Effect of Exogenous Natural Plant Growth Regulators (Pgrs) on the Morphology, Growth, and Nutrient of Sea Grapes (Caulerpa Racemosa)

This study aimed to determine the effectiveness of aqueous extract from various plants as an exogenous plant growth regulator (PGRs) in the morphology, growth, and nutrition of C. racemosa. The natural PGRs used were coconut water (CW), shallots and coconut water (AESCW), maize (AEM), moringa leaves and mung beansprout (AEMBS), and E. cottonii (AEEC). A 50g of C. racemosa was soaked on the natural PGRs and was cultivated for 30 days with a sandy mud substrate. The results showed that the soaking of C. racemosa on natural PGRs had a signi�cant effect (P<0.05) on absolute growth, speci�c growth rate, and nutrient. C. racemosa soaked in aqueous shallot and coconut water showed the best absolute growth (214.32±7.68 g) and SGR (5.55±0.08%/day), meanwhile C. racemosa soaked in coconut water had the best nutrition (protein: 9.54±0.16%; �bre: 1.37±0.21%; and fat 0.42±0.04%). The morphological analysis showed that soaking C. racemosa on natural PGRs resulted in long fronds, bigger stolons, a greener and fresh colour, and more ramulli.

In Indonesia, locally known as "Latoh", it is used as "pecel" or salads.C. racemosa cultivation has been carried out in Indonesia, including in Sulawesi, Nusa Tenggara and Jepara, Central Java.According to DJPB data (2017), the demand for Caulerpa increases yearly, both nationally and internationally.National seaweed data for ve years (2011)(2012)(2013)(2014)(2015) shows a positive upward trend, with an average increase of 22.25%.Increased demand must be accompanied by increased production as well.The problem that arises is that C. racemosa has not been maximized.Improving the quality of seeds to boost growth is one strategy to solve this challenge.Seaweed grows due to the processes of respiration and photosynthesis that it performs.Synthetic plant growth regulators alter the plant's quality and potentially harm human consumption.
Plant growth regulators (PGRs) can improve physiological e ciency, including photosynthetic ability, thereby helping in effective ower formation, seed, and fruit development and ultimately enhancing the productivity of the crops (Solaimalai et al. 2001).However, the excessive use of PGRs and their degradation products have side effects on human health, animals, and plants.The residues of PGRs in agricultural products are seriously detrimental to human health because they have been found with hepatotoxicity, neurotoxicity, carcinogenicity, and other side effects.Furthermore, PGRs are suspected of disrupting human and animal reproductive systems (Xu 2018;Chen et al. 2022).
Currently, efforts are being made to boost growth by making plant growth regulators from natural materials.The role of growth regulators has been documented in hundreds of herbaceous crops (Kavina et al. 2011).Some marine natural materials have been used as PGRs, such as Ulva, Padina, and Sargassum (Garcia et al. 2020).In addition to using marine natural materials, natural PGRs from land plants are widely used.Coconut water has auxins, gibberellins, and cytokinins (Mamaril 1988

Materials And Methods
Experimental setup and natural plant growth regulators preparation C. racemosa used was from Jepara, Central Java, weighing 50g/treatment.C. racemosa seeds are selected, which are still fresh, healthy, and clean of dirt.The PGRs used were coconut water, shallots, maize, moringa leaves, mung bean sprouts, and Eucheuma cottonii.Coconut water (CW) added as much as 25% and 75% seawater.An aqueous extract of shallots and coconut water (AESCW) was made by blending shallots with distilled water (150 g/L).The solution is ltered and taken as 10% plus 25% coconut water and 65% seawater.Seagrapes were soaked in each natural PGRs with the following treatment: aqueous extract of maize (AEM) was made by mixing 50g of young maize kernels, 50g of sugar, and distilled water and fermented for 24 hours, and the crude extract was taken and then mixed with seawater.Aqueous extract of moringa leaves and mung bean sprouts (AEMBS) was made by blending 250g of sprouts and adding 500ml of distilled water and ltering; 100 g of Moringa leaves were blended added 500 ml of distilled water and ltered.The two solutions were mixed, and 5mL was taken to be mixed into 1L of seawater.Aqueous E. cottonii (AEEC) extract was made by blending E. cottonii with distilled water (1:1), ltering, and extracting.The aqueous extract was taken 5% and mixed into 1L of seawater.
TC: without any PGRs (treatment control) CW: C. racemosa was soaked in coconut water for 40 minutes AESCW: C. racemosa was soaked in an aqueous extract of shallots + coconut water for 40 minutes AEM: C. racemosa was soaked in an aqueous extract of maize for 40 minutes AEMBS: C. racemosa was soaked in an aqueous extract of moringa leaves + mung beansprout for 120 minutes AEEC: C. racemosa was soaked in an aqueous extract of Eucheuma cottonii for 90 minutes Previously, a preliminary test was carried out for each treatment to determine the concentration and duration of immersion that did not make C. racemosa wither or even die.After soaking, C. racemosa was cultured in cement ponds with sandy mud substrate (Windarto et al. 2021) using the off-bottom method with a 6:1 concentration of nitrate and phosphorus (Harwanto et al. 2020).The cultivation process was carried out for 30 days.

