Effect of Dissolved Oxygen and Chemical Scarification on Andrographis paniculata Seed Germination in Macrobubble Conditions

Andrographis paniculata is used in Tai traditional medicine. Tis plant contains a bitter compound called andrographolide, which is highly efective in the prevention of many diseases. It is an efective treatment for infectious diseases and has a prophylactic efect owing to its powerful immunity-boosting benefts. Recently, it has been widely used to treat COVID-19. However, commercial planting of A. paniculata is performed by seeding, which leads to seed germination problems. Te seed germination is relatively low and not efcient under normal conditions for various reasons, such as a combined dormancy of physical and innate nature, the diversity of the seeds in diferent lots, and the fact that the germination duration was not uniform in the same lot. An easily applied and inexpensive method for farmers to develop mass plantings to stimulate germination is by using macrobubble conditions by aerating seeds in sterile water in collaboration with chemical scarifcation, which is the idea of creating a hard seed coat that causes seed dormancy to break while root germination occurs at 25 ° C. Germination was completed after 16days. Te dissolved oxygen (DO) concentrations in this environment were 5, 6, 7, 8, and 9 mg · L − 1 . Te oxygen intensity of 9mg · L − 1 showed the highest germination percentage (26.33%). It was found to be optimal for macrobubble conditions. Seedlings were treated with chemicals (PEG, NaCl, H 2 SO 4 , KCl, KNO 3 , NaHClO 3 , and GA 3 ) after soaking in macrobubbles with optimum DO. Te results showed that NaHClO 3 conc. (30min) showed a generation percentage reaching 92%, which could greatly promote up to 3.63 folds compared with the control in the macrobubble aeration system.


Introduction
Andrographis paniculata, also known as Fah Talai Jone, is a popular traditional Tai medicine. Tis plant belongs to the family Acanthaceae and is widely used in medicinal and pharmaceutical applications. It is generally known as "the King of Bitters," and its secondary metabolite, andrographolide (AG), has various medical applications [1,2]. In Asia, America, and Africa, A. paniculata has been used for centuries to cure a variety of diseases, such as diabetes, high blood pressure, cancer, ulcers, leprosy, dysentery, dyspepsia, fatulence, and malaria [2]. Andrographolide is a major active constituent of this plant, and it has a lot of diferent biological activities. Some of these activities are antiinfammatory, antibacterial, antitumor, antidiabetic, antimalarial, hepatoprotective, and antiviral for HIV [3,4]. COVID-19 has recently emerged as a global health threat with a rapid global spread and high mortality rates. Terefore, it is critical to identify new treatments as soon as possible. According to the integrative medicine, some herbs can help cure COVID-19 when used in combination with traditional therapies. Tailand is afected by epidemics, and infected patients are being treated with herbs. For example, some patients are administered a diferent dose of A. paniculata than those prescribed for fever and sore throats [5]. Te COVID-19 therapeutic potential of these plants was chosen. Integrating Tai traditional medicine concepts with modern COVID-19 treatment mechanisms would almost certainly result in a more efective clinical treatment [6]. Because of its importance in the treatment of a variety of ailments, A. paniculata is in high demand owing to its potent immune-boosting abilities [7]. However, seed germination remains a signifcant issue because this plant has such a wide variety. Te seeds were very small and dormant between 5 and 6 months after dispersal, germinating at an extremely low rate. Despite germination issues, A. paniculata is commonly propagated through seeds, which may indicate the presence of physical and innate dormancy, a usual survival strategy of plants for the efective spread on this planet [8,9].
One fundamental issue with the production of this plant from seeds is its destitute seed germination execution. Under normal conditions, the germination percentage and germination rate of this seed are generally poor [8,10]. Te dormancy of A. paniculata seeds is primarily ascribed to the hard seed coat. Te seed coat secures the embryo and its environment from water and any outside dangers. It is a physical obstruction that actuates the seed dormancy [9]. Te seeds of A. paniculata contain mainly alkaloids, saponins, and monounsaturated fatty acids. Seed storage is a big problem in countries with high temperatures and humidity. Tis can cause seeds to age quickly, which can make them less viable [11]. Seed dormancy can take the form of physical, morphological, physiological, morphophysiological, or a combination of physiological and physical both [12]. Seed dormancy is an internal state that prevents seeds from germinating, even under ideal temperature and gaseous and hydric conditions [13]. Dormancy is a feature of plant seeds that prevents germination and must be overcome by exogenous stimuli [14].
For breaking seed dormancy, scarifcations using physical techniques or chemical agents can be applied to overcome dormancy. According to reports, sunfower seed priming had a substantial impact on increasing germination percent, germination speed, and seedling dry weight and had the opposite efect in drought conditions, producing anomalous seedling decrement [15]. Several crops, including maize, wheat, rice, and canola, can beneft from seed priming treatments that improve seed germination and establishment [16,17]. Some plant seed dormancy is susceptible to chemical agents, for example, potassium nitrate (KNO 3 ), plant growth regulators, gibberellic acid (GA 3 ), and osmotic solutions such as polyethylene glycol (PEG) and salt solutions such as sodium chloride (NaCl), sulphuric acid (H 2 SO 4 ), potassium chloride (KCl), and sodium hypochlorite (NaHClO 3 ) [9,18,19]. From early research, seed germination of Kalmegh (A. paniculata) was enhanced by PEG and NaCl [19]. A. paniculata seeds were soaked in GA 3 at 100 ppm for 4 hours, which showed noticeably better seed germination and feld emergence [11]. Te A. paniculata seeds showed the highest germination percentage of 57.20 at 20°C while utilizing the top of a paper substrate after being treated with 0.5% potassium nitrate (KNO 3 ) for 24 hours [20]. Seed germination of A. paniculata was observed to be enhanced by the 25% sulphuric acid (H 2 SO 4 ) pretreatment before planting [21]. Soaking the seeds in 1 and 2% KCl for 10 min was also quite efective in promoting seed germination of A. paniculata [9]. Te study of Kumari et al. [18] found that almost all of the seeds germinated; KNO 3 and GA 3 made the seeds germinate faster, and the seeds germinated quickly in the presence of NaHClO 3 .
Tere are insufcient scientifc reports available on the seed quality and germination of A. paniculata. As a result, the current study is required to generate data on the standardization of germination tests to improve germination [18]. Germination starts with the seed absorbing water, known as imbibition, and ends with the elongation of the embryonic axis. It is a complicated procedure in which the seed must physically recover from maturation drying, restart a sustained intensity of metabolism, complete the necessary cellular activities required for the embryo to emerge, and prepare for subsequent seedling development. Seeds require moisture, a suitable temperature, and an aerobic environment to germinate [22]. In A. paniculata, a temperature of 25°C was optimal for seed germination [23]. Some of the dissolved oxygen (DO) in the water is caused by stream turbulence, which occurs when air is trapped by moving water, resulting in the dissolution of oxygen into the water [24,25]. A high DO concentration has been maintained and enhanced using macrobubbles [26].
Tis study hypotheses that chemical and macrobubble aeration in a water system saturated with dissolved oxygen and moisture can activate seed germination. Our purpose was to fnd the basic system by using a normal air pump connected with a bubble air stone. It was the equipment that cost the lowest for breaking dormancy and improving the germination for commercial production of A. paniculata in small farming.

