil Palm Flowers (Elais Guineensis Jac.) in Some Extraction Methods for Identification of the Flowering Gene

Palm oil production is related to the number of fruit and is thought to be determined since the beginning of the flowering phase as the initial stage of fruit formation. Identification of oil palm flowing genes needs to be done as the first step of knowing the mechanism of flowering in oil palm molecular. Oil male and palm females contain high polysaccharides and polyphenolic compounds so they can inhibit the process of molecular identification. Molecular studies provide accurate and fast information about the potential of oil palm genetics as a commercial commercial commodity. The purity and concentration of DNA and the temperature of annealing are the main requirements for PCR-based molecular studies. The purpose of this study obtained the extraction method of oil palm flowers which produced high DNA concentration and purity and optimized the temperature of annealing to identify the flowering gene. DNA from male flower spike and female flowers is extracted using the A (SDS) method, method B (CTAB), method C (CTAB + PVP), and method D (commercial kit). Primary BMS annealing temperature for amplification of optimized flowering genes using PCR gradients. The extraction of male flowers and female flowers using the C (CTAB + PVP) method produces the best DNA concentration and purity compared to other methods. The best annealing temperature for male flower DNA amplification and female flowers using the BMS primer is 61.1 o C. Amplification of male flowers and female flowers using Primary BMS produces 1200 PB products. The results of DNA extraction and PCR amplification using the BMS primers in female flower samples are better than male flowers


A. Introduction
Palm oil (Elaeis Guineensis Jacq.) Is one of the national superior commodities because it contributes greatly to the Indonesian economy. Based on Badan Pusat Statistik (2020) the area of oil palm plantations in Indonesia reached 14.33 million hectares with production reaching 42.9 million tons in 2018. The area of oil palm plantations is estimated to increase by 1.88% to 14.60 million hectares with Production increase of 12.92% to 48.42 million tons in 2019. This caused

B. Methology
The study was conducted in biomolecular, PTPP, Laptiab, BPPT, Serpong laboratories. Plant sampling is carried out in Puspitek, Serpong. The material used in this study was the spikelet of male and individual flowers from the spike of the teneral female flower varieties taken from the Puspiptek trial garden, Serpong (Figure 1). Flower samples after being removed from the midrib are immediately stored at -20 oC for DNA extraction.

DNA Extraction
As many as 0.5 grams of spicles of male flowers and individual flowers from female flower spiketets are crushed using liquid nitrogen until smooth. The method used is as follows: Method A (SDS). Sample powder is inserted into a centrifuable tube that contains 5 ml extraction buffer (10% SDS; EDTA 0.5 M pH 8; Tris HCl 1 m pH 8; NaCl 5 m; DDH2O; 1% MercaptoenaTOL) that has been incubated earlier at 65 OC.
3. Method C CTAB + PVP). Samples are crushed by adding 0.1 grams of PVP, then the powder is inserted into a centrifuge tube which contains 5 ml extraction buffer (CTAB 10%; EDTA 0.5 M pH 8; Tris HCl 1 m pH 8; NaCl 5 m; DDH2O; 1 % Mercaptoenatol) which had been incubated earlier at a temperature of 65 oC

The extraction procedure method A to the C method
The solution is homogenized with vortex then incubated at 65 oC for 30 minutes and turned back every 5 minutes. The solution was incubated at room temperature then added 1 Volume CI (Chloroform: isoamil alcohol = 24: 1) and centrifuged 14000 rpm for 20 seconds at 4 oC. This step is repeated three times. The supernatant was taken then added 1 volume of isopropanol that had been incubated at 4 oC. After being incubated at a temperature of -20 OC for 30 minutes, the solution was centrifuged at 14000 rpm for 10 minutes 4 oC. The supernatant was discarded then the pellet was dried, then added 500 μL, 1/10 volume nach3COO3 3 m pH 7 cold, 2 volume ethanol absolute that has been incubated at 4 oC, then stored at -20 OC overnight. Disentrized solution at 4 oC at a speed of 14000 rpm for 10 minutes. DNA pellets were washed using 400 μL 70% ethanol that had been incubated at 4 o C, then centrifuged at 4 o C at a speed of 14000 rpm for 5 minutes. The dried pellets are then added 100 μL DDH2O and 1/10 volume of RNASE, then incubated at 37 o C for 1 hour to actively in the RNase. (Commercial Kit). The sample powder was crushed by adding 0.1 grams of PVP, subsequently extracted using a commercial DNA extraction kit in accordance with the procedure recommended by the manufacturer. Each extraction method is repeated three times.

