Enhancing the Growth Performance, Cellular Structure, and Rubisco Gene Expression of Cadmium Treated Brassica chinensis Using Sargassum polycystum and Spirulina platensis Extracts

Abstract

Cadmium (Cd) is one of the highly toxic, non-essential heavy metals that inhibit plant growth and development by prompting chlorophyll loss and affecting photosynthetic activities. This study investigated the efficacy of Spirulina platensis and Sargassum polycystum extracts in alleviating Cd stress in Pak Choi at morpho-biochemical, anatomical, and molecular levels. Different concentrations (0, 25, 50, and 100 mL/L) of S. polycyctum (SAR), S. platensis (SPI), and a mixture of both extracts (SS) were exposed to 100 mg/kg Cd-contaminated Pak Choi seedlings. Non-Cd-treated Pak Choi and Cd-contaminated Pak Choi without algal extracts were assigned as positive and negative controls, respectively. The results showed that the application of algal extracts increased the plant height, fresh weight (FW), and dry weight (DW) as the extract level increased. This was greatest in 100SS with 37.51% (shoot length), 68.91% (root length), 110.8% (shoot DW), and 216.13% (root DW), while an increase of 176.7% (shoot FW) and 256.9% (root FW) was seen in the 100SPI treatment. Chlorophyll a, b, carotenoid, and chlorophyll fluorescence increased significantly after the treatment with 100SS. Antioxidant enzymes CAT, APX, POD, and protein were significantly increased in 100 mL/L extracts by 28.13% (100SS), 36.40% (100SAR), 46.92% (100SS), and 153.48% (100SS), respectively. The same treatment was also identified to dominate the development of root structures such as total length, surface area, projected area, diameter, volume, tips, and fork number. The highest reduction of Cd content in the root and shoot of Pak Choi was observed in 100SS with a 53.8% and 39.88% decrease, respectively. Increasing algal extract concentration also improved the leaf histological characteristics substantially, such as stomatal size and opening, mesophyll tissues, and vascular bundles. In addition, the fold change ratio of the Rubisco gene at 100SS treatment was noticeably greater than other algal extract treatments, with a 0.99 fold change when compared with the untreated sample. This present study illustrated that Sargassum polycyctum and Spirulina platensis extracts have the potential to effectively alleviate Cd stress in Pak Choi plants, especially with the application of 100 mL/L of an algal extract mixture. These findings contribute to the development of sustainable strategies for mitigating Cd toxicity in crops.

1. Introduction

Cadmium is a chemical element denoted by the symbol (Cd) that is classified as a non-essential heavy metal, is commonly found in zinc ores, and is naturally extracted as a byproduct of zinc mining [1]. Cd and its compounds are highly toxic and can be harmful to human health and the environment [2]. Cd has been shown to have negative effects not just on human health but also on the surrounding natural ecosystem. Human activities are the primary cause of Cd pollution. These activities include metallurgical operations, mining, electroplating, paints, combustion emissions, and excessive use of fertilizers and pesticides [1]. Equally, the use of synthetic phosphate fertilizers that include Cd as a contaminant is a prevalent reason for the rise in Cd content in groundwater and soil [1]. The culmination of years of progress made in agriculture as well as industry has seen an increase in the amount of Cd found in agricultural soils [3]. As a result of its high solubility and relative mobility in comparison to that of other metals in soil, it is rapidly absorbed by plants [4]. After being taken in, Cd then moves to other areas of the plant and begins to accumulate there, presenting significant risks to human health and the plant itself [5]. It is non-essential since it inhibits plant growth and development, causes chlorophyll loss, and affects photosynthetic activities [2]. Additionally, because of its high mobility in the soil, its bioaccumulation and subsequent accumulation in the food chain surpass all other elements [6].

Cadmium’s toxicity has a number of negative effects on plant life, including a reduction in chlorophyll content and activity, an inhibition of carbon fixation, and an overall slowing of photosynthesis. Plants that have been exposed to cadmium in the soil develop osmotic stress, which reduces the amount of relative water content, stomatal conductance, and transpiration in the leaves. This ultimately leads to plant physiology being impaired [7]. The harmful effects of cadmium originate in the excessive formation of reactive oxygen species (ROS), which in turn causes damage to plant membranes as well as the destruction of cell macromolecules and organelles [8]. As a consequence, Cd can inhibit growth and reduce crop yields [9]. It can also cause chlorosis, necrosis, and other visible symptoms on the leaves and stems of plants [10]. Generally, leafy vegetables have a relatively high potential for Cd uptake, translocation, and accumulation. As a result, Cd absorption and accumulation in food plants, as well as its potential impacts on human health, have received a lot of attention in recent decades. Vegetable consumption is predicted to provide 70–90% of total Cd intake by humans [11]. Therefore, it is necessary to control the uptake, translocation, and accumulation of Cd in plants, particularly in their edible parts, to ensure food safety and security. Furthermore, to limit its exposure to humans and minimize the effects of Cd toxicity on plants and crops, it is important to control the levels of Cd in soil and water with appropriate crop management practices.

There are several management strategies that have been practiced to alleviate heavy metals in soil. Physical strategies such as soil replacement, capping, and thermal desorption are considered effective in alleviating heavy metals. Nonetheless, these techniques involve the use of expensive technology, require the exploitation of other areas that are not impacted by pollution, and only cover a limited spatial area [12]. Phytoremediation has been practiced with promising biological methods, and it is an economically viable and effective in situ technology for mitigating pollutants from soil. However, this approach has limitations on application in elevated contaminated areas, and the plants used would likely be affected by heavy metal toxicity [13]. Chemical leaching is another common chemical approach to heavy metal removal in soil. However, the level of soil washing expense is directly proportional to how thoroughly polluted areas are cleaned up. Additionally, the mineral dissolution that occurs during intense acid washing causes a decline in soil productivity and other undesirable changes to the soil’s chemical and physical composition, affecting plant production [14]. Since the removal of pollutants from soil has become an issue with the limitations mentioned, other strategies and approaches must be established to reduce the toxicity effects of Cd on plants and preserve the security of crops and food supplies.

Therefore, the current study is making use of microalgae and macroalgae extracts to demonstrate their effectiveness in reducing the translocation of Cd in plant parts, thus reducing the toxicity effects on plants and increasing crop production. As far as we are concerned, there is no research on the potential of Sargassum polycystum and Spirulina platensis extracts in mitigating Cd toxicity, enhancing plant growth performance, or enhancing the gene expression of Pak Choi. In fact, information on Sargassum polycystum and Spirulina platensis extracts as bioremediation reagents is scarce in the literature. Macroalgae and microalgae are the two broad categories into which algae are most divided. Macroalgae are grouped into three main families according to their color: Phaeophyceae (brown), Chlorophyta (green), and Rhodophyta (red). It is estimated that there are more than 1500 different species of brown algae globally. With a reported total of more than 400 known species around the globe, Sargassum is one of the brown algae species that is most prevalent and widespread [15]. Sargassum sp. has garnered much attention because it contains more phytohormones, macronutrients, and micronutrients than species in other phyla [16]. Consequently, it has been continually applied as biofertilizers and growth stimulants in agricultural and horticultural settings. A liquid extract of Sargassum wightii was found to enhance the root and shoot lengths of Vigna radiata [17]. According to Prasedya et al. [18], Sargassum crassifolium extracts, both solid and liquid, boosted rice plant development and production. Sargassum horneri extract showed potential for enhancing abiotic stress, antioxidant activity, and the growth of Neopyropia yezoensis [19]. Meanwhile, microalgae are believed to be an excellent alternative due to their beneficial properties as bioremediators. It has strong binding affinity, high tolerance, grows easily, provides a large surface area, is eco-friendly and cost effective [20]. Microalgae of various species have shown remarkable resistance to Cd toxicity tests, metal absorption, alterations in cell shape, and impairments in internal photosynthetic activities [21]. Soil fertilization and foliar spray of S. platensis improved the sugar content, anthocyanin content, and flower numbers of Begonia semperflorens [22]. Foliar spray of S. platensis extract had a significant and marked effect on the plant morphologies and mineral content of Dutch fennel [23]. S. platensis was also tested as a good candidate for the removal of heavy metals from aquatic environments [24], as it is able to chelate some heavy metals from wastewater up to 95% [25]. Hence, algal extracts can eventually play a significant role in crop improvement while mitigating the negative effects of abiotic and biotic stresses, particularly Cd stress. Therefore, this study used Sargassum polycystum and Spirulina platensis extracts to mitigate Cd in Pak Choi seedlings. S. polycystum, which belongs to the Sargassaceae family, has been recognized as a common species in Malaysia’s coastal areas. S. polycystum biomass was discovered to be a reliable biosorbent of industrial metals Cd and Zn with a removal efficiency of 86.20% and 92.90%, respectively [26].