A. paniculata Seeds and Chemicals.
A. paniculata seeds were collected in October 2021 from the Experimental Garden of Walailak University (8°38′42.7″N 99°54′04.4″E). Te seeds were dehydrated at 25 ± 2°C for a week and kept in a zipper bag under ambient temperature control at 4°C until the experiment was conducted in November-December 2021. Tese seeds were selected using a light microscope, and the large seeds with a dark brown color were selected. All of the chemicals utilized in the treatments were of analytical grade.

Generation of Macrobubble Water for the Germination
Experiment. Te macrobubble system was set up in an Erlenmeyer fask with a capacity of 500 mL, flled with 300 mL of water, which was autoclaved for sterilisation at 121°C for 15 min (Autoclave SX-700, Tomy Seiko Co., Ltd., Japan). Air was pumped into the water by a pump (Twin Air Pump Magic-8800, A.S. Union UNION Co., Ltd., Tailand) and circulated through a perforated stone with continuous aeration to generate macrobubbles at 25°C to obtain "watercontaining macrobubbles." After the seed was germinated, the seedling will be moved to a Petri dish that is flled with 10 mL of autoclaved water and the water was changed every day until completion (Figure 1).

Oxygen Concentration for the Seed Germination Test.
A. paniculata seeds' germination was carried out using a completely randomised design (CRD). Germination tests were performed in three replicates with fve seed groups. Each sample consisted of 100 seeds. Te seeds were frst disinfected in 10% sodium hypochlorite for 30 seconds and then washed with sterile autoclaved water three times before being immersed in macrobubble water. One fask was flled with autoclaved water as the control, and the others were flled with diferent concentrations (6, 7, 8, and 9 mg·L −1 ) of DO under macrobubble conditions.

