The effect of parica seed water immersion time on the success of parica seed initiation (Schizolobium amazonicum Huber ex Ducke)

Parica culture has the potential to overcome problems in parica seed germination. One factor that influences the successful initiation of parica culture is the process of sterilization and pre-treatment in the form of breaking seed dormancy. This paper aims to analyze the effect of the immersion time of parica seeds on the germination rate of parica seeds. The explants used were parica seeds with a size of ± 2 cm. Seeds are given pre-treatment, the seeds are immersed in hot water at around 70°C for 10 minutes, the immersion time in hot water is 3, 6, 12, and 24 hours according to treatment. The variables observed were the first time the parica seeds germinated, the percentage of germinated seeds, the percentage of contamination (fungi, bacteria), and the percentage of browning. The results showed that the immersion time for pre-treatment affected the success of parica seed initiation, especially seed germination. The 3-hour immersion treatment used resulted in the highest germination of parica seeds (55%) and significantly differed from the germination rates in other immersion times. Based on the high germination rate and low level of contamination, this becomes prospectively to provide optimal in vitro parica germination.


Introduction
Vegetative and generative propagation by tissue culture in Indonesia has yet to be widely carried out.Tissue culture is a method of plant propagation using aseptic principles by isolating or initiating explants such as cells, tissues, or organs in the room being repaired.Plant parts or explants will be planted in aseptic media containing macro and micronutrients, vitamins, and PGR to help plants regenerate and multiply to become complete individuals.Propagation by tissue culture can produce seeds in large numbers without requiring many mother plant and in a relatively short time.The tissue culture technique was initially used for fast plant propagation, but now tissue culture is also used to support plant character improvement programs.
Parica culture has the potential to overcome problems in parica seed germination.Many factors influence the success of parica culture.One of the factors that influence the successful initiation of parica culture is the process of sterilization and breaking of seed dormancy.The dormancy breaking process carried out at the time of sterilization can determine the length of time the parica seeds that are cultured germinate.The obstacle in the sterilization process is contamination.If contamination occurs, culture 1315 (2024) 012075 IOP Publishing doi:10.1088/1755-1315/1315/1/012075 2 death can occur.Therefore, sterilization needs to be done for parica seeds that will be planted aseptically to minimize contamination of the culture.It is necessary to know the proper sterilization method to obtain highly sterile parica seed cultures.
Parica seeds or seeds have a flat, oval shape, hard and water-resistant skin, and are included in physical dormancy.An effective way to increase parica germination is high and uniform by using sulfuric acid, hot water, and mechanical scarification using sandpaper [3].Parica seeds in nature have low germination and a long germination time.Several methods have been used to overcome seed dormancy.The long germination time is strong evidence that parica seeds have physical dormancy [2].This study aimed to analyze the effect of immersing parica seeds in water on initiation success, precisely to determine the duration of water immersion during the dormancy-breaking process.This research is expected to increase the success of the initiation of explants in the form of parica seeds.

Methodology 2.1 Time and Place
This research was conducted from September 2022 to November 2022.The laboratory used was the Esha Flora laboratory which is located at Jalan Kemuning 6 No. 9, Taman Cimanggu, Kedung Waringin Village, Tanah Sareal District, Bogor City.