Quality and quantitative DNA analysis
The quality of DNA was analyzed using an electrophoresis of gel agarose 0.8% with a tae buffer at a 100 volt voltage for 30 minutes. The electrophoresis results visualized using Gel Doc UV Transilluminator. Analysis of the quantity of DNA extraction was carried out using a 2000 thermo scientific production spectrophotometer. Data of purity and concentration are analyzed using R studio.

Amplification of flowering genes
The PCR reaction consists of DNA male flower genomes and female flowers as much as 150-180 ng as templates, 1 μl of Mix 2 mm, 1 μl 10 x Dream Taq Buffer, 0.1 Uream Taq DNA polymerase, 0.5 μl Primary Forward 0 , 2 μm, 0.5 μL primer reverse 0.2 μm, ddh2o nuclease-free to 10 μL. The condition of the PCR used is the initials denaturation of 95 oC 5 minutes, denaturation of 95 oC 30 minutes, the annealing temperature is 65.4 ± 06 oC 30 seconds, the 72 OC extension is 1 minute, this condition is repeated to 35 cycles, and the final extension 72 OC 5 minutes. The primary used for the amplification of the flowering gene is the Primary BMS F AgncacyaCcagnaggcn / r acncactyayggytcccacn for amplification of squamosa flowering genes

C. Result and Discussion
Palm oil flowers are very easy to oxidize, causing brown (browning) that causes degraded DNA, this causes the extraction of DNA to oil palm flowers is quite difficult. The solution to avoid browning on samples of male flowers and palm oil is a room, mortar and pestle must be cold when the sample fleeces. Addition of liquid nitrogen when the grade can prevent degraded oxidation and DNA occurrence. Fresh samples of male and female flowers are soaked in liquid nitrogen and crushed quickly to help break cell walls mechanically, this is done to keep DNA degraded. According to Xin and Chen (2012) (2009) The DNA extraction process consists of several stages, namely the preparation of the material to be used, the process of destruction of cells (lysis), removal of contaminant compounds, and DNA collection. The phase of the destruction of plant cells greatly affects the purity of DNA so that the appropriate technique and reagent material is needed so that the resulting DNA has a high purity. SDS and CTAB are detergent solutions that function to reduce the surface voltage of the liquid and dissolve lipids at the lysis stage, this causes cell membranes to degradate, so the cell organelets can come out. According to the Bintang (2010), CTAB is a cationic detejen that is destroying cells, unraveling proteins, and separating carbohydrates from nucleic acid. The interaction between SDS and cell membrane proteins affects cell lyzers in DNA extraction, so that cell membrane proteins do not hinder the next DNA process (Perumal, N.V., X. Zhang, M. Yuki, I. Fumio, dan F. Wang, 2016). The addition of SDS and Positively charged CTAB serves to separate the polysaccharide from DNA by binding negatively charged DNA.
Buffer extraction method A, B and C heat is heated at a temperature of 65 oC, because the ability of SDS and CTAB to melisis the cell will be active in conditions of 65 oC. According to Lade, B.D., A.S. Patil, & H.M. Paikrao (2014) Addition of extraction buffers incubated less than 65 oC causes imperfect denaturation in proteins that can produce contamination at the next extraction stage. The addition of heated extraction buffers in frozen samples by liquid nitrogen causes the network to change temperatures quickly so as to avoid damage to DNA. This is in accordance with the opinion of Purchooa (2004) that the addition of a heated CTAB buffer on frozen tissue by liquid nitrogen causes the network to experience a temperature difference from -80 oC to a temperature of 60 oC quickly resulting in high quality DNA.
Analysis of the quality of male and female DNA extraction using the A (SDS) method, method B (CTAB) and method C (CTAB + PVP) on agile gel 0.8% shows the quality of DNA is quite good even though there is still contaminant, while DNA extraction uses a kit Commercial shows degraded DNA ribbons (  Extraction using the D method (Kit method) produces degraded DNA shown by the existence of smears on the DNA band. The use of kits to extract DNA male and female flower organs is not recommended because this organ contains a fairly high phenolic compound. The reagents used on kits cannot reduce phenolic compounds produced by flower organs so that DNA is degraded. Commercial DNA extraction kit is an ideal procedure for DNA extraction because it does not use dangerous reagents, but in some commercial research kits produce low DNA results and varied quality ( Quantitative analysis shows that as much as 50% of male flower samples and extracted female flowers use the A (SDS) method, method B (CTAB), and the C (CTAB + PVP) method produces a concentration of DNA more than 500 ng / μL and 50% more DNA purity Of the 1.5 (Figure 3). Aboul-Maaty and Oraby (2019) recommended the ratio of DNA purity greater than 1.5 and better close to 1.8, while according to Healey, A., A. Furtado, A. Cooper, & R.A. Henry (2014) for NGSbased Molecular activities (next generation sequencing) needed DNA with a comparison of A260 / A280 between 1.8 to 2.0. The DNA extraction using the D method (Kit Method) is not quantified due to degradation.