In light of this, the purpose of this research was to evaluate the specific extracts from Sargassum polycystum and Spirulina platensis that have the potential to successfully mitigate the unfavorable effects of Cd stress on Pak Choi plants. It was postulated that the administration of these algae extracts would result in improved morpho-biochemical parameters, such as enhanced plant growth, lowered oxidative stress, and higher photosynthetic efficiency, in comparison to Cd-stressed plants that do not include the algal extracts. In addition, it is anticipated that the algal extracts would produce anatomical changes in the Pak Choi plants, such as increased stomatal density and changed leaf structure, which would then lead to improved Cd tolerance. It is predicted that the algal extracts could induce changes at the molecular level, and regulate the expression of genes linked with photosynthetic activities that are greatly impacted by Cd toxicity, therefore boosting the plant’s capacity to endure Cd stress. By carrying out this research, it is hoped that the findings could provide valuable insights into the potential of Sargassum polycystum and Spirulina platensis extracts as effective Cd stress alleviators and unravel the underlying mechanisms involved in Cd toxicity and tolerance in Pak Choi plants.

2. Materials and Methods

2.1. Algae Collection and Identification

The algae collected (Sargassum polycystum) were handpicked during low tide from the coastal areas of Blue Lagoon (2°24′56.5″ N 101°51′17.5″ E), Port Dickson, a district of Negeri Sembilan, Malaysia. The algae were washed thoroughly with seawater to remove all the unwanted impurities, adhering sand particles, and epiphytes. The thallus of algal biomass was packed in new polythene bags, maintained in an ice box containing slush ice, and immediately transported to the laboratory for meticulous evaluation of morphological characteristics for species identification, according to Yip et al. [27].

A selected strain of Spirulina was bought from Algaeliving SDN. BHD, an established microalgae producer in Malaysia and Southeast Asia. The isolated S. platensis was cultured in Zarrouk’s medium [25]. Spirulina platensis was cultured at 25 °C under continuous illumination with cool white fluorescent light (1020 lux). Cultures were continuously bubbled with an atmospheric air filter sterilized, and the pH of the medium was adjusted using NaOH or 1 M HCL. The optical density of the culture was measured at 730 nm to monitor the cell growth conditions of S. platensis. After 30 days of culture, the total biomass of S. platensis was harvested by centrifugation at 5000 rpm for 10 min. The harvested biomass was covered with 10 mm transparent glass, sun-dried for 4 h, and stored at −18 °C.

2.2. Molecular Identification of Sargassum polycystum

2.2.1. DNA Extraction

The fresh Sargassum and Spirulina were finely ground using liquid nitrogen, and DNA extraction was subsequently conducted using the conventional method, according to Phillips et al. [28]. The sample was lysed using 1 mL of CTAB buffer with an additional 10 µL of mercaptoethanol. The lysing process was conducted by incubating the sample for 90 min at 65 °C. After incubation, 500 µL of CIA reagent was then added for the washing step with centrifugation at 13,000 rpm for 10 min. The supernatant was transferred to new microcentrifuge tubes and subjected to a second washing with the same reagent and centrifugation for 5 min. Once again, the supernatant was transferred into new tubes and mixed with ice-cold isopropanol. The sample was refrigerated at −20 °C overnight. After overnight refrigeration, the sample was centrifuged at 13,000 rpm for 10 min. The supernatant was discarded, and the pellet was mixed with 70% ethanol and centrifuged for 5 min. The pellet was dried for 1 h at room temperature. An amount of 50–60 µL of sterile, nuclease-free water was added to dilute the pellet. The extracted DNA was kept at −20 °C.

2.2.2. PCR and DNA Sequencing

A polymerase chain reaction (PCR) was carried out to amplify extracted DNA prior to DNA sequencing. The KOD OneTM PCR Master Mix (Toyobo, Osaka, Japan) kit was used to amplify the targeted gene of mitochondrial cytochromeoxidase I (COI) as a molecular marker to assign the Sargassum sample to a species. A forward primer, GazF2 = 5′-CCA ACC AYA AAG ATA TWG GTA C-3′ and reverse primer, GazR2 = 5′-GGA TGA CCA AAR AAC CAA AA-3′, were employed, targeting 700 bp of DNA size [29]. Meanwhile, for spirulina, the 16srDNA gene was used with a forward primer, 27F1 = 5′ AGA GTT T GA TCC TGG CTC AG-3′, and a reverse primer, 809R = 5′-GCTTCGGCACGGCTCGGGTCGATA-3′ [30], with a target of 800 bp of DNA size. The thermal cycling conditions for Sargassum were 1 cycle of initial denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 30 s, extension at 72 °C for 1 min, and 1 cycle of final extension at 72 °C for 7 min. PCR conditions for 16srDNA were 1 cycle of initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. The amplified gene was sent to the 1st Base company for DNA sequencing. The sequence was blasted with NCBI Blast to identify the Sargassum and Spirulina species.

2.2.3. Algae Extraction

A portion of the fresh algal samples were air-dried (20 °C) prior to extract preparation. The extract preparation was conducted according to Pise et al. [31] with slight modifications. An amount of 50 g of dried algae biomass was added to 500 mL of distilled water in a 1 L flask. The solution was heated and stirred at 70 °C for 2 h using a hot plate stirrer. The sample was then centrifuged at 4000 rpm for 10 min, and the filtrate was further filtered using a vacuum filtration set. The pH and color of the extracts were observed subsequently. The liquid filtrate was taken as 100% concentration extracts of the two algae species and diluted as per the treatment of the experiment.

2.3. Experimental Design and Plantation

The study was conducted in the Plant Growth Chamber (PGC) of the Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, Terengganu, Malaysia, under manipulated conditions of 26 ± 2 °C with a relative humidity of 70–80% during the 16/8 h photoperiod, and the photon flux density was 400 μmol m⁻² s⁻¹. The seeds of Brassica rapa subsp. chinensis were bought from an authorized dealer and germinated in the dark for 4 days at 26 °C. The soil was taken from the field of an agricultural farm (Jerteh, Terengganu, at a soil depth of 0–30 mm. It was later air-dried and sieved (2 mm) before being placed into pots. The soil characteristics were pH 6.2, 0.12% N, 1.48% C, 18 mg/kg P. 11.7 cmol (+)/kg CEC, 1.3 cmol (+)/kg of K, 1.47 cmol (+)/kg of Ca and 1.09 cmol (+)/kg of Mg, 36.7% coarse sand, 9.8% fine sand, 42% clay, and 11.5% silt, and prepared for plantation. Each pot was filled with 1 kg of soil after being artificially mixed with a 100 mg CdCl₂ solution. One single seedling was transplanted into each pot. Eighty-eight uniform Pak Choi seedlings were selected and transplanted into the prepared soils. Eleven treatments with controls were conducted in five replications. The seedlings were treated twice a week for two weeks through manual foliar spray and soil injection with 25 mL of different concentrations of algal extract: 25, 50, and 100 mL/L of S. platensis (25SPI, 50SPI, and 100SPI), S. polycystum (25SAR, 50SAR, and 100SAR) and a mixture of S. platensis and S. polycystum (25SS, 50SS, and 100SS) extracts. Cd-treated and non-Cd-treated plants without algal extracts were assigned as the negative and positive control, respectively. The experiment was arranged in a Completely Randomized Design (CRD).