Morphological analysis
The samples were taken out of the pond one at a time, cleaned, and preserved in wet seawater until weighed.After the study, morphology and appearance were noted, particularly ramulli, fronds, stolon, rhizoid, colour, fouling, and disease signs.

Analysis of C. racemosa nutrient
A standard technique was used to determine the samples' proximate chemical composition (AOAC 2005).
The parameters for proximate analysis were protein, fat, ash, crude bre, and moisture.Protein analysis was performed using the Kjeldahl method.Lipid samples were analyzed using the gravimetric method using the weight of the sample to nd the lipid levels.
Water Quality Different devices were used to determine the water quality for each characteristic.A water quality checker (YSI environmental 550A) was used to assess DO, temperature, and pH; a refractometer (Atago S/Mill-E) was used to detect salinity.

Data Analysis
The data collected throughout the trial was evaluated using Analysis of Variance (ANOVA) and the Duncan test to see signi cant differences between treatments.Water quality was analyzed descriptively.

Morphology of C. racemosa
The result of the morphology of C. racemosa under treatment can be seen in Fig. 1.Parameters observed were ramulli, fronds, stolon, rhizoid, colour, fouling, and disease signs.Overall, C. racemosa was in good condition and clean, with no signs of disease.
The fronds and stolons of each C. racemosa have different sizes.Based on observations made after 30 days of the study, data on the longest fronds and the largest stolon size were obtained, as seen in Table 1.The result showed that C. racemosa soaked in coconut water (CW) had the longest fronds and the most oversized diameter of stolons; meanwhile, the C. racemosa without any treatments showed the shortest fronds and stolons.

Absolute Growth of C. racemosa
The initial weight of C. racemosa planting was 50g; during 30 days of maintenance, the weight increased.
The initial weight and nal weight data can be seen in Table 2.The result showed an increase of C. racemosa during the culture (30 days); treatment AESCW showed the best result; on the other hand, the TC showed the slightest result.
The absolute growth of C. racemosa under different treatments can be seen in Fig. 2.

Speci c Growth Rate (SGR) of C. racemosa
The speci c growth rate (SGR) of C. racemosa under different treatments can be seen in Fig. 3.

C. racemosa Nutrient
The nutrient composition of C. racemosa under treatment after 30 days is presented in Table 3.The nutrient analysis showed that the soaking of C. racemosa on natural PGRs had a signi cant effect (P < 0.05) on the nutrient.The highest protein was found in the treatment CW (9.54 ± 0.16%), and the highest bre was found in the treatment AEEC (1.58 ± 0.06%); meanwhile, the lowest nutrient was found in the treatment TC.

Water quality
Results of water quality measurement for C. racemosa culture during the research were presented in Table 4.The data showed that the water quality during research was suitable for culturing the C. racemosa.

