Growth of Eucheuma cottonii seaweed from tissue culture with different time acclimatization in Hurun Bay

. Constraints on the seaweed culture in Indonesia until now still rely on seeds obtained by vegetative propagation and have poor quality, which often causes a decrease in genetic variability. Planting seaweed seedlings from tissue cultures that have been acclimatized in a greenhouse laboratory is believed to improve the quality of the seaweed seeds. This research aimed to observe the growth of tissue culture acclimatized Eucheuma cottonii seaweed seedlings to different rearing times in greenhouses applied to floating raft cages. This study used a completely randomized design (CRD) with three treatments and three replications. Acclimatization treatment was conducted for 2 weeks, 4 weeks, and 6 weeks. Seaweed seedlings acclimatized in the greenhouse are reared on bamboo floating rafts in the sea, with an initial weight of 5 grams each. The length of maintenance at sea is 28 days. The resulting daily growth rate in two-week acclimatization was 2.92%, the four-week treatment was 2.93%, and the six-week treatment was 2.57%. in raising seedlings at sea, the highest daily growth rate value was in the four-week treatment of 7.25%, followed by the two-week treatment of 7.11% and the six-week treatment of 6.43%.


Introduction
Seaweed culture has an important role in increasing fishery production to fulfill food and nutrition needs, fulfill domestic and foreign market needs, expand employment opportunities, improve the well-being of fishermen and fish farmers, and maintain the sustainability of aquatic biological resources [1].The success of seaweed culture is inseparable from several factors, such as the environment, seed quality, the method used, the availability of nutrients, and the initial density or weight in maintenance.Using superior seaweed seeds is expected to provide a good harvest and high production [2].
The bottom of the bay waters in the Southwest and South are generally sloping with a depth of less than 5 m, and the northern part is covered by mangrove forest, while the southern part has several traditional floating net cages.Hurun Bay is a place that has the potential to produce seaweed.
One of the common Rhodophyceae species cultured by seaweed farmers is Eucheuma cottonii.According to [3], E. cottonii is also called Kappaphycus alvarezii because the carrageenan produced is included in the kappa-carrageenan fraction.However, E. cottonii is generally better known and commonly used in national and international trade.In general, E. cottonii grows well in coastal areas (reef).
Typical habitats are areas with a steady flow of seawater, small daily temperature variations, and dead coral substrate [1].Furthermore, [2] stated that the water conditions suitable for the culture of Eucheuma cottonii seaweed are waters protected from wind and large waves, water depth 7.65-9.72m, salinity 33-35 ppt, sea water temperature 28-30 °C, brightness 2.5-5.25 m, pH 6.5-7, and current speed 22-48 cm/second.
The application of tissue culture techniques to seaweed appeared in the 1970s to respond to market needs and seaweed culture practices [4].Tissue culture is a method of isolating parts of plants (in seaweed, it is thallus) and growing them under aseptic conditions (free from infection) in closed containers, so that these parts can multiply and regenerate into complete plants again like their parents [5].This method is considered fast and effective in seaweed culture because it does not require land for nurseries [6].
Tissue culture is a technique for growing plant parts in the form of cells, tissues or organs in aseptic conditions in vitro, which is characterized by aseptic culture conditions, the use of artificial media containing complete nutrients, Growth Regulatory Substances (ZPT) and the conditions of the culture room, [7] controlled temperature and lighting, in contrast to conventional vegetative propagation techniques, tissue culture involves the separation of biological components and a high level of control in spurring the process of tissue regeneration and development.Each process sequence can be manipulated by selecting plant material, culture medium, and environmental factors, including eliminating microorganisms such as fungi and bacteria.This is intended to maximize the final product in the form of quantity and quality of propagules based on the principle of cell totipotency.Plant propagation by tissue culture has the following advantages: 1.To propagate certain plants that are difficult or very slow to propagate conventionally.Plant propagation by tissue culture offers an excellent opportunity to produce many plant seeds in a relatively short time, so that it is more economical.2. Plant propagation by tissue culture does not require a large area.3. Plant propagation techniques by tissue culture can be carried out throughout the year without depending on the season.4. The seeds produced are healthier.5. Allows for genetic manipulation [7].
In plant propagation by tissue culture, explants are an important factor in determining success.Explants are plant parts that are used as material for the initiation of a culture.Physiological age, ontogenetic age, explant size, and plant parts taken must be considered when selecting explants to be used as starting material for culture.Generally, plant parts used as explants are young tissues actively growing.Young plant tissue has a higher regeneration power, cells are still actively dividing, and are relatively cleaner (containing fewer contaminants) [7].According to [5], explants must be free of contaminants.Contaminants must be removed without killing plant cells.Clean material is reduced to a specific size.This size must be larger than the planned explant size.Examples of explants include embryos, stems, shoots, applical meristems, leaf tissues, seeds, root tips, calluses, single cells, and pollen.
The culture environment results from interactions between plant materials, culture containers, and the culture space's external environment, which greatly influences a tissue culture system.Theoretically, all variables in each culture container in the same rooms are uniform.Consequently, the same thing happens in culture containers in other culture spaces.For uniform culture growth, uniformity of environmental factors must be sought, not only in the culture room but also in all culture containers by using uniform containers [8].Several studies on tissue culture methods on E. cottonii have been carried out, including using growth regulators (ZPT), various types of fertilizers, photoperiods, and different containers [9].
This study was conducted to determine the effect of the growth of E. cottonii seaweed from tissue culture at bamboo floating rafts in the sea that had been acclimatized to different times in a greenhouse laboratory.The research is expected to be used as a reference material in producing excellent and sustainable tissue culture seaweed seedlings from a greenhouse laboratory scale to a marine aquaculture scale.