Tool sterilization
Tool sterilization activities include sterilization of glassware equipment, sterilization for planting, and LAF (laminar air flow) sterilization.The sterilization of planting equipment includes sterilizing scalpel knives, tweezers and scissors, while the sterilizing glassware includes the sterilization of petri dishes and bottles.
Glassware and planting equipment are washed first.Planting equipment and petri dishes before being put into the autoclave are wrapped using newspaper.Planting equipment and petri dishes wrapped in newsprint are given information to determine the type of blade to use such as P4 for scalpel no.4 with blade pair number 23. Equipment that has been given a description, is sprayed with 70% alcohol.The glassware that has been washed is then put into the autoclave, wet-sterilized at 121°C with a pressure of 1 atm for 1 hour.The sterilized bottles are then put into plastic sprayed with alcohol and arranged.Tools that have been sterilized are placed in a storage area so they are not exposed to dust.
Contaminated media is disposed of in the waste plastic provided.Bottles cleaned of contamination are then immersed in water containing detergent and sodium hypochlorite for approximately 30 minutes.After immersing, the bottle is taken to be rubbed and cleaned on the surface and bottom of the bottle using wood that has been given a sponge at the end.Then, the bottle is rubbed clean and no stains are left on either the bottom of the bottle or the mouth of the bottle.Bottles that have been rubbed, then rinsed using running water to remove the foam.
Bottles that have been washed clean, and stored in a boiler to be sterilized in an autoclave at 121°C and 1 atm pressure for ± 1 hour.After 1 hour, the fire was turned off and allowed to stand for a few moments until the autoclave temperature dropped and then the pot containing the bottles was removed using heat-resistant gloves.The bottle is put into a sterile plastic with 70% alcohol.The bottles are neatly arranged, and then the plastic ends are tied with rubber so that no air enters.The plastic containing the sterile bottles is placed on the shelf provided.

Media preparation
Making 1 L of MS media was done by adding 4.43 g/L of MS, 1.5 g/L of activated charcoal and 25 g/L of sugar.These ingredients are mixed in a plastic measuring cup and then added with distilled water until it reaches 1 liter and stirred until evenly distributed.The stirred solution is then measured for its pH to make sure the pH is already worth 6, if the pH has been measured then the solution is given as much as 6 g/L of agar.The solution is heated using a stove over medium heat until it boils.The solution was removed when it was boiling, poured into a sterile bottle, and closed not too tightly.
Bottles filled with media are arranged in a chamber to be sterilized in an autoclave at 121 °C, 1 atm pressure for approximately 30 minutes.The boiler was removed using gloves after 30 minutes.The bottle containing the media is tied back using rubber so that no air enters.Bottles containing media are arranged in plastic sprayed with 70% alcohol, the plastic is tied using rubber.Bottles containing media are then stored in the media storage room.

Sterilization process and initiation of explants
The explants used were parica seeds.The seeds are ± 2 cm long.The seeds have dark brown skin with a smooth surface and are difficult to peel.Seeds are given pre-treatment, seeds are immersed in hot water with a temperature of around 70°C for 10 minutes, immersed in water for 3 to 24 hours according to treatment.The explants in this treatment were not given an external sterilization stage, only immersed in water, and entered the internal sterilization stage.The immersed seed explants were then placed in LAF to be rinsed 2 times using sterile water for 3 minutes.The explants were immersed in 15% sodium hypochlorite for 7 minutes, then immersed in 5% sodium hypochlorite for 7 minutes while gently shaking.Rinsing with sterile water was repeated 2 times with 5 minutes for each rinse.

Planting
The sterilized explants were then planted into the media using tweezers.The tweezers are burned over the bunsen until warm.The rim near the bottle cap was burnt.The bottle is closed again with plastic and rubber.

Maintenance, Observation and Data collection
Culture observation was carried out for 30 days (one month) by observing its daily growth.Observation and data collection was carried out for 30 days.The variables observed were the first time the parica seeds germinated, the percentage of germinated seeds, the percentage of contamination (fungi, bacteria), and the percentage of browning.
Germination of sterile seeds (%).Measurement of germination of sterile seeds is obtained from the comparison results of sterile seed cultures that germinate with seed cultures that are planted according to the following formula: Germination = germinated seeds total seeds

𝑥 100%
Fungal contamination (%).Measurement of the percentage of cultures contaminated with fungi was obtained from the results of comparing cultures contaminated with fungi with all cultures according to the following formula: The experiment was performed in 5 repetitions and each consisting of 8 bottles.Each bottle contains 1 parica seed so there are 160 units of observation.
Data analysis was carried out in 2 ways, namely qualitatively and quantitatively.Qualitative data analysis, namely by descriptive; quantitatively, the data were analyzed with formulas, tables and graphs.Data obtained from the observations, tabulated and analyzed using the ANOVA test using the help of the IBM Statistics SPSS 26 program to determine whether there is a treatment effect on the observed variables.If the P-value > α (0.05), then the treatment does not significantly affect the observed variables.If the P-value < α (0.05), then the treatment significantly affects the observed variables.Differences between treatments were identified through a follow-up test with Least Significant Differences (LSD) at an error level of 5%.