2.4. Morphological Parameters

The morphology of Pak Choi was measured by choosing the three third layers of the plant. Plant samples were washed with distilled water. The root parts were washed in ice-cold 5 mM CaCl₂ for 15 min to displace extracellular Cd [32]. The seedlings were washed again with DW and blotted gently on paper towels. Shoots and roots were stored separately for subsequent use. The leaf area, shoot and root length, fresh weight and dry weight, leaf number, and stalk diameter were measured prior to plant harvest.

2.5. Chlorophyll a, b and Carotenoid, Chlorophyll Fluorescence

The chlorophyll content was determined according to Lichtenthaler and Wellburn [33], using 80% acetone as the extraction solvent. An amount of 200 mg of fresh leaf was weighed and homogenized with 80% acetone. The extract was filtered and centrifuged subsequently to collect the supernatant. Finally, the extracted chlorophyll was diluted in up to 10 mL of 80% acetone and subjected to a UV-visible spectrophotometer (Shimadzu UV mini-1240, Kyoto, Japan). The absorbances of 470, 645, and 663 nm were taken and substituted into the following equations:

Chl a = [12.7 (OD663 − 2.69 (OD645)]

(1)

Chl b = [22.9(OD645 − 4.68 (OD663)]

(2)

Carotenoid contents = [1000A470−3.27 (Chl a) − 104 (Chl b)/229]

(3)

to quantify the chlorophyll content. While chlorophyll fluorescence was measured using a PEA meter.

2.6. Gas exchange

Changes in gas exchange of Cd-treated Pak Choi were assessed to determine the effects of different concentrations of algal extracts on the mitigation of Cd stress. The C1-340 Handheld Photosynthesis System (CID Bio-Science, Camas, WA, USA) was utilized for the collection of data pertaining to the net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (C), and internal carbon dioxide (Ci) of leaf samples. The conditions in the leaf chamber were set to provide a photosynthetic photon flux density (PPFD) of 1000 µmol m⁻² s⁻¹, chamber temperature was maintained at 25 °C, and CO₂ inflow concentration was set at 410 µmol mol⁻¹ with a flow rate of 500 mL s⁻¹ and a relative humidity of 50 ± 3%.

2.7. Stress Biomarker

2.7.1. Catalase (CAT), Ascorbate Peroxidase (APX) and Peroxidase (POD) and Protein

Crude protein and enzyme extracts were obtained by crushing 0.2 g of fresh leaf samples using a mortar and pestle in liquid nitrogen, as described by Chen and Zhang [34]. The powdered leaf was homogenized with 3 mL of 50 mM sodium phosphate buffer (pH 7.0) and followed by refrigerated centrifugation at 10,000 rpm for 30 min. The supernatant was collected and kept at 4 °C for antioxidant enzyme activity and protein content analysis [35].

At a wavelength of 240 nm, the CAT activity (EC 1.11.1.6) was analyzed according to the method of Thomas et al. [36] by measuring the absorbance of H₂O₂. The reaction mixture was composed of 50 mM potassium phosphate buffer (pH 7.0), 12.5 mM H₂O₂, and 0.1 mL of enzyme extract. The degradation at 240 nm for 2 min was recorded. A change of 0.01 in the absorbance at 240 nm per minute is considered one unit of enzyme activity.

The activity of the APX enzyme (EC 1.11.1.11) was determined as described by Nakano and Asada [37]. The reaction solution consisted of 50 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, and 0.5 mL of enzyme sample. The reaction was initiated by the addition of 0.1 mM H₂O₂, and the absorbance at 290 nm was taken for 2 min at 15 s intervals. The specific activity of APX was calculated by using E.C. 2.8 mM⁻¹ cm⁻¹.

The estimation of guaiacol peroxidase (POD) activity (EC 1.11.1.7) was measured according to Aebi [38] using guaiacol as a substrate. POD was determined using a spectrophotometer to determine the amount of rise in absorbance at 470 nm that occurred over the course of three minutes as a result of the oxidation of guaiacol. The reaction mixture consisted of 100 mM sodium phosphate buffer, 0.2% guaiacol, 5 mM H₂O₂, and 0.1 mL of enzyme extract. Absorbance was recorded spectrophotometrically at 470 nm against the reagent blank. One unit of POD activity was calculated as a 0.1-unit change in absorbance per minute.

2.7.2. MDA and Proline Content

Malondialdehyde (MDA) was quantified as a nonenzymatic stress biomarker for the estimation of lipid peroxidation levels. MDA content was measured according to Du and Bramlage [39]. Plant tissue samples (0.4 g) were homogenized with inert sand in 1:25 (g FW: mL) 80% ethanol. Samples were centrifuged at 6000 rpm for 5 min. 1 mL of diluted sample was added into the test tube containing 1 mL of thiobarbituric acid (TBA) solution in 20% trichloroacetic acid and butylated hydroxytoluene (BHT). The samples were mixed vigorously and heated in a block heater at 95 °C for 25 min. The sample was cooled and centrifuged at 3000 rpm for 10 min. Absorbance at 440 nm, 532 nm, and 600 nm was recorded using a spectrophotometer.

Proline content was identified in Pak Choi seedlings using the method as described by Bates et al. [40] with a slight modification. 0.2 g of fresh leaf was homogenized in 6 mL of 3% sulpho-salicylic acid and centrifuged at 10,000 rpm for 10 min. In a test tube, 1 mL of supernatant was mixed with a ninhydrin reagent consisting of 1.25 g of ninhydrin in 30 mL of glacial acetic acid and 20 mL of 6 M orthophosphoric acid. Samples were then incubated at 100 °C in a boiling water bath for 1 h. The reaction was inactivated by immediately placing the samples on ice for 5 min. Toluene (2 mL) was added prior to the absorbance reading to extract the developed color. The absorbance was read at 520 nm using a UV-visible spectrophotometer (Shimadzu UV mini-1240, Kyoto, Japan).

2.8. Cadmium Quantification in Pak Choy

The dried samples were weighed (0.3 g) and crushed into powder in a lab mixer mill. Samples were then digested in 10 mL of 65% HNO₃ using a microwave digestion system (Anton Paar MULTIVAWE 3000, Graz, Austria). The sample solution was diluted at 1:40 in mili-Q water and subjected to ICP-MS (Bruker Aurora M90 ICPMS, Billerica, MA, USA) to measure the Cd contents. The translocation factor, root retention, and tolerance index were calculated using the following equations:

Cd TF = (Cd concentration in shoots)/(Cd concentration in roots)

(4)

Root retention % = (Cd amounts in roots)/(Total amount of Cd in plants) × 100

(5)

TI % = (Mean DW of plant under Cd treatment)/(Mean of plant under control) × 100

(6)

2.9. Root Structure and Measurement

Root structure of Cd-treated Pak Choi, with exposure to algal extracts, was subjected to a root scanner or imager using established WinRHIZO software (Pro ver. 2008b). The root of each treatment was cut and separated. All roots were carefully washed to remove dirt and soil particles before being subjected to the waterproof tray. The waterproof tray containing root samples was subsequently analyzed under root scanner. WinRHIZO software scanned for root images, total length, surface area, projected area, root diameter, root volume, tips, and fork number.