Morphology
Based on the morphology analysis, C. racemosa was in good condition and clean, with no signs of disease.Each treatment's ramulli was good, with lots of ramulli on the branches and healthy.However, TC treatment found some rotten parts.C. racemosa soaked in natural PGRs showed more ramulli than the TC.Fronds and stolon were normal; treatment CW showed the longest and thickest stolon, while TC showed the shortest and thinnest stolon.The morphological differences between C. racemosa that were not immersed in PGRs (TC) and C. racemosa that were immersed in PGRs were due to coconut water, shallots, maize, moringa leaves, mung beansprouts, and E. cottonii possessing growth hormones such as auxin, gibberellin, cytokinins and its derivatives which are effective for stimulating growth, strengthening stems, and regulating cell development.Coconut water's use focuses on the content of the auxin hormone in coconut water.The hormone auxin affects the size and length of cells in plants, so it will signi cantly affect seaweed growth.The TC treatment, which did not involve soaking, resulted in more stems that did not grow fronds, while the existing fronds did not have ramulli at the ends of the fronds.In other treatments, there is a tallus with lots of ramulli at the ends of the fronds.2005).The morphology condition showed that the natural PGRs affect the ramulli, fronds, stolon, rhizoid, and colour.
In addition to the role of hormones, the environment is an external factor that causes seaweed to have a good morphology.A suitable environment will make seaweed absorb the nutrients in the media, either in the form of fertilizers or hormones derived from natural ingredients.Brightness is an important factor related to sunlight's availability that affects photosynthesis to support its growth so that rhizoids, assimilators (fronds), and seaweed ramulli can grow well (Yuniarsih et al. 2014).Sandy mud substrate is a suitable medium because C. racemosa can stick and propagate well so that the morphology of the C. racemosa is not damaged (Windarto et al. 2021).

Growth Performances
Adding hormones through soaking in natural PGRs produces signi cant effects on seaweed growth.This is thought to be because coconut water, shallots, maize, moringa leaves, mung beansprouts, and E. cottonii possess growth hormones such as auxin, gibberellin, cytokinins, and its derivatives which are effective for stimulating growth, cell division, elongation, and cell development.
Based on the result of the study, soaking C. racemosa in the natural PGRs had a signi cant effect (P < 0.05) on the absolute growth and speci c growth rate.C. racemosa soaked in aqueous shallots and coconut water (AESCW) showed the best result (G: 214.32 ± 7.68; SGR: 5.55 ± 0.08%/day), followed by treatment CW, AEM, AEMBS, AEEC, and TC.AESCW showed the best result because shallot and coconut water contain auxin, gibberellin, and cytokinin.Auxin in coconut water is absorbed by seaweed.Auxin stimulates cells to divide rapidly and develop into shoots and stems (Ariyanti et al. 2018;Aisa et al. 2020).The hormone auxin itself in uences the formation of fronds and cell division in seaweed.Auxin hormone helps accelerate the occurrence of cell division in the growth process.The auxin concentration of 0.0039% in young coconut water causes differentiation and shoot formation because coconut water has better auxin and cytokinin reserves.The content of auxin and cytokinin in coconut water has an essential role in cell division, thus helping the formation of shoots and stem elongation.Coconut water contains 60% auxin and 20% cytokinin.

Conclusion
The results showed that the soaking of C. racemosa on natural PGRs had a signi cant effect (P < 0.05) on absolute growth, speci c growth rate, and nutrient.C. racemosa soaked in an aqueous of shallot and coconut water at 40 minutes showed the best absolute growth (214.32 ± 7.68 g) and SGR (5.55 ± 0.08%/day), but C. racemosa soaked in coconut water had the best nutrition (protein: 9.54 ± 0.16%; bre: 1.37 ± 0.21%; and fat 0.42 ± 0.04%).The morphological analysis showed that soaking C. racemosa on natural PGRs resulted in long fronds, bigger stolons, a greener and fresh colour, and more ramulli.

Statements And
The speci c growth rate of C. racemosa (%/day) during research During the research, the speci c growth rate (SGR) of C. racemosaresulted in C. racemosa, which soaked in an aqueous extract of shallots and coconut water had the best result (5.55±0.08%/day),followed by CW, AEM AEMBS, AEEC, and TC.The statistical test showed that the soaking of C. racemosa on natural PGRs had a signi cant effect (P<0.05) on its speci c growth rate.
Absolute growth of C. racemosa Absolute growth of C. racemosa has used the formula of Togatorop et al. (2017): G = W t -W 0 Where: G: absolute growth (g) W 0 : weight at the start of culture (g) W t : weight at the end of culture (g) Speci c Growth Rate The following formula (Syahlun et al. 2013) was used to calculate speci c growth rate (SGR) values: x 100% Which: SGR: speci c growth rate (% day − 1 ); SGR = LnW t−LnW 0 t W t : weight of C. racemosa at the end of culture (g); W 0 : weight C. racemosa at the start of culture (g); t: Culture duration (days).