The effect of parica seed immersion time on the success of parica seed pre-treatment
Parica seed immersing time affects the success of parica seed pretreatment, so the hypothesis is accepted.The immersion time of the seeds applied also affects the germination of the seeds.The overall results showed that germinated parica seeds were higher than contaminated seeds, but not higher than nongerminated seeds (Figure 1).Immersing the seeds using hot water is one method used to increase the germination percentage in parica seeds.Hot water can break the physical dormancy in leguminosae through stress so that the palisade breaks or damages the strophiolar cap [5].Seed germination process depends on internal conditions, namely the seeds internal conditions and external factors, namely the water, temperature, oxygen, light and medium.Water plays a role in softening the seed coat, facilitating the entry of O2, and transporting food.Fast germination affects initiation success.
The percentage of germination can influence the success of seed pre-treatment.Several factors influence germination.The immertion time for seed pre-treatment affects the success of parica seed initiation, especially seed germination.The percentage of parica that germinated was close to half of the total planted.Based on the results of the study [4] the percentage of parica germination in the treatment in the form of immersing in hot water for 10 minutes resulted in a value of 32%, while if parica was not treated, only 3% percent of parica germination.Seed immersing time resulted in differences in the percentage of sprouts.The long immersing treatment significantly affected the vigour growth rate parameter.
The germination process of parica seed explants was marked by the radicle's emergence on the culture medium's surface.At the beginning of germination it only looks like a slight white bulge (Figure 2a), but then continues to grow in length and is increasingly lifted to the surface of the media (Figure 2b).After that, the cotyledons will lift and divide into 2 (Figures 2c and 2d).The growing cotyledons are light green and the growing plumule is greenish white.The sterilization method (A1) used resulted in the highest parica seed germination (55%) (Figure 3).Observations showed that parica seed germination had begun in the first week after planting.Immersing the seeds before germinating is one way to speed the germination process.Immersing for 3 hours has the lowest percentage of contamination.Immersing for 3 hours has the highest germination percentage.The germination rate in 3 hours of immersion significantly differed from other immersions.[8] The best immersing time for seeds is characterized by the highest germination capacity because the time for water absorption is maximum enough to germinate properly.One of the advantages of a high germination percentage (3 hours of immersing) is that growing cultures can be saved from contamination by acclimatization or used as a source of culture explants by saving the top part that has not been contaminated so that the more that germinate, the higher the culture or parica planting material.Generated either through the salvage process or not.
Immersing for 6 hours and immersing for 12 hours had a reduced composition of germinated seeds and non-germinated seeds.Immersing for 24 hours had the lowest proportion of germinated ore and the highest proportion of ungerminated ore caused by reduced endogenous hormones due to being dissolved in the immersing water.In addition, immersing for too long can cause physiological damage to the seeds.Immersing too long can cause oxygen loss or anoxia inhibiting seed respiration and germination [7].
The results showed that most germinated parica seeds produced normal sprouts and only a few grown abnormal sprouts (3.33%).The criteria for abnormal sprouts were that some hypocotyl and cotyledon colors turned brown and the parica seed coat did not come off (Figure 4).The split test results that most of the parica seeds that did not germinate produced normal sprouts.The parica seeds that were split tested still showed viable seeds with the characteristics of the seeds being light green and having embryos that were still white (Figure 5).Parica seed cultures where the seed size did not change (no imbibition process) and the seeds remained sterile were found in each treatment.At the end of the observation, the percentage of parica seed cultures did not germinate was 51.25% (Figure 6).Parica seeds that do not germinate are seeds that do not or have not undergone the germination process.Seeds that do not germinate can occur because the imbibition process is not optimal or does not even occur.The imbibition process does not run optimally or even does not occur because the seed coat is difficult to penetrate by water.Seeds that do not germinate can also occur due to a lack of substances that stimulate germination such as endogenous hormones.The immersing process that is carried out can result in leaching or washing of substances that can help germination, which is indicated by the color change that occurs in the immersing water to become brownish.The leaching process resulted in a decrease in the percentage of germinating cultures.Seed physiological factors also affect seed germination.Physiologically immature seeds can result in seeds not germinating because food reserves are insufficient and the state of the embryo is not perfect [10].Seeds that do not germinate but bulge are caused by water absorption but cannot push the seed coat until it breaks.[6] Water absorption is the initial process of seed germination, followed by softening of the seed coat and seed development.The seed coat carries out water absorption through imbibition and osmosis processes.This process causes swelling of the embryonic structure and endosperm.The swelling process forces the softened seed coat to crack and opens the roots to emerge [9].