2.10. Leaf Histology

Leaf histology was observed under scanning electron microscopy (SEM) (Model JEOL JSm-636OLA, JEOL, Tokyo, Japan) at the Electron Microscope Scanning Laboratory, Institute of Oceanography and Environment (INOS), Universiti Malaysia Terengganu (UMT), Terengganu. The leaf samples treated with the highest (100 mL/L) and lowest (25 mL/L) algal extract concentrations were selected for histological study. Preparation was carried out according to standard procedure by Rohini et al. [41]. Both treated and untreated leaves were collected and preserved in ethanol at a concentration of 80% for 12 h. After preservation, the leaf samples were cut into standard sizes (1 cm × 1 cm) before being fixed in 2.5 g glutaraldehyde in 0.1 M sodium cacodylate for 3 h. Next, the sample was washed using 0.1 M sodium cacodylate buffer (pH 7.2) three times with a 15 min interval between each rinse. The dehydration process was later conducted by soaking the samples in a series of ethanol solutions (35%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%). The samples were ready for gold coating after final dehydration with hexamethyldisilazane (HMDS). The coated samples were observed under SEM by focusing on stomata size and leaf cross-section.

2.11. Rubisco Gene Expression

The leaves of Cd-treated Pak Choi with the highest (100 mL/L) and lowest (25 mL/L) concentrations of algal extract treatments were chosen for further gene expression analysis. The RNA extraction was conducted according to the procedure described in the NucleoSpin® RNA Plant Kit (Macherey-Nagel, Düren, Germany) manual. The final yield of extracted RNA was measured at 260 nm using the spectrophotometer. Subsequently, extracted RNA was subjected to cDNA synthesis using a RevertAid First Strand cDNA Synthesis kit. The samples in reaction tubes were incubated in a heat block at 42 °C for 60 min. After incubation, the reaction was terminated by heating at 70 °C for 5 min. Aliquots of the single-strand cDNA of the algal extract-treated plants, either at 0, 25, or 100 mL/L, were subjected to quantitative RT-PCR analysis on the rbcL gene. The expression of the Co-GAPDH (glyceraldehydes-3-phosphatedehydrogenase) gene was considered the reference gene. The expression of the rbcL gene was determined using a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems^TM, Waltham, MA, USA).

2.12. Statistical Analysis

A Completely Randomized Design was followed to assign the treatments, which were replicated five times. The SPSS-17 statistical software was employed to analyze the data as per the procedure of one-way ANOVA to know the significant difference between the parameters, and the Tukey’s test (HSD) was used to compare different concentrations of algal extract in their effects on the parameters studied at p = 0.05.

3. Results

3.1. Molecular Identification of Sargassum polycystum and Spirulina platensis

Figure 1a shows the DNA of both Spirulina and Sargassum before being amplified through the PCR method. The DNA samples were subjected to PCR, where the Cytochrome oxidase subunit 1 (COI) gene was used for identification of Sargassum species with an estimated product size of 700 bp (Figure 1b). While Spirulina was identified using the 16srDNA gene with an estimated size of 800 bp (Figure 1b). The product of PCR amplification was sequenced and aligned before being substituted in the BLAST system for identification. The data and accession numbers of both algal species are tabulated in

Table 1. The results of the BLAST search after sequencing and alignment show the similarity between GenBank sequences and sample sequences.

Scientific Name	Query Cover (%)	Percentage Identity (%)	Accession Number
Spirulina platensis	98	99.48	ON775378.1
Sargassum polycystum	97	98.00	KT280278.1

.

From the BLAST algorithm, it is understood that the more than 98% similarity (Table 1) obtained matches the sample with the correct species.

3.2. Effects of Cd on Growth and Physiological Parameters

This study found that the plant height, fresh weight, and dry weight were significantly reduced in the 100Cd treatment over control. However, exposure of plant algal extract greatly promoted the growth and development of Cd-treated Pak Choi. The increased pattern of plant height, fresh weight, and dry weight can apparently be seen with the increased concentration of algal extracts of Sargassum, Spirulina, and a mixture of both Sargassum and Spirulina (

Table 2. The effects of algal extract treatment (Spirulina (SPI), Sargassum (SAR), and a mixture of Spirulina + Sargassum (SS)) on the growth characteristics of Brassica chinensis L. (Pak Choi) plants grown in 100 mg/L of CdCl₂ media. All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	Shoot Length (cm)	Root Length (cm)	Shoot FW (g)	Root FW (g)	Shoot DW (g)
Control	16.38 ± 0.55 a	42.50 ± 3.33 a	27.85 ± 0.83 a	6.74 ± 0.04 a	2.96 ± 0.16 a
100Cd	11.73 ± 0.38 c	24.67 ± 6.64 c	10.04 ± 0.55 d	1.67 ± 0.36 c	1.39 ± 0.14 c
25SPI	11.75 ± 0.19 c	39.40 ± 3.53 ab	16.00 ± 3.19 cd	3.77 ± 1.25 abc	1.76 ± 0.10 bc
50SPI	12.43 ± 0.55 bc	39.97 ± 2.92 ab	17.87 ± 0.80 c	4.95 ± 0.09 ab	2.27 ± 0.05 ab
100SPI	14.75 ± 0.37 bc	41.33 ± 4.06 a	27.78 ± 1.73 a	5.96 ± 0.14 a	2.48 ± 0.14 ab
25SAR	12.90 ± 0.17 bc	32.67 ± 3.76 abc	17.87 ± 1.18 c	3.49 ± 0.14 bc	1.89 ± 0.16 bc
50SAR	13.58 ± 0.72 bc	35.83 ± 1.59 ab	18.56 ± 1.65 bc	4.27 ± 0.19 ab	2.43 ± 0.27 ab
100SAR	13.63 ± 0.25 bc	37.33 ± 1.20 ab	27.61 ± 3.35 a	5.27 ± 0.16 ab	2.45 ± 0.12 ab
25SS	13.33 ± 0.49 bc	31.67 ± 2.60 bc	20.43 ± 1.87 abc	4.58 ± 0.59 ab	2.17 ± 0.07 abc
50SS	14.18 ± 0.36 bc	35.67 ± 1.45 ab	25.38 ± 4.79 ab	5.36 ± 0.45 ab	2.57 ± 0.14 ab
100SS	16.13 ± 2.85 ab	41.67 ± 0.33 a	25.63 ± 2.54 ab	5.73 ± 0.18 ab	2.93 ± 0.32 a

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

). Higher readings were observed in 100SPI, 100SAR, and 100SS for each extract group with no significant difference in root length, FW, or DW. However, 100SS had been observed to have the greatest influence on the growth parameters of Cd-treated Pak Choi. The highest reading of shoot length and root length was shown by 100SS with 37.51% and 68.91% over 100Cd respectively. However, there was no significant difference between algal extract treatments and 100Cd in terms of shoot length, excluding 100SS. In contrast, almost all algal extract treatments distinctly increased compared to 100Cd except for 25SAR and 25SS which were the lowest dilutions of algal extracts applied, as shown in Table 2.

