Biostimulant activity of Eucheuma cottonii extract on early growth of Elaeis guineensis Jacq

Seaweed extract contains many bioactive compounds that boost the metabolisms of various plant commodities, ranging from germination, growth, and productivity. This study aimed to increase the early growth of oil palm germinated seeds by applying Eucheuma cottonii extract. The study used a completely randomized design (CRD) with four treatments (0%, 0.5%, 1%, and 1.5%) to test the optimum dose of E. cottonii methanol extract on oil palm growth stimulation (22 oil palm germinated seeds/treatment with equal growth performance). The germinated seeds were soaked with E. cottonii extract for one hour, then germinated for seven days. Observations were made on plumula length, radicula length, and the number of lateral radicula that performed daily. The data were analyzed statistically using ANOVA with Tukey HSD as the further test (p>95%). E. cottonii extract was dominated by auxin, 32.24 mg Kg-1, with a dose of 0.5% significantly increased radicula length by 50.85%, followed by an increased number of lateral radicula by 70.97% compared to control. Thus, 0.5% of Eucheuma cottonii extract could be used as an alternative plant growth regulator in stimulating the early growth of oil palm germinated seeds.


Introduction
Over the last 20 years, Indonesia's seaweed industry has expanded significantly, supplying 66% of the world's seaweed production.South Sulawesi has the most significant production, namely more than a third of the National production and more than 20% of the world's carrageenan seaweed supply [13].In recent years, seaweed has gained global interest, so numerous seaweed-based products have been developed for agri-horticultural, food, pharmaceutical, and nutraceutical purposes [6].
It was reported that since the 19 th century, coastal residents began to use seaweed as a raw material for fertilizer and understood the benefits of seaweed application for cash crops commodities.Seaweed contains great macro and micronutrients, amino acids, vitamins, and plant growth regulators [10].Seaweed extract has been widely used as a biostimulant in plants due to its growth-accelerating effect and increased resistance to stress [3].Biostimulants are ingredients containing one or more substances or microorganisms that can stimulate the absorption and efficient use of nutrients by plants, increase plant's tolerance to abiotic/biotic stresses, and improve the quality of plants that applied in small amounts [17].Brown algae such as Ascophyllum nodosum, Laminaria spp., Turbinaria spp., Sargassum spp., and Fucus spp.have been extensively studied as a source of biostimulants [10].
Eucheuma cottonii, a member of the Rhodophyta group, has green, yellowish gray, or red color.If dried, it becomes brownish yellow.It is mainly cultivated in tidal areas up to 15 m deep and only takes between 45-50 days before harvesting [14].Besides Gracilaria and Kappaphycus, Eucheuma is a group widely used to produce carrageenan and agar [13].Only a few have reported the potencies as a plant growth regulator.In this study, E. cottonii methanol extract was tested for phytohormone content, then performed bioassays using oil palm germinated seeds.The research aimed to increase the early growth of oil palm germinated seeds by applying Eucheuma cottonii extract.

Material and method
2.1 Materials.The raw material, Eucheuma cottonii, was obtained from Bontang, West Borneo, Indonesia.The seaweed was washed with tap water to remove sand, salt, and another debris, then dried using oven (60°C) until it reached the constant weight (Figure 1), while the commercial oil palm germinated seed var.Simalungun from Indonesian Oil Palm Research Institute (IOPRI), Indonesia, was used for bioassay.The seaweed extraction and bioassay were conducted at the Laboratory of Biochemistry, IOPRI Bogor, Indonesia.The phytohormone analysis was performed at Agrochemical Material Residues Laboratory "Balingtan", Bogor.The research was conducted from November 2022 to January 2023.

Eucheuma cottonii extraction.
The Eucheuma cottonii extraction method was maceration adopted from Dolorosa et al. [14] with a few modifications.The dried seaweed of E. cottonii was milled mechanically using a crusher tool.A 10 gr of ground E. cottonii was soaked with 100 mL of methanol (grade PA) solvent on Erlenmeyer covered with aluminum foil (alufo).Sample maceration coupled with shaking at 200 rpm was carried out for 30 hours at room temperature (25-27°C).The extract was filtered using filter paper to obtain filtrate and residue.Crude extract of E. cottonii in liquid phase was stored at 4°C for further analysis.