The effect of parica seed immersion time on parica contamination
All treatments of the sterilization method used in this study resulted in a low level of contamination (Figure 7).The percentage of contamination does not exceed 8% of each treatment.The highest percentage of contamination was found in treatment A4.The causes of contamination are fungi and bacteria.Fungal contamination occurs more frequently than bacterial contamination.The contamination in treatments 3 and 4 was caused by a fungus marked by grayish-white hyphae that covered the outside of the seed and the media (Figure 8a).Bacterial contamination in the treatment was caused by bacteria with signs in the form of whitish slime spots (Figure 8b).The long process of immersing time has no significant effect on the contamination percentage.However, the germination process can result in opportunities for endophytic microbes to emerge from the seeds.These endophytic microbes can invade parica growths.Endophytic fungi are fungi that can associate with plant tissues and do not cause harm to plants [1].Fungi mainly cause contamination that occurs.The fungi found had the same characteristics in all cultures contaminated with the fungus.These same characteristics indicate that there are fungi that specifically contaminate parica.The fungi found in parica are also indicated as endophytic fungi because they are found in the body parts of the parica culture still, no fungi are found in the culture media.

The effect of parica seed immersion time on parica browning
The release of secondary metabolites causes the browning that occurs.Anticipation of browning is done by adding activated charcoal to the media.Activated charcoal was used to absorb secondary metabolites produced by parica so no browning was found in this study.Activated charcoal is also known to eliminate the adverse effects of phenol oxidation on the growth of Disa spp.[11].Giving activated charcoal in the media can also help root growth by reducing the intensity of light entering the media so that it can stimulate endogenous growth substances to work.[12].

Conclusion
The immersing time of parica seeds affects the percentage of germination.Germination of parica seeds affects the success of initiation.Immersing seeds in water for 3 hours had the highest germination percentage.Immersing for too long can inhibit seed respiration and germination.The level of contamination is not affected by the immersion time.Contamination that occurs is the contamination of endophytic fungi.The use of activated charcoal prevents browning.b a Fungus contamination =fungus contaminated cultures whole cultures 100%Bacterial contamination (%).Measurement of the percentage of bacteria-contaminated cultures is obtained from the results of comparisons of bacteria-contaminated cultures with all cultures according to the following formula: ).The measurement of the percentage of browning is obtained from the results of a comparison of the culture affected by browning with the whole culture according to the following formula: Design and Data Analysis Research topic 1 used a completely randomized design (CRD) with 1 factor, namely the duration of water immersion.The long water immersion treatment consisted of 4 levels, namely: (1) Duration of 3 hours (A1), (2) Duration of 6 hours (A2), (3) Duration of 12 hours (A3), (4) Duration of 24 hours, (A4).

Figure 1 .
Figure 1.The effect of parica seed immersion time on the success of parica seed pre-treatment

Figure 4 .
Figure 4. Abnormal sprouts in parica seed culture in the form of the cotyledons which are not separated from the parica seed coat.

Figure 5 .
Figure 5. Results of the seed split test that did not germinate (-= 1 cm).

Figure 6 .
Figure 6.Percentage of parica seeds that did not germinate in all treatment parica seed water immersion time at 2 months of age.A1 = Duration of 3 hours, A2 = Duration of 6 hours, A3 = Duration of 12 hours, A4 = Duration of 24 hours.

Figure 7 .
Figure 7. Contamination that occurs in all treatment of parica seed water immersion time.A1 = Duration of 3 hours, A2 = Duration of 6 hours, A3 = Duration of 12 hours, A4 = Duration of 24 hours.