Shoot and root fresh weight have been noted to have the greatest increase in 100SPI treatment by 176.7% and 256.9%, respectively (Table 2). However, other treatments also showed an increase as the concentration of algal extracts became more concentrated. There was no significant difference demonstrated by 25SPI, 25SAR, and 25SS in both shoot and root dry weight. This means that lower concentrations of algal extract were not sufficient to improve the plant’s dry weight. Nevertheless, 50 and 100 mL/L of algal extract were significantly found to have a certain effect on Pak Choi’s dry weight, where 100SS showed the highest reading for both shoot (Table 2) and root dry weight (

Table 3. The effects of algal extract treatment (Spirulina (SPI), Sargassum (SAR), and a mixture of Spirulina + Sargassum (SS)) on root dry weight and chlorophyll content of Brassica chinensis L. (Pak Choi) plants grown in 100 mg/L of CdCl₂ media. All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	Root DW (g)	Chl a (µg/L)	Chl b (µg/L)	Carotenoid (µg/L)	Quantum Yield (Fv/Fm)
Control	1.25 ± 0.06 a	9.04 ± 0.80 d	3.90 ± 0.54 b	2.41 ± 0.32 bc	0.82 ± 0.02 a
100Cd	0.31 ± 0.01 c	4.37 ± 0.31 e	2.50 ± 0.11 c	1.86 ± 0.23 d	0.73 ± 0.02 d
25SPI	0.39 ± 0.17 c	9.97 ± 0.37 cd	3.14 ± 0.26 bc	2.07 ± 0.28 c	0.75 ± 0.06 cd
50SPI	0.85 ± 0.06 ab	8.53 ± 0.27 d	3.48 ± 0.50 bc	2.21 ± 0.31 bc	0.77 ± 0.04 abcd
100SPI	1.07 ± 0.12 a	11.98 ± 0.68 bc	3.86 ± 0.61 b	2.38 ± 0.45 bc	0.80 ± 0.03 abc
25SAR	0.52 ± 0.09 bc	9.01 ± 0.17 d	3.26 ± 0.15 bc	2.14 ± 0.36 c	0.77 ± 0.02 bcd
50SAR	0.87 ± 0.03 ab	12.34 ± 0.36 ab	5.09 ± 0.50 ab	3.15 ± 0.51 b	0.76 ± 0.05 bcd
100SAR	1.08 ± 0.03 a	13.93 ± 0.15 ab	5.14 ± 0.48 ab	3.19 ± 0.49 b	0.79 ± 0.09 abc
25SS	0.70 ± 0.06 abc	8.93 ± 0.23 d	3.62 ± 0.47 bc	2.26 ± 0.42 bc	0.78 ± 0.05 abc
50SS	0.84 ± 0.08 ab	12.70 ± 0.51 ab	4.69 ± 0.43 b	2.42 ± 0.37 bc	0.79 ± 0.03 abc
100SS	0.98 ± 0.08 a	14.63 ± 0.66 a	7.12 ± 0.53 a	5.10 ± 0.55 a	0.81 ± 0.02 ab

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

) with a 110.8% and 216.13% increment, respectively.

3.3. Chlorophyll a, b and Carotenoid, Chlorophyll Fluorescence

The chlorophyll pigments were significantly reduced in 100Cd with 51.66%, 35.90%, and 22.82% reductions of Chl a, Chl b, and carotenoid, respectively, when compared to control (Table 3). A decrease in the amount of chlorophyll found in a plant would have a disastrous effect on the process of photosynthesis, which in turn would slow down the growth and development of the plant. However, the application of algal extracts was discovered to essentially boost the chlorophyll contents, confronting the Cd toxicity. An increasing pattern of Chl a, Chl b, and carotenoid content can be seen with the increasing algae concentration. A higher increment was shown by the 100 mL/L concentration for each extract. As described in Table 3, 100SS has revealed the greatest increase with 234.78% (Chl a), 184.80% (Chl b), and 174.19% (carotenoid).

The range of the unstressed Fv/Fm value is between 0.79 and 0.84. The study found that 100Cd showed the lowest Fv/Fm ratio (0.73), indicating the plants were stressed as compared to control. All treatments were significantly increased, excluding 25SPI, 50SPI, 25SAR, and 50SAR. The highest Fv/Fm was observed in 100SS (0.81), followed by 100SPI (0.80), 100SAR and 50SS (0.79), 25SS (0.78), 25SAR (0.77), 50SAR (0.76), and 25SPI (0.75) over 100Cd, as shown in Table 3.

3.4. Gas Exchange

The graphs in Figure 2 revealed that 100Cd has noticeably reduced the gas exchange parameters. Compared to the control, the Pn value was greatly reduced in 100Cd with a 58.17% decrease. Algal extracts showed great potential for increasing the Pn value (Figure 2a) with 28.80% (25SPI), 32.12% (50SPI), 66.23% (100SPI), 14.50% (25SAR), 26.49% (50SAR), 77.48% (100SAR), 15.89% (25SS), 27.15% (50SS), and 76.82% (100SS). However, only 50SPI, 100SPI, 100SAR, and 100SS were significant over 100Cd, with 100SAR giving the highest effect.

Stomatal conductance (C) was seen to drop with 100Cd treatment alone, with a 78.46% decrease (Figure 2b). An increasing pattern of C value with increasing algal extract was detected over 100Cd. However, there was no significant difference between 25SPI, 25SAR, 50SAR, 25SS, and 50SS. Algal extracts were capable of increasing the C value up to 122.22% (50SPI), 144.05% (100SPI), 130.16% (100SAR), and 215.48% (100SS). The result suggested that 100SS has the most substantial effect on C value among all treatments. Figure 2c demonstrates the reduction of internal CO₂ (Ci) in 100Cd at 19.09%. Even though there was no significant difference in Ci between algae extract treatments in Cd-treated Pak Choi, the increment was significantly greater than 100Cd. 100SS showed the highest Ci value with 30.80%.

The results show that 100Cd greatly reduced the transpiration rate (E) of Pak Choi by 52.70% (Figure 2d). The highest E value can be seen in 100SS (214.29%), followed by 100SPI (185.71%), 100SAR (171.43%), 50SAR (155.71%), 50SS (141.43%), 25SS (112.86%), and 50SPI (100.00%). Meanwhile, 25SPI and 25SAR showed no significant effect on the E value of Cd-treated Pak Choi.

3.5. Stress Biomarker

3.5.1. Catalase (CAT), Ascorbate Peroxidase (APX) and Peroxidase (POD) and Protein

Antioxidant enzyme activity varied between the Pak Choi cultivated in non-contaminated soil, Cd-contaminated soil without algal extract exposure, and Cd-contaminated soil with algal extract treatments. It is demonstrated in

Table 4. The effects of algal extract treatments (Spirulina (SPI), Sargassum (SAR) and a mixture of Spirulina + Sargassum (SS)) on antioxidant enzymes, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), and protein content of Brassica chinensis L. (Pak Choi) plants grown in 100 mg/L of CdCl₂ media. All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	CAT (µmolmin−1·g−1 Protein)	APX (µmolmin−1·g−1 Protein)	POD (µmolmin−1·g−1 Protein)	Protein (μmol·g−1 FW)
Control	0.71 ± 0.04 c	0.24 ± 0.02 e	0.85 ± 0.04 e	6.23 ± 0.10 a
100Cd	0.96 ± 0.04 b	0.33 ± 0.03 cd	1.30 ± 0.02 cd	2.3 ± 0.10 c
25SPI	1.05 ± 0.03 ab	0.31 ± 0.03 cd	1.39 ± 0.06 cd	3.07 ± 0.27 bc
50SPI	1.08 ± 0.03 ab	0.35 ± 0.03 bc	1.47 ± 0.09 bc	4.11 ± 0.57 abc
100SPI	1.05 ± 0.06 ab	0.37 ± 0.01 abc	1.72 ± 0.07 ab	4.20 ± 0.89 abc
25SAR	0.93 ± 0.02 bc	0.30 ± 0.02 cd	1.16 ± 0.05 d	4.24 ± 0.08 abc
50SAR	1.06 ± 0.07 ab	0.36 ± 0.02 bc	1.31 ± 0.07 cd	4.82 ± 0.14 ab
100SAR	1.08 ± 0.06 ab	0.45 ± 0.01 a	1.86 ± 0.03 a	4.44 ± 0.30 abc
25SS	0.93 ± 0.04 bc	0.39 ± 0.04 abc	1.38 ± 0.05 cd	4.61 ± 0.69 abc
50SS	1.05 ± 0.06 ab	0.36 ± 0.02 bc	1.71 ± 0.04 ab	5.56 ± 0.49 a
100SS	1.23 ± 0.06 a	0.43 ± 0.01 ab	1.91 ± 0.04 a	5.83 ± 0.29 a