Figure 3. Histogram concentration and purity DNA extraction results using the A (SDS) method, B (CTAB) method, and method C (CTAB + PVP)
The male flower DNA extraction using the B (CTAB) method produces the lowest concentration and purity of DNA compared to samples and other methods (Figure 4). The extraction of female flower DNA uses the B (CTAB) method and the C (CTAB + PVP) method shows good purity, while the male flower extraction using the C (CTAB + PVP) method produces better DNA purity than male interest extracted using other methods. Method C (CTAB + PVP) produces the concentration of DNA from male flowers and the highest female interest compared to the A (SDS) method and B (CTAB) method. This shows the method C (CTAB + PVP) is able to produce the concentration and purity of male flower DNA and oil palm female flowers higher than other methods. This result is in accordance with the results of Murtiyaningsih's research (2017) that the extraction of DNA pineapple leaves using CTAB and PVP buffers show good purity between 1.8 -1.9. The purity of female flowers DNA differs significantly with the purity of male flowers DNA, while DNA purity is no different between method A (SDS), method B (CTAB) and C method (CTAB + PVP) ( Figure 5). The concentration of male flower DNA is no different from the concentration of female flower DNA, but the sample extracted using the C (CTAB + PVP) method produces a significant different concentration with other methods. Addition of PVP when the grade is thought to be able to inhibit the oxidase polyphenol enzyme. The polyphenol enzyme can degrade DNA and cause the oxidation of phenol compounds. Oxidation is characterized by the formation of brown color on the plant tissue to be isolated. This is in accordance with the opinion of Utami, A., R. Meryalita, N.A. Prihatin, L. Ambasari, P.A. Kurniatin, & W. Nurcholis (2012) Insulation with CTAB as an extraction buffer requires additional PVP to eliminate phenol contamination. Extraction of male flowers and female flowers use method B (CTAB) and C methods (CTAB + PVP) show the same pattern, while the A (SDS) method shows a different direction in the parameters of purity and DNA concentration ( Figure 6). The extracted female flowers using the B (CTAB) and C (CTAB + PVP) method shows higher purity and concentration than male flowers, while extracted female flowers using the A (SDS) method shows lower purity and concentrations than male flowers. This shows a CTAB buffer that is applied to a single and modified with the addition of PVP capable of producing the concentration and purity of oil palm female DNA better than the SDS buffer application. The extracted male flowers using SDS buffers produce purity and concentration of DNA better than Buffer CTAB and CTAB + PVP.  The results of the Gradient PCR show amplification of male flower DNA using the BMS primary produces PCR products at 59.4 oC annealing temperatures; 60.2 oC: 61.1 OC and not produced PCR products at annealing temperature 62.1 oC, whereas in DNA female flower PCR products are produced at all annealing temperatures. These results show a type of sample and annealing temperature allegedly influencing the quality of PCR products. The best annealing temperature for male and female DNA amplification using the BMS primer is 61.1 oC. Amplification of male flowers and female flowers using Primary BMS produces 1200 PB products.
In general, the results of this study show extraction of DNA and PCR reactions to identify the flowering gene from female flower samples producing purity and concentration and and PCR products are better than male flowers, because they are suspected of male flowers containing more contaminants that can reduce DNA purity and concentration, thus disrupting the PCR reaction. One source of contaminants in male flowers is the presence of pollen composed of polysaccharide compounds. The high polysaccharide male flower causes male flower DNA extraction and the PCR reaction is more difficult than female flowers.

D. Conclusion
The extraction of male and female flowers DNA uses the CTAB + PVP method produces the best DNA concentration and purity compared to the SDS method and the CTAB method. The best annealing temperature for male and female DNA amplification using the BMS primer is 61.1 oC. Amplification of male flowers and female flowers using Primary BMS produces 1200 PB products. DNA extraction and PCR amplification using primary BMS on female flower samples is better than male flowers.

E. Acknowledgement
The author expressed gratitude to the Agricultural Production Technology Center -Deputy Technology of Agroindustry and Biotechnology -The Assessment Agency and Application of Technology that facilitate this research.