Study of Seed Germination after Chemical Treatment.
Eight parallel groups were prepared for each seed type to evaluate the efects of seven chemical treatments on daily germination, which were estimated by the percentage of germinated seed number to the total number of macrobubble conditions with optimum DO. Te chemical scarifcation method for breaking seed dormancy was based on soaking seeds with seven diferent chemical agents: 2% (v/v) sulphuric acid (H 2 SO 4 ) for 10 min, 2% (w/v) potassium chlorite (KCl) for 10 min, 10% polyethylene glycol (PEG6000) for 24 h, 25 mM sodium chloride (NaCl) for 24 h, 150 mM potassium nitrate (KNO 3 ) for 30 min, conc. sodium hypochlorite (NaHClO 3 ) for 30 min, and 200 ppm gibberellic acid (GA 3 ) for 30 min. Among all groups, the control group was treated without any chemicals. Each group had three replicates and each with 100 seeds. Tey were germinated in macrobubble water using a randomised laboratory design. After germination, the seedlings were separated and submerged in 10 mL of autoclaved water in a nonsterilised Petri dish. Te dishes were kept under natural light conditions at 25°C. During the test, every 24 h, the macrobubble water and autoclaved water were replaced.

Observations.
Seed germination was recorded daily, and seven germination parameters were evaluated. Te formulas of calculating the parameters for (1a) (germination percentage: GP) and (1b) (germination energy: GE) followed Kumar et al. [23], (1c) (germination index: GI) and (2) (germination rate index: GRI) followed Al-Mudaris [27], (3) (mean germination time: MnGT) followed Ellis [28], mean germination time is a measure of the rate and time-spread of germination, it is an index of germination speed [29] and the fnal germination time was estimated by the following formula (4) (maximum generation time: MxGT), while seedling secondary roots (SSR) and cotyledons (SR) were calculated from the mean of the number of seedlings that generate secondary roots and cotyledon, respectively [7].
where n1, n2,. . .n16 = No. of germinated seeds on the 1 st , 2 nd and following days to the 16th day, 16, 15,. . ., and 1 = weights of the number of germinated seeds on the 1 st , 2 nd and following days: where G1 = the germination percentage on 1 st day, G2 = the germination percentage at 2nd day, and Gi = the germination percentage at i day.
where n = No. of germinated seeds on each day, d = No. of days from the beginning of the test, and N = total No. of germinated seeds at the termination of the experiment. MxGT where X = No. of new germinated seeds on each day and f = No. of day after seeds germinated.

Data Analysis.
Analysis of variance (ANOVA) was performed on the results. Te comparison was followed Duncan's multiple range test (DMRT) and the diferences were reported at P < 0.05.