Table 1
The longest fronds and the largest stolon size of C. racemosa

Table 2
The initial weight and nal weight of C. racemosa

Table 4
(Fu et al. 2000;Costa et al. that coconut water contains auxin, a leaf rot inhibitor.Auxins can react in plants to produce inhibitors that function as inhibitors of the formation of ethylene, which causes the rotting of plant organs.Ariyanti et al. (2020) and Indriyanti et al. (2020) stated that Auxin regulates cell enlargement and elongation and stimulates plant growth.The rhizoid was normal, and the colour of each treatment was green.C. racemosa soaked in natural PGRs showed greener colour than TC (without soaking).It is presumably due to the interaction of growth regulators auxin and cytokinins with C. racemosa, which can increase the amount of chlorophyll and slow down chlorophyll degradation in C. racemosa.Using growth regulators auxins and cytokinins can increase the amount of chlorophyll in aging plant tissues, thereby slowing down the degradation of chlorophyll and the aging process of plants(Fu et al. 2000;Costa et al. (7)mba(9)da et al. 2019;George 1993;Satyavathi et al. 2004logical processes and stimulates plants to grow at low levels.This is con rmed byAyyubi et al. (2019)that coconut water is also known as a natural growth stimulant in cuttings propagation.Shallot was known to contain growth hormones such as auxin and gibberellin, which could enhance the growth of seeds.Plant growth and development were stimulated by the environment or hormones(Yunindanova et al. 2018).The combination (AESCW) will work synergistically compared to treatments that use only one ingredient (CW).C. racemosa soaked in an aqueous extract of maize (AEM) showed better results compared to the control because the crude maize extract contains the hormone gibberellins, which causes thickening of the thallus, the cytokinin group that functions in cell division, inhibits chlorophyll degradation, and aging increasing stem height(Damiska et al. 2015; Pandeey et al. 2017; Sundari et al. 2016).C. racemosa soaked in an aqueous extract of moringa leaves and mung beansprout (AEMBS) and soaked in an aqueous extract of E. cottonii had a better result than the control moringa leaves, mung beansprout, and E. cottonii containing exogenous growth hormones, such as auxin, cytokinin and gibberellin.The auxin content in mung beansprouts is 3.24 ppm (Sukmadi 2013).Moringa leaves have a high cytokinin content; the cytokinin content in Moringa leaves ranges from 5-200 ppm(Culver et al. 2012).Adding auxin or cytokinin to the culture media can increase the concentration of endogenous growth regulators in cells to become a "trigger factor" in growth and tissue development(Poonsapaya et al. 1989).Cytokinins are not able to carry out their functions properly alone.Cytokinins can play an active role in shoot formation when they work together with auxin.Shoot formation and differentiation occur if there is an interaction between auxin and cytokinin (Widyastuti and Tjokrokusumo 2007).Natural growth regulators play an important role in controlling biological processes in plant tissues(Gaba 2005; Davies 2010).The role of growth regulators, among others, regulates the growth rate of each tissue.The activity of growth regulators in growth depends on the type, chemical structure, concentration, plant genotype and plant physiological phase(Dodds and Roberts 1982;George 1993;Satyavathi et al. 2004).obtained a value of 26-28 o C, which is considered optimal to support the growth of C. racemosa; according toGao et al. (2018), the optimal temperature for the growth of C. racemosa is 26 o C-30 o C this is because at that temperature C. racemosa will form new stolon growth.The salinity value obtained during the maintenance period was 30-32 ppt.The salinity range suitable for the growth of Caulerpa sp. is 25-30 ppt(Bambaranda et al. 2019; Hui et al. 2015), a relatively high growth rate is in the salinity range of 29-37 ppt (Rabia 2016) and will not experience growth if the salinity is below 25 ppt (Mosquera et al. 2016; Rabia 2016).The measurements in the rearing pond showed that the average dissolved oxygen value obtained was 5.3-5.83mg/L.This value follows the opinion of Sunaryo et al. (2015) that the value of dissolved oxygen in the cultivation of Caulerpa sp. is preferably more than 3.5 mg/L.The pH measured during the study was 7-8, where the range was still suitable for the growth of C. racemosa.Illustrisimo et al. (2013) stated that the pH 7-8.3 growth of Caulerpa sp. has increased, Susilowati et al. (2012) said that alkaline waters(7)(8)(9)are productive waters and play a role in encouraging the process of changing organic matter in water into minerals that can be assimilated by phytoplankton.
Caulerpa spp is native to warm tropical waters and is relatively easy to propagate.Several factors in uence Caulerpa spp growth, including age, phenotype, genotype, salinity, temperature, nutrient availability, and light (Mosquera and Salamanca 2016; Kasim et al. 2017; Aris et al. 2021).The results of temperature measurements