Phytohormone analysis.
E. cottonii crude extract in liquid phase was sent to Balingtan, Bogor, for phytohormone analysis.The analysis was carried out using a protocol from Barendse [3] in HPLC instrument, and the hypersil gold C18 column was used as compound separation.A standard solution of auxin, gibberellin, kinetin, and zeatin was prepared at 0,1%.The methanol : water with the ratio 65 : 35 (v/v) was used as the mobile phase, and the flow rate was 0,5 mL min -1 .A 5 µL of the sample was loaded into the column.The column temperature at 40°C was maintained.The wavelength detection was performed at λ254 nm.In advanced of analysis, the sample was homogenized with methanol 65%, then centrifuged at 4000 rpm, 30 min.The supernatant was filtered using a syringe filter 0,45 µm, then the filtrate was ready to examined.Phytohormone content was measured by comparing the chromatogram peak of the standard.

Bioassay in oil palm germinated seed.
The methanol extract of E. cottonii was tested for early growth stimulation of oil palm germinated seeds.The experimental design was a completely randomized design (CRD) with four serial concentrations (0%, 0,5%, 1%, and 1,5%) of E. cottonii extract in 22 experimental units (replications) of oil palm germinated seeds.First, the initial oil palm germinated seed with the average size of plumulae and radiculae around 8-10 mm and 12-15 mm, respectively, was selected (Figure 2).Oil palm germinated seeds were soaked according to the treatment for 1 hour [6], then air dried for 30 minutes.The seedling growth was performed on a tray covered with moist cotton for seven days.The moisture was maintained by adding aquadest (150-200 mL per day) with the same volume for each treatment.A 2 g/L dithene solution was sprayed on day 4 to avoid fungal growth [5].Seedling growth observation, including plumula length, radicula length, and the number of lateral radicula, was performed until day 7.The growth increment was calculated by the ratio between the growth on day 0 and day 7.The data were analyzed statistically using ANOVA with Tukey HSD as the further test (p>95%).