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

that CAT, APX, and POD increased in 100Cd compared to the positive control with 35.21%, 37.50%, and 52.94%. Enzyme activities increased substantially after the SPI, SAR, and SS treatments, and this trend persisted at all concentrations of the algal extracts. Conversely, compared to 100Cd, other treatments include 25SPI, 50SPI, 100SPI, 25SAR, and 50SAR. 100SAR, 25SS, and 50SS were found to boost CAT activity to an insignificant degree. Nonetheless, 100SS has appeared to be effective, showing a 28.13% rise. Table 4 also shows higher levels of APX activity after the algal extract treatment. The results indicate that 100 mL/L of algal extract had the largest impact on generating APX activity compared to the lower doses examined (50 and 25 mL/L) for each group of algal extracts, with 100SAR showing the highest activity at 36.40%, followed by 100SS (30.30%) and 100SPI (12.12%). It was discovered, however, that only 100SAR and 100SS had a statistically significant effect on APX activity. As for POD activity, 100SS has the greatest activity with a 46.92% rise, followed by 100SAR, 100SPI, and 50SS with a 43.08%, 32.31%, and 31.54% rise, respectively.

Protein content was observed to decrease in the 100Cd treatment over control, with a reduction of 63.08%. It was noticed that algal extract treatments increased the protein content of Pak Choi leaves in every extract group with an increment in extract concentration. Nevertheless, there was no significant difference in 25SPI, 50SPI, 100SPI, 25SAR, 100SAR, or 25SS. It was revealed that the protein content was most positively influenced by 100SS with a 153.48% increment, followed by 50SS (141.74%) and 50SAR (109.57%), as shown in Table 4.

3.5.2. MDA and Proline Content

Measuring malondialdehyde (MDA) levels in Cd-treated Pak Choi allowed us to quantify the level of lipid peroxidation, and hence the degree of membrane damage caused by Cd stress, and the efficacy of algae extracts in mitigating that damage. The data showed that following Cd treatment, there was a 67.00% rise in MDA levels (Figure 3a), indicating a high level of membrane damage and lipid peroxidation. Exposure to algal extracts through soil injection and foliar spray reduced the MDA content substantially to 1.80% (25SPI), 8.18% (25SAR), and 12.97% (25SS). Higher reductions can be observed in 50 mL/L algal extracts with 9.78% (50SPI), 22.55% (50SAR), and 15.57% (50SS). While 100SPI, 100SAR, and 100SS showed a drastic fall in MDA at 32.34%, 18.16%, and 33.93%, respectively (Figure 3a). Data analysis revealed that algal extracts at concentrations of 25, 50, and 100 mL/L were able to reduce MDA content, hence mitigating Cd detrimental effects (membrane damage). However, 100SS was found to be the most effective extract treatment for ameliorating the Cd influence.

Exposure to abiotic stresses causes plants to respond physiologically by generating large amounts of proline (Hnilickova et al., 2021). Cd-treated Pak Choi were tested for their proline content after being exposed to several concentrations of algal extracts. The data showed a 52.87% increment of proline content in the 100Cd treatment over control (Figure 3b). A significant drop in proline can be seen in algal extract treatments at 17.29% (25SPI), 20.30% (25SAR), 18.04% (25SS), 44.36% (50SPI), 25.56% (50SAR), 21.80% (50SS), 40.60% (100SPI), 33.08% (100SAR), and 29.32% (100SS). Compared to 100Cd, proline content was significantly affected by all treatments, with 50SPI showing the most efficient treatment, followed by 100SPI, 100SAR, 100SS, 50SAR, 50SS, 25SAR, 25SS, and 25SPI (Figure 3b).

3.6. Cadmium Quantification in Pak Choi

Figure 4 demonstrates that the Cd content was higher in the root part than the shoot part. Explaining this, translocation or absorption of Cd occurred in other arial parts of Pak Choi but was mostly retained in the root system. 100Cd showed the highest Cd content in the root and shoot. It was determined that an enhanced reduction of Cd content in the root and shoot occurred as the concentration of algal extracts increased. 100SPI, 100SAR, and 100SS show higher reductions of Cd content in each of the extract groups, as shown in Figure 4. The highest reduction in root was observed in 100SS (53.8%), followed by 50SS (46.73%), 100SPI (42. 1%), 50SPI (42.07%), and 29.90% (100SAR). Other treatments showed no significant difference over the 100Cd treatment (Figure 4a). The highest reduction of shoot Cd content (Figure 4b) can be seen in 100SS with a 39.88% decrement, followed by 25SAR (29.76%), 100SAR (26.61%), 50SS (26.01%), 25SS (24.23%), 50SAR (22.80%), 100SPI (18.63%), 50SPI (14.46%), and 25SPI (1.19%). Excluding 25SPI, all other treatments employing algal extracts showed a substantial influence on Cd-treated Pak Choi, as shown in Figure 4b.

Cd retention in roots (RI) was determined to be a good indicator of how much Cd roots were able to hold on to despite environmental stresses. While the tolerance index (TI) is the capacity or tolerance of plants to grow under Cd stress.

The results demonstrate (

Table 5. The value of the translocation factor, root retention, and tolerance index of Cd-treated Pak Choi after treatment with different concentrations of algal extract exposure. All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	Translocation Factor	Root Retention (%)	Tolerance Index (%)
Control	0.68 ± 0.04 a	57.84 ± 3.16 f	100.00 ± 5.43 de
100Cd	0.39 ± 0.01 cde	72.10 ± 0.70 abc	69.01 ± 5.86 f
25SPI	0.43 ± 0.04 bcde	70.29 ± 2.14 abcde	87.01 ± 7.45 ef
50SPI	0.54 ± 0.03 bc	64.77 ± 1.37 def	126.52 ± 4.44 bc
100SPI	0.56 ± 0.09 b	64.42 ± 3.85 ef	144.11 ± 10.48 ab
25SAR	0.32 ± 0.02 e	75.76 ± 1.04 a	97.83 ± 4.81 de
50SAR	0.37 ± 0.04 de	73.35 ± 2.24 ab	133.96 ± 8.45 bc
100SAR	0.40 ± 0.01 bcde	71.27 ± 0.69 abcde	143.44 ± 5.01 ab
25SS	0.39 ± 0.06 bcde	71.76 ± 3.22 abcd	116.51 ± 3.05 cd
50SS	0.54 ± 0.04 bc	65.05 ± 1.93 cdef	138.57 ± 8.26 abc
100SS	0.51 ± 0.08 bcd	66.60 ± 3.92 bcde	158.86 ± 16.27 a

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

) an increment of TF with the addition of algal extract. Nevertheless, there is no significant difference in TF values between algal extract treatment and 100Cd except for 100SPI treatment. Inversely, the RT value specifically dropped with algal extract concentration increments. The significant RT value can be seen in the 50SPI (64.77%) and 100SPI (64.42%) treatments. Whereas other treatments insignificantly affected the RR value. TI values are exhibited in Table 5, where the increment of algal extract dose increased the value over 100Cd. All treatments substantially increased the TI value except for 25SPI. The highest increase was observed in 100SS, followed by 100SPI, 100SAR, 50SS, 50SAR, 50SPI, 25SS, and 25SAR. Low Cd deposition and translocation in shoots or aerial parts have previously been correlated with higher Cd tolerance.