Material and Method
Research were carried out for six weeks in the greenhouse laboratory and one month in a bamboo floating raft of the sea in Hurun Bay Fig 1 .using RAL (randomized design) 3 treatments and 3 replications.The treatment used was acclimatization treatment of young thallus seedlings resulting from tissue culture, namely 2 weeks acclimatization (treatment 1), 4 weeks acclimatization (treatment 2), and 6 weeks acclimatization (treatment 3).The container used is an aquarium (10-liter volume), which has been sterilized as many as 9 pieces.The seawater used is seawater that has been sterilized and added with 5 ml/l PES media with the frequency of once a week.The tissue culture of E. cottonii seaweed seeds came from the Tissue Culture Laboratory at the Center for Marine Aquaculture Lampung, and the seeds were cleaned of adhering dirt, weighed 5 grams each per treatment, and then rinsed repeatedly with sterile seawater.Tissue culture seeds were put into an aquarium container, which had been filled with sterile seawater plus PES media.Thallus was reared for 2 weeks, 4 weeks, and 6 weeks according to different acclimatization treatments in a greenhouse laboratory, and then the seedlings were reared on a bamboo floating raft in the sea for one month.Growth weight absolute is known by Weighing the seeds at the start of research and end of the study with analytical scales (accuracy 0.0001g).
Growth weight absolute can count with equation [10]  The growth rate daily can count with the formula [10]: G = Growth rate per day (%) W0 = initial weight (grams) Wt = Weight at moment measurement (grams) t = Research time (day i) The variables of water quality measured during the study were temperature, acidity degrees (pH), oxygen dissolved, salinity, ammonia, and nitrites.
Obtained data during research and conducted an ANOVA test with a level of 5% confidence.Then the data is declared no own influence real when the sig value > 0.05, and the data is stated as influential real when the sig value < 0.05.The results obtained are then processed using the SPSS application and Microsoft Office software to make it easier to process the ANOVA test data of Excel 2007.