Efect of Diferent DO Concentrations on Germination
Percentage and Seed Germination Energy. Reducing the germination time and increasing the germination percentage of A. paniculata seeds are important goals in commercial cultivation. In addition to being essential for plant establishment in both natural and agricultural settings, seed germination is a critical stage in the life cycle of seeds and plants [30]. Te seeds quickly recover physiologically from maturation drying during germination, resume a sustained International Journal of Agronomy level of metabolism, fnish crucial cellular processes that enable the embryo to emerge, and become ready for following seedling growth [31]. According to a study by Liu et al. [32], nanobubble (NB) water can produce exogenous reactive oxygen species (ROS) ofering physiological promotion and oxidation efects of seed. However, stimulating seed germination in aerated water requires consideration of optimal ROS concentration because in high density of bubble was beyond their toxic threshold, and negative efects were shown on hypocotyl elongation and chlorophyll formation. In order to confrm whether water containing macrobubble can enhance physiological processes, a germination test is a suitable procedure. Using distilled water and water that had macrobubble created from each batch of distilled water, comparison experiments were conducted. We proved, to the best of our knowledge, that air macrobubbles encourage seed germination as well.
One hundred seeds were recorded by the maximum generation time within 16 days of seed culture. Following seed germination in under macrobubble aeration with the dissolved oxygen (DO) 6.0, 7.0, 8.0, and 9.0 mg.L −1 (Figures 2(a)-2(e)). Te control is nonmacrobubble aeration that DO 5.0 mg·L −1 . After germination at stage ii in volumetric fask, seedlings were transferred to Petri dishes for growth to stages ii-iv (Figure 2(f )). Figure 2(e) shows the highest amount of seed germination in dissolved oxygen 9.0 mg·L −1 while Figure 2(g) shows that the number of secondary seedling root (SSR) growth was accelerated by DO 9 and 8 mg·L −1 , making no signifcant diference followed by DO 7, 6, and the control, respectively. Figure 2(h) presents the number of seedlings with cotyledons (SC) under different kinds of DO concentration for germination. Te cotyledon numbers increase with exposure to most of the tested DO 7.0 mg·L −1 with no signifcant diference with DO 8.0. Treatment DO 7.0 mg·L −1 SC 5.5 seedlings is the frst treatment generation, followed by treatment DO 8.0 mg·L −1 SC 5.0, the second seedling, respectively (Table 1). So, this result indicates that increasing of the macrobubble aeration tended to increase the amount of seed germinations and the number of seeds that occur secondary root (SSR) increased correspondingly, while number of seedling cotyledon (SC) formation was not directly proportional to the macrobubble concentration, but the growth of cotyledons depends on the germination period by pregerminated seeds produce cotyledons before later-germinated seeds.
After 16 days of submersion in macrobubble, the highest germination percentage and germination energy of DO 9.0 mg·L −1 reached 26.33 and 6.58, respectively, as presented in Table 1, followed by DO 8, 7, 6, and control. Tus, DO 9.0 mg·L −1 signifcantly promoted seed germination compared to other treatments that had a higher germination percentage (GP) and germination energy (GE) than immersion seed in distilled water without aeration or control at 11.3 folds. As supported by the study of Chauhan et al. [7], seed germination can occur in any substrate that allows for enough aeration. More detailed fndings on fne bubbles have been reported in previous studies. It has been found that fne bubbles can add air and oxygen to the water needed for respiration to oxidize starch, fat, and other food reserves and induce metabolic activity in seeds [33]. Corresponds to the study of Purwanto et al. [34] that allows ultrafne bubble to be infused into liquids for extended periods of time that the application of water containing ultrafne bubbles has a positive efect on seed germination rate. Ultrafne bubble water treatment can also improve the germination of seeds with poor physiological quality [35]. Soaking seeds in bubbled water is one form of seed priming. Seed preparation by priming is an ancient, simple, and efective technique for improving germination rate and speed, achieving uniform plant stands, and improving yields in a variety of environmental conditions to improve low seed viability and vigor [36].
Four levels of dissolvable oxygen consistently promoted higher seed germination than the control treatment (Figure 3). After 3 days of submersion, seed germination started 3 folds faster than in the control treatment, which started germination after 9 days of submersion. After a week of submersion, germination percentage (GP) and germination energy (GE) (Figures 3(a) and 3(b)) were stable, and regeneration began to decline at DO 6, 7, and 8 mg·L −1 , but at 9 mg·L −1 , it was still increasing. According to the study of Vashisth and Nagarajan [37], the higher molecular mobility of the bulk and hydration water fractions, and the increased activity of germination-related enzymes (amylase, dehydrogenase, and protease), including the early hydration of the membrane, may all contribute to the earlier germination. Corresponding to the study in barley seeds, fne bubbles can promote seed bioactivity and activate germination-related enzymes, and high dissolved oxygen is important to increase the germination rate [38,39]. Moreover, fne bubbles signifcantly improved the germination rate of Chinese celery and sweet corn seeds compared with conventional distilled water. In addition, fne bubbles also extended the root length of sweet corn seedlings [40]. However, although in this experiment, the germination percentage of A. paniculata was highest at a dissolved oxygen concentration of 9 mg·L −1 , further studies are needed at higher DO concentrations to determine the optimum germination percentage.