Phytohormone content in Eucheuma cottonii extract.
Phytohormones content consisting of auxin, gibberellin, kinetin, and zeatin of E. cottonii extract was shown in Table 1.The data showed a higher concentration of auxin, 32.24 mg Kg -1 sample, followed by zeatin, gibberellin, and kinetin, namely 11.52 mg Kg -1 , 10.71 mg Kg -1 , and 7.04 mg Kg -1 , respectively.Sunarpi et al. [9] reported that Sargassum polycystum extract produced the highest levels of gibberellin and kinetin, 8 mg L -1 and 10 mg L -1 , compared to S. cristaefolium and S. crassifolium.Fitriyah et al. [6] added that the different extraction methods for Sargassum sp., namely acidic, alkaline, and water extraction, showed different levels of phytohormone such as IAA, gibberellin, zeatin, and kinetin.Zeatin showed the highest levels, around 34.92 mg L -1 to 38.25 mg L -1 , followed by gibberellin, IAA, and kinetin.Thus, different species of seaweed and extraction methods produce different phytohormone profiles.
E. cottonii contains much chlorophyll, around 74.92%, followed by xanthophyll 7.72%, and carotene 0.95% [12].It also contains vitamin E of 158.07 µg mL -1 , moisture 76.15%, ashes 5.62%, proteins 2.32%, lipid 0.11%, and carbohydrates 15.8%.Moreover, it contains phenol, flavonoid, hydroquinone, and triterpenoid [15].E cottonii methanol extract contains total phenol 141 mg GAE gr - 1 and flavonoids 17.78 mg QE gr -1 [20].Sedayu et al. [19] reported that E. cottonii liquid extract contains several growth hormones, such as gibberellins in the form of GA3 and GA7 of 128 ppm and 110 ppm, respectively.In addition, cytokinin comprises zeatin and kinetin, 117 ppm and 73 ppm, respectively, and auxin (IAA) of 160 ppm.After germination for seven days, the physical appearance of oil palm seedlings in each treatment is shown in Figure 3. Visually, there was a significant difference in the radicula length followed by the number of lateral radicula, while there was no difference in plumula length.Stimulation of the oil palm germination, growth, and development could be conducted through several approaches, such as heat treatment and hormone treatment.Fondom et al. [7] reported that heat treatment at 39°C for 60 days was the optimum treatment to promote oil palm seedling growth recorded at the leaf production rate, stem diameter, leaf area, and plant growth increment.Farhana et al. [5] reported that heating at 80°C for 72 hours, followed by immersion in 0.4% etephton, increased seed germination with a maximum growth potential of 52%.Daza et al. [4] added that applying 2,4-D and NAA can potentially promote parthenocarpic fruit development of oil palm plants with an equal oil-to-bunch (O/B) ratio to assisted pollination.
On day 0, the plumula length for each treatment and control was different, that is 10.49 mm (ME 0.5%), 9.79 mm (ME 1%), and 9.13 mm 1.5%), while for the control, it was 8.57 mm (Figure 1A).Plumula length in all treatments increased slowly until day 3, while on days 4 to 7, it increased exponentially.On day 4, the increase in plumula length in the ME 1% treatment was faster than others, namely 12.43 mm, while in ME 0.5% treatment, it was 12.73 mm.From day 5 to 7, there was an equal increase in plumula length between the ME 0.5% and ME 1% treatments, while ME 1.5% treatment showed a decreasing trend compared to others, which was almost the same as the control (0% ME).On day 7, the plumula length of ME 0.5%, ME 1%, ME 1.5%, and ME 0% treatment were 15.00 mm, 14.29 mm, 13.32 mm, and 13.18 mm, respectively.However, although not significantly different, each treatment showed a lower increase in plumula length than the control (Figure 4B), that is 4.51 mm, 4.50 mm, 4.19 mm, and 4.61 mm for ME 0.5%, ME 1%, ME 1.5%, and 0% ME treatments, respectively.Thus, it is confirmed that E. cottonii extract at 0.5%, 1%, and 1.5% did not show an increase in the plumula length of oil palm compared to the control.The effect of immersing oil palm germinated seeds on radicula length increment is presented in Figure 5B.On day 0, radicula lengths for ME 0.5%, ME 1%, ME 1.5%, and 0% ME were 14.78 mm, 14.21 mm, 15.57mm, and 12.69 mm, respectively.Seedling growth on day 1 to day 3 for ME 1%, ME 1.5%, and 0% ME treatment showed a uniform trend, while the ME 0.5% treatment showed a dramatic growth increment.On day 4, the ME 0.5% treatment showed the highest radicula length, 17.59 mm, slightly longer than the ME 1.5% treatment (17.09 mm).Until day 7, the ME 0.5% treatment showed faster growth than the other treatments, including the control.On day 7, radicula length for ME 0.5%, ME 1%, ME 1.5%, and 0% ME treatment were 19.50 mm, 17.43 mm, 18.59 mm, and 15.83 mm, respectively.Thus, a ME 0.5% treatment significantly increased the early growth of oil palm germinated seeds as observed in the radicula length increment, which is 4.72 mm (50.85%), while in ME 1%, ME 1.5%, and 0% ME were 3.22 mm, 3.02 mm, and 3.13 mm, respectively.The effect of E. cottonii extract on the increase in the number of lateral radicula is presented in Figure 6.On day 0, the number of lateral radicula of oil palm germinated seeds in the ME 0.5% and ME 1.5% treatments were lower than the ME 0.5% (4.32) and ME 0% (4.04), namely 3.69 and 3.52, respectively.On days 1 to 3, the increase in the number of lateral radicula for each treatment showed a similar trend, while on days 4 to 7, it showed an exponential trend.On day 5, the number of lateral radicula in the ME 0.5% and ME 1.5% treatment was almost the same as the control, which was around 5.20, while on day 6, they showed faster growth.On day 7, the number of lateral radicula treated with ME 0.5% and ME 1.5% was higher than those treated with ME 1% (6.10) and 0% ME (5.68), 6.5 and 6.23, respectively.The increase in the number of lateral radicula in ME 0.5%, ME 1%, ME 1.5%, and ME 0% treatments for seven days of germination were 2.81, 1.78, 2.70, and 1.64, respectively (Figure 6B).Although not significantly different, the number of lateral radicula in ME 0.5% and ME 1.5% treatments showed an increase of 70.97% and 64.52% compared to the control, respectively.Thus, 0.5% and 1.5% of methanol extract are potential to increase lateral radicula growth compared to the control.Based on the results, a 0.5% of E. cottonii extract significantly increased the initial growth of oil palm germinated seeds.The result is in accordance with previous studies that the biostimulant activity of seaweed is effective at low concentrations [11; 18; 6].The application of biostimulant produced from Ascophyllum nadusum on Arabidopsis at a dose of 3% showed faster plumula and radicula growth [11].Application of S. tenerrimum extract at a dose of 0.8% showed the highest germination rate, followed by maximum leaves number, radicula length, and dry weight on tomato, while a dose of 0.6% increased the plumula length and plant height [18].Fitriyah et al. [6] also reported that Sargassum sp.alkaline extract at a dose of 3.5% showed the highest germination percentage, while at a dose of 1.5%, it showed the highest plumula height and radicula number.A 0.5% acidic extract dosage causes the highest plumula and radicula weight.
Seaweed-based biostimulants have been widely reported to increase plant growth, productivity, and stress resistance.Mondal et al. [16] reported that a biostimulant (K-enriched) from K. Alvarezii containing GA3 and IAA applied through a foliar spray can increase photosynthetic activity as shown in a 26% increase in plant biomass compared to control.Gomathi et al. [8] stated that seaweed extract accelerates plant growth and health because it can supply N, P, K, and minerals such as Zn, Mn, Mg, and Fe.These elements are available and ready to be used by plants to accelerate photosynthesis, resulting in healthier leaves.In addition, the extract contains natural growth hormones, such as auxins, gibberellins, and cytokinins, resulting in increased nutrient absorption from the soil and antioxidant activity.Meanwhile, Almaroai & Eissa [1] added that seaweed extract applied to onion plants during drought significantly increased the uptake of N, P, and K by up to 116%, 113%, and 93% compared to the control.
The bioactive compounds in seaweed are diverse, ranging from single compounds, such as amino acids and phytohormones, to complex compounds, like polysaccharides and polypeptides.Crude extract of seaweed-based biostimulants can increase plant growth by increasing cell elongation, nutritional value, and sugar content, resulting in higher productivity and quality.In addition, seaweed extract stimulates cell division and improves plant physiology and metabolic activity, enhancing radicula growth, flowering, and fruit development [10].Indole-3-Acetic Acid (IAA), or auxin, is a hormone commonly found in seaweed extracts such as Sargassum sp.[9].According to the analysis, the methanol extract of E. cottonii was dominated by auxin, 32.24 mg Kg -1 , so it was suggested responsible for a significant increase in the radicula length of the oil palm germinated seeds compared to the control.Auxin is vital in plumula and radicula growth because it stimulates apical meristem cell division and elongation.At the same time, cytokinin content, kinetin and zeatin, are thought to be responsible for lateral radicula growth.In order to gain a more comprehensive understanding, analysis of other bioactive compounds such as peptides, amino acids, and polysaccharides needs to be carried out in further research.