Result and Discussion
The result of absolute growth rate, daily growth rate (DGR), weight gain, and total harvest of E. cotonii seaweed from tissue culture with different acclimatization in Hurun Bay (Table 1) Table 1 shows that according to the findings, the 4-week treatment had the best Weight gain (30.5 g), with no significant difference (P>0.05) from treatment 2-week (29.2 g), but a significant difference (P<0.05) from treatment 6-week (23,6 g).The same trend was seen in the Absolute growth rate parameter, which indicated a significant difference (P<0.05) between treatment 6-week (0,84 g) with treatment 2-week (1,04 g) and 4-week (1,09 g), but no significant difference (P>0.05) between 2-week treatment and 4-week treatment.Observation of seaweed growth rate tissue culture seaweed that was acclimatized in the greenhouse laboratory experienced growth starting from the 1st week of cultivation with the characteristics of increasing the length of the thallus tip and the growth of young thallus.The black thallus changes to brown when it enters the second week.The growth of the thallus begins to be clearly visible from 3 weeks to 4 weeks.At 6 weeks, many thallus branches are visible.The growth of seaweed that has been acclimatized becomes more visible after being reared on floating rafts at sea.According to [20], plant tissue is generally formed due to cell division activity.One cell undergoes a continuous process of division.From one cell, it becomes two cells, and so on.According to [17], the average length of seaweed shoots will increase with increasing rearing time.
Based on the sampling of test E. cottonii seaweed from tissue culture during the research activities, the weight growth of tissue culture seedlings that have been acclimatized and reared on floating rafts at sea, data presented in Fig. 2 Fig. 2. Graph of thallus weight growth of tissue culture that have been acclimatized and reared on floating rafts at sea.From Fig. 2, the highest weight growth of tissue culture seedlings that have been acclimatized and reared on floating rafts at sea was in the 4-week treatment at 35,5 g, followed by the 2-week treatment at 34,2 g, and the lowest was the 6-week treatment at 28,65 g.Acclimatization for 4 weeks in the greenhouse is sufficient acclimatization so that the seedlings can grow and develop in the sea because the thallus is strong enough and the thallus branches have grown a lot, so it doesn't take long to adapt and grow.At 2 weeks of acclimatization, the thallus is still too young to be kept in the sea because it breaks easily and is weak, thus inhibiting growth, whereas at 6 weeks of acclimatization treatment, there may be a lack of nutrition in the aquarium container.so that the growth of the thallus to grow and develop becomes slow.According to [18], the first week to the third week is a phase of vegetative growth, where the plant is at that time carry out the growth of tissue cells mature so that growth is obtained the weight is getting bigger, entering the fourth week to six weeks Eucheuma cottonii seaweed growth rate begins to decline.The reason for decreased growth is thought to be due to starting in the sixth week of cells meristematic has reached phase level mature to vegetative growth progresses very slowly and is replaced by generative growth, namely the formation of reproductive organs for the need for plant regeneration, the consequences growth rate decreases.
Based on the sampling of test E. cottonii seaweed from tissue culture during the research activities, the daily growth rate (DGR) of tissue culture seedlings that have been acclimatized and reared in the greenhouse, data presented in Figure 3.In Fig. 3, The tissue culture seaweed seeds that were maintained in the greenhouse, experienced different daily growth rates.The highest daily growth rate was at 4 weeks of acclimatization, was 2,93%, followed by 2 weeks of acclimatization, was 2,92%, and the lowest at 6 weeks of acclimatization, was 2,57%.The result obtained on treatment was still within the appropriate daily growth range.According to [20], the daily growth of seaweed ranges from 2% -3% per day; this depends on the supply of sunlight, climate, and geographical conditions of the waters that are measured by somatic growth and are expressed as weight growth and length growth of thallus.
Based on the sampling of test E. cottonii seaweed from tissue culture during the research activities, the daily growth rate (DGR) of tissue culture seedlings that have been acclimatized and reared on floating rafts at sea data presented in Fig. 4 Fig. 4. Graph of the daily growth rate of acclimatized tissue culture seedlings for 2 weeks, 4 weeks, and 6 weeks on floating rafts at sea In Fig. 4, the tissue culture seaweed seeds acclimatized and reared on floating rafts at sea had different daily growth rates.The highest daily growth rate was at 4 weeks of acclimatization, which was 3,51%, followed by 2 weeks of acclimatization, which was 3,47%, and the lowest at 6 weeks of acclimatization, which was 3,27%.The daily growth rate of acclimatized seaweed maintained on floating rafts in the sea shows good daily growth according to the standard daily growth rate of seaweed.Based on daily growth results in tissue culture seaweed maintained on floating rafts at sea, It is known that the daily growth value of seaweed during the research experienced growth well according to quality standards.according to [18] the rate of good daily growth for grass marine Eucheuma cottonii is no less than 3% per day Based on the observations that have been made, the water quality test results are presented in following Table 2.In Table 2, the temperature obtained from the measurement results for each treatment during the study ranged from 28.6-29.6°C.The temperature range is sufficient to support the growth of tissue culture E. cottonii seaweed.The results of the study's dissolved oxygen (DO) measurements were 4.77-5.31mg/L.The DO content is still within the optimum limit for seaweed growth.[3], stated that for the growth of E. cottonii seaweed, 2-4 ppm of dissolved oxygen in the water is needed, but growth is better if dissolved oxygen is above 4 ppm.According to [14], DO content of 2.18 and 2.84 has supported seaweed growth.Salinity during the study was in the range of 32-34 ppt.According to [1], a good range of salinity for E. cottonii seaweed is around 30-37 ppt.The pH measurement results ranged from 7.77 to 8.09.This range is still within the limits suitable for seaweed growth.[11] stated that pH 8.5 positively responded to an increase in the number of E. cottonii medulla cells.[1] states that the maximum pH range for seaweed life is 6-9, with an optimum range of 6.8-8.2.Ammonia levels during the study ranged from 0.112-0.366ppm, and nitrite content ranged from 0.04-0.09ppm.The range of ammonia and nitrite at the beginning of the rearing was still below the seawater quality standard, but after one month or at the end of the maintenance, the ammonia and nitrite levels were above the seawater quality standard, but this did not interfere with thallus growth.The results [19] study on Kappaphycus alverezii culture showed that the ammonia content from day 0 (zero) to day 15 of the rearing period, the fluctuations were very small then decreased on the 30 th day of rearing and increased at the end of the rearing period, but still lower than the seawater quality standard for marine biota, which is 0.3 ppm.According to [15], the nitrite content of 0.06 mg/L is still below the threshold for aquaculture activities and can still be tolerated by seaweed.
Different acclimatization times significantly affected the growth of the absolute weight of Eucheuma cottonii tissue culture seaweed reared in a greenhouse laboratory aquarium.The longer the acclimatization time, the lower the thallus weight growth and daily growth rate.Four weeks of acclimatization is the acclimatization that gives the best growth of thallus weight and daily growth rate.

Fig. 3 .
Fig. 3. Graph of the daily growth rate of acclimatized tissue culture seedlings for 2 weeks, 4 weeks, and 6 weeks in the greenhouse

Table 1 .
Absolute growth rate, Daily growth rate (DGR), weight gain, and total harvest of Eucheuma cotonii seaweed from tissue culture with different acclimatization in Bay.

Table 2 .
Water quality in the rearing of seaweed seedlings with different acclimatization