Efect of Chemical Scarifcation for Seed Germination in
Macrobubble Aeration. One hundred A. paniculata seeds were soaked in solution to investigate the efects of chemical treatments, namely, GA 3 , NaHClO 3 , H 2 SO 4 , KNO 3 , KCl, PEG, and NaCl. Seeds respond diferently to chemical treatments. Te highest seed germination was observed with NaHClO 3 , followed by H 2 SO 4 and control, respectively, as shown in Figures 4(a), 4(c), and 4(d). But the number of seed germinations was reduced less than control by KNO 3 , KCL, GA 3 , PEG, and NaCl, respectively, as shown in Figures 4(b), 4(e)-4(h). We can verify this by the highest generation percentage of NaHClO 3 at 92%, generation energy at 23, germination energy in a week at 57.33%, the germination index at 897, and the germination rate index at 15.3, a signifcant diference from other treatments. Treatment with NaHClO 3 shows the mean germination time at 7.25 days; it isn't signifcantly longer than the control at 6.00 days or PEG at 5.72 days. (Table 2). Te results of this experiment were consistent with those reported by Kumari [18]: NaHClO 3 -treated seeds took on a green color, which had not been observed under laboratory conditions, and full germination was recorded in only 8 days in the nursery test. Tus, it confrms the role as a stimulatory agent.
Te high emergence and uniform seedling size required for agricultural production of A. paniculata are not always achieved due to vigor diferences among commercial seed lots. However, low vigor and a high mean germination time were caused by seed ageing, as indicated by mean germination time [41]. Te mean germination time is a suitable index for the vigor evaluation of seeds [42]. In order of seed strength and seed age, it was found that NaCl had the highest seed strength and the lowest seed age compared to NaH-ClO 3 . By the way, the seed sample from treatment NaHClO 3 was vigour and seed ageing show same level with control and PEG because mean germination time is not signifcant. Anyway, mean germination time from other treatments wasn't signifcantly diferent from control except for NaCl but NaCl wasn't signifcantly diferent from other treatments except for control and NaHClO 3 . So, it meant that the selected seeds were similar in age and vigor in all treatments.
For growth, development can be observed based on the number of seedlings that develop from secondary roots and cotyledons. Te secondary roots of the NaHClO 3 treatment showed the highest number, followed by H 2 SO 4 and the control, with no signifcant diference. Te cotyledons of the NaHClO 3 treatment showed the highest number and were no signifcantly diferent except PEG and NaCl (Table 2). According to the fndings of Yadav et al. [43], scarifcation increases germination rate, greater hypocotyl elongation, and radicle growth, and produced in a substantial reduction in the time for germination. Table 1: Comparison parameter of means and variation for germination of dissolved oxygen concentration treatment.

Dissolved oxygen (DO)
Germination percentage (GP ± SD) Germination energy (GE ± SD) No. of secondary seedling roots (SSR ± SD) No. of seedling cotyledon (SC ± SD) Control (NB) * 2.33 d ± 0.58 0.58 d ± 0.14 1.33 c ± 1.15 0.00 c ± 0.00 6 mg·L −1 6.67 c ± 0.58 1.67 c ± 0.14 5.00 bc ± 2.00 2.50 bc ± 0.71 7 mg·L −1 12.00 b ± 2.00 3.00 b ± 0.50 9.33 b ± 2.52 5.50 a ± 0.71 8 mg·L −1 14.33 b ± 2.08 3.58 b ± 0.52 10.33 ab ± 3.21 5.00 ab ± 1.41 9 mg·L −1 26.33 a ± 1.53 6.58 a ± 0.38 15.67 a ± 1.53 2.00 bc ± 0.00 * Control was conditional on nonmacrobubble aeration with dissolved oxygen (5.0 mg·L −1 ).  International Journal of Agronomy Te best indicator of the depth of dormancy is thought to be the germination index [44]. Moreover, high germination index values indicate great seed quality and homogeneity [45]. Te NaHClO 3 treatment showed the highest germination index and germination rate index, meaning that the seeds had a shorter dormancy and better quality than other treatments. Te germination index and germination rate index of sulfuric acid were comparable to those of the control but other treatments show lower than control (Figures 5(e) and 5(f )). So, treatment with H 2 SO 4 had no efect on seed dormancy destruction or seed quality improvement. On the other hand, other treatments tend to increase seed dormancy and decrease seed quality.

Control (NB
Most seeds remain dormancy related to the hard seed coat. It results in impermeable to water and gases. Dormancy in A. paniculata is infuenced by both physical properties and physiological mechanisms (unknown protein). Chemical treatments in A. paniculata seeds can reduce seed coat layer or dissolve some inhibitor proteins that might be benefcial in breaking seed dormancy and improving germination [9]. Tus, this experiment concluded that NaHClO 3 was able to destroy proteins on the hard seed coat, allowing water and oxygen to penetrate into the seed, thereby stimulating seed germination in macrobubble aeration systems that are full of enough dissolved oxygen and water for seed germination. According to the research of Coelho et al. [46], physical  scarifcation can speed up the germination index of seeds. It is reported that using sodium hypochlorite as a disinfectant slowed the rate of decomposition of tamarind seeds during germination [47].

Conclusions
Te destruction of A. paniculata seed dormancy by chemical scarifcation of the seed coat by immersing the seeds in a combination of chemicals and physical methods in swirling macrobubble water is a more efective method than using only macrobubble soaking. Tis is efective in stopping dormancy, stimulating seed germination, and also improving the quality of the seeds. Tis method also afects the physiological development of secondary root formation. Te conduct of this study may serve as useful information in the production and improvement of germination because it allowed us to better understand the seed germination process of this plant.

Data Availability
All data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.