Conclusion
In conclusion, a 0.5% of Eucheuma cottonii extract is the optimum dose for early growth stimulation of oil palm germinated seeds.Therefore, E cottonii methanol extract could be an alternative oil palm growth regulator.

Figure 4 .
Figure 4.The effect of liquid methanol extract of E. cottonii on plumula length (A) and plumula length increment (B) of oil palm germinated seed.The bar represents the mean of the growth parameter with the corresponding standard error (n = 22).A significant value is indicated by

Figure 5 .
Figure 5.The effect of liquid methanol extract of E. cottonii on radicula length (A) and radicula length increment (B) of oil palm germinated seed.The bar represents the mean of growth parameter with the corresponding standard error (n = 22).A significant value is indicated by the letter on top of each bar.P < 0,05.ME 0%: control, ME 0.5%: Methanol extract 0.5%: ME 1%: Methanol extract 1%, and ME 1.5%: Methanol extract 1.5%.

Figure 6 .
Figure 6.The effect of liquid methanol extract of E. cottonii on lateral radicula number (A) and lateral radicula number increment (B) of oil palm germinated seed.The bar represents the mean of growth parameter with the corresponding standard error (n = 22).A significant value is indicated by the letter on top of each bar.P < 0,05.ME 0%: control, ME 0.5%: Methanol extract 0.5%: ME 1%: Methanol extract 1%, and ME 1.5%: Methanol extract 1.5%.

Table 1 .
Phytohormones content of E. cottonii methanol extract 3.2 Eucheuma cottonii extract promotes the early growth of oil palm germinated seeds.