3.7. Correlation among Studied Parameters

The plant growth parameters, which include plant height, fresh weight, and dry weight, were found to have a positive correlation with the physiological parameters (chlorophyll a, carotenoid, and quantum yield) (Figure 5). On the other hand, it appears that morphological and physiological factors have a negative correlation with the malondialdehyde and proline content in leaves and the Cd content in the root and shoot. From the correlations of the tested variables, antioxidant enzymes (CAT, APX, and POD) have a substantial positive correlation with plant growth and photosynthetic components. Additionally, the net photosynthetic rate was seen to positively correlate with plant growth but negatively correlate with Cd content in the root and shoot as well as root retention of Pak Choi grown in a pot of 100 mg/kg of Cd with exposure to algal extracts (S. polycyctum and S. platensis) through soil injection and foliar spray.

3.8. Root Structure and Measurement

Scientific research into complicated root structures and their function under stress settings was greatly aided by the WinRHIZO optical scanner and software system. The study found qualitative differences in scanned images of Cd-treated Pak Choi’s root in terms of root size and volume, as exhibited in Figure 6. Figure 6b illustrates the root image of 100Cd-treated Pak Choi, where the roots were much smaller and shrank compared to the control group (Figure 6a). Magnificently, treatment with algal extracts, particularly 100SPI (Figure 6e), 100SAR (Figure 6h), and 100SS (Figure 6k), resulted in considerably more robust root development.

In addition to depicting the root system graphically, WinRHIZO can also analyze and quantify a selection of vital root metrics, including the total root length, root surface area, root projected area, average root diameter, root volume, root tip numbers, and root fork numbers. Upon exposure to 100Cd, drastic reductions were detected with 61.99% (total length), 66.00% (root surface area), 66.01% (root projected area), 10.62% (average root diameter), 69.86% (root volume), 44.57% (number of tips), and 69.31% (number of forks) as shown in

Table 6. Root growth and structural properties of Cd-treated Pak Choi as affected by algal extracts (0, 25, 50, 100 mL/L mg/L) measured by WinRHIZO software. All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	Total Length (cm)	Surface Area (cm2)	Projected Area (cm2)	Diameter (mm)	Root Volume (cm3)	Tips	Forks
Control	353.78 ± 46.31 e	125.63 ± 25.21 cd	39.99 ± 8.21 cd	1.13 ± 0.35 cd	3.55 ± 0.40 cd	2457 ± 320 d	9882 ± 1055 cde
100 Cd	134.47 ± 5.77 f	42.71 ± 3.06 d	13.59 ± 1.21 d	1.01 ± 0.12 bc	1.07 ± 0.21 c	1362 ± 153 d	3033 ± 88 f
25 SPI	283.76 ± 11.32 c	64.79 ± 3.58 cd	20.62 ± 3.05 cd	0.72 ± 0.09 c	1.17 ± 0.23 c	1899 ± 144 cd	6505 ± 139 d
50 SPI	216.99 ± 6.90 de	54.81 ± 2.77 d	17.44 ± 2.12 cd	0.80 ± 0.12 c	1.10 ± 0.10 c	1858 ± 173 cd	4393 ± 125 e
100 SPI	356.2 ± 6.36 a	84.97 ± 4.11 bc	27.04 ± 3.74 cd	0.75 ± 0.20 c	1.61 ± 0.14 bc	3111 ± 166 a	7491 ± 166 c
25 SAR	243.94 ± 6.15 d	58.28 ± 2.77 cd	18.55 ± 3.42 cd	0.76 ± 0.10 c	1.10 ± 0.06 c	1917 ± 159 cd	5091 ± 155 e
50 SAR	194.02 ± 5.60 e	44.24 ± 3.25 d	14.08 ± 3.11 d	0.72 ± 0.14 c	0.80 ± 0.06 c	1879 ± 118 cd	3451 ± 149 f
100 SAR	294.85 ± 6.11 c	66.14 ± 3.18 cd	21.05 ± 3.64 cd	0.71 ± 0.11 c	1.18 ± 0.12 c	2099 ± 124 bc	6346 ± 121 d
25 SS	301.46 ± 9.12 bc	101.78 ± 5.17 b	32.39 ± 4.16 bc	1.07 ± 0.17 abc	2.73 ± 0.35 b	1926 ± 107 cd	7738 ± 126 c
50 SS	336.82 ± 8.03 ab	155.77 ± 8.66 a	49.58 ± 5.03 ab	1.45 ± 0.23 ab	5.73 ± 0.52 a	2490 ± 102 abc	9729 ± 180 b
100 SS	356.06 ± 9.47 a	162.58 ± 10.40 a	51.75 ± 3.73 a	1.47 ± 0.21 a	5.90 ± 0.49 a	2680 ± 115 ab	10474 ± 168 a

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

.

The introduction of algal extracts from S. platensis, S. polycystum, and a mixture of both S. platensis and S. polycystum was found to greatly boost the root structures (Table 6) and morphologies (Figure 6). Both 100SPI and 100SS were capable of increasing root total length up to 164.00% growth. Meanwhile, 50SS and 100SS both led to an increase in the root surface area of 264.72% and a 280.66% expansion, respectively. Other significant treatments were 100SPI and 25SS, with lower measurements of root surface area as compared to 100Cd. Almost similar trends can be observed in the root projected area, root diameter, and root volume, where 100 SS had the greatest impact on the Cd-treated Pak Choi, leading to a 180.79%, 45.54%, and 451.4% increase, respectively, followed by 50SS and 25SS. Other groups of S. platensis and S. polycystum extracts with 100Cd showed no apparent differences. Contrary to the mentioned parameters, 100SPI was found to show the highest root tip numbers when compared to other treatments, being 128.41% greater than 100Cd. However, 50SS, 100SS, and 100SPI did not differ much from one another. The greatest influence on root fork numbers was demonstrated by 100SS, with a 245.33% increase. To a lesser extent, other treatments also gave a great spike in root fork numbers except for the 50SAR treatment, as shown in Table 6.

3.9. Leaf Histology

As seen in Figure 7, the size of the stomatal opening was smaller compared to the control. The stomata seemed bigger, and the aperture was much greater at the lower concentrations of algal extracts (25SPI, 25SAR, and 25SPI) compared to 100Cd. Nevertheless, at higher doses (100SPI, 100SAR, and 100SS), both stomatal aperture size and permeability improved to a greater extent. Stomata that are larger and have wider openings allow for more of the gas and vapor exchange that is especially essential for photosynthesis to take place. To that end, it was discovered to counteract Cd’s detrimental effects on Pak Choi’s growth and development. The images of cross section and leaf thickness were observed under SEM to see the effects of algal extracts on the mesophyll tissue of Cd-treated Pak Choi (Figure 8). At lower concentrations, 25 mL/L of algal extract showed thinner mesophyll tissue compared to the positive control, yet it was thicker than 100Cd. The findings demonstrate that even at lower concentrations, algal extracts have a great potential to mitigate Cd’s harmful effects. The thickest mesophyll as affected by algal extracts was shown by 100SS (134.5–144.0 µm), followed by 100SAR (128.2–141.5 µm), and 100SPI (80.3–116.2 µm) (Figure 8). The enhanced palisade and spongy cell structure of algal-treated Pak Choi boost the plant’s photosynthesis. This bodes well for the efficacy of algal treatments on Cd-exposed plants. Therefore, there will be a favorable effect on plant growth and development from the increased photosynthesis in Pak Choi.

Phloem, xylem, and the sheath are all shown in Figure 9 as they make up the vascular bundle component. A twisted configuration of the vascular bundles is readily apparent after 100Cd treatment. With the application of algal extracts, the structure of tissues was effectively established, as can be seen in Figure 9 (100SPI, 100SAR, and 100SS).

3.10. Rubisco Gene Expression

The Rubisco gene was used to study the effect of different concentrations of algal extracts on the plant, specifically whether they changed the expression of the gene. Figure 10 shows the results of the qualitative effect of algal extracts on rbcL. The bands of rbcL gene fragments were clearly seen in 100SPI, 100SAR, 25 SS, and 100 SS treatments compared to 100Cd, 25SPI, and 25SAR. The bands of the GAPDH gene were unclearly seen in most treatments except for 25SAR and 25SS. This might be due to protein dimer or sample contamination.

From

Table 7. The value of threshold cycle (CT) rbcL and GAPDH genes of Cd-treated Pak Choi as affected by algal extracts (0, 25, and 100 mL/L). All the data are a mean of five replicates. Different letters in the same column represent the significant difference at the 5% level according to the Tukey HSD test at α = 0.05.

Treatment 1	rbcL CT Mean	GAPDH CT Mean	ΔCT	ΔΔCT	2−(ΔΔCT)
Untreated	24.25 ± 0.15	22.47	1.78 (0.15)	0.00 (0.15)	1.00 (0.85–1.15)
100Cd	26.03 ± 0.17	23.25	2.77 (0.15)	1.00 (0.15)	0.50 (0.35–0.65)
25Spi	24.99 ± 0.13	22.81	2.18 (0.17)	0.41 (0.17)	0.75 (0.58–0.92)
100Spi	26.17 ± 0.16	24.11	2.07 (0.13)	0.29 (0.13)	0.82 (0.69–0.95)
25Sar	25.79 ± 0.11	23.20	2.59 (0.16)	0.81 (0.16)	0.57 (0.41–0.73)
100Sar	25.23 ± 0.17	23.20	2.03 (0.11)	0.25 (0.11)	0.84 (0.73–0.95)
25SS	25.28 ± 0.09	23.13	2.15 (0.17)	0.37 (0.17)	0.77 (0.60–0.94)
100SS	26.23 ± 0.15	24.45	1.79 (0.09)	0.01 (0.09)	0.99 (0.90–1.08)

¹ 100Cd = 100 mg/kg Cd, 25SPI = 25 mL/L S. platensis, 50SPI = 50 mL/L S. platensis, 100SPI = 100 mL/L S. platensis, 25SAR = 25 mL/L S. polycystum, 50SAR = 50 mL/L S. polycystum, 100SAR = 100 mL/L S. polycystum, 25SS = 25 mL/L S. platensis + S. polycystum, 50SS = 50 mL/L S. platensis + S. polycystum, 100SS = 100 mL/L S. platensis + S. polycystum.

, it is clearly illustrated that the fold change ratio of the 100SS treatment is noticeably greater than other algal extract treatments when compared with the untreated sample (reference sample). However, the ratio is less than 1 (reference sample), showing that the expression of the gene was downregulated in Cd-treated Pak Choi. Graph a in Figure 11a shows that fold change decreased in 100Cd. However, an increase in the ratio closer to 1 was observed in 100SS (0.99), showing the potential of extracts to induce the rbcL gene expression level. Figure 11b shows downregulation of the rbcL gene as proofed by Log2(FC) values. Cd was seen to deviate farther from the 0 value. Meaning that the gene is less expressed in 100Cd treated plant compared to the Cd-treated plant with algae treatment. The lowest value can be seen in 100SS treatment, which indicates an enhanced gene expression level.

4. Discussion

As a result of heavy metal exposure, plants experienced profound changes in their development, photosynthetic pigments, and antioxidant defenses [42]. This study demonstrates that Cd significantly affected the growth and development of Pak Choi. Exposure to 100 mg/kg of Cd showed an apparent reduction in plant height, fresh weight, and dry weight of Pak Choi. In previous studies, potato (Solanum tuberosum L.) seedlings cultivated in pots experienced a reduction in shoot and root length as well as dry weight when subjected to Cd stress at 60 mg/kg compared to the control group [43]. Despite this, the study identified that the negative effects of Cd stress on Pak Choi’s development could be mitigated with the application of algal extracts (S. polycyctum, S. platensis, and a combination of the two). Sargassum sp. extracts from water-based extraction methods have been reported to have good biostimulant activity on the early growth of Zea mays L. by improving the shoot and root growth [16]. A considerable increase in rice plant growth and yields has also been observed after the application of Sargassum extracts [44]. Sargassum angustifolium extract has been reported to enhance the plant height, specific leaf area, root length and volume, and root and shoot dry weight of salt stress-treated milkweed seedlings at 1% concentration [45].

The growth and development of plants are highly manipulated by their physiological functions, specifically photosynthesis activities. The process of photosynthesis is an essential physiological function that plants must consistently carry out. Cd stress, on the other hand, can persistently prevent plants from engaging in this obligatory action [46] by altering physiological traits [47]. These physiological functions are greatly affected since the processes through which Cd is absorbed by plant roots typically include competition for absorption sites with other nutritional minerals with comparable chemical properties. Cd in mineral form would substitute for other essential minerals with identical charge, ionic radius, and chemical behavior [48]. As a result, Cd may inhibit the uptake of Mg, Fe, K, and P from soil, thus constraining the creation of leaf porphyrin rings and leading to a drastic decrease in chlorophyll synthesis and changes in chloroplast structure [45].

In the present study, photosynthesis attributes were investigated to observe the effects of Cd stress. It was found that Cd substantially reduced chlorophyll a, b, carotenoid, quantum yield (Fv/Fm), and gas exchange parameters (net photosynthetic rate, stomatal conductance, internal CO₂, and transpiration rate) in Pak Choi. Fenugreek plants grown in the same concentration of Cd contaminated soil have shown similar results [49]. Farooq et al. [50] observed that stomatal conductance and the transpiration rate of Gossypium hirsutum decreased by 74% and 70%, respectively, after Cd treatment in a hydroponic system. Reduced net photosynthetic rate and stomatal conductance may result from Cd-induced increases in ROS that damage chloroplast ultrastructure and the thylakoid membrane [47]. Nonetheless, the study revealed that adding 100 mL/L of algal extracts (100SPI, 100SAR, and 100SS) to Cd-treated Pak Choi increased its chlorophyll a, b, carotenoid content, quantum yield, as well as gas exchange parameters. Extracts from algae have improved photosynthetic pigments by raising both stomatal conductance and photosynthetic capacity [45]. The presence of bioactive compounds in algal extracts such as amino acids, betaines, and minerals is capable of inhibiting chlorophyll deterioration and stimulating the photosynthetic capacity of plants [51]. Phytohormones such as auxins, abscisic acid, cytokinins, ethylene, and gibberellins were found in Arthrospira, Chlamydomonas, Chlorella, Phormidium, Protococcus, and Scenedesmus extracts that react as chemical messengers that help in stimulating plant growth and regulating the cellular activities in crops as well as responses to stress conditions [52,53,54]. These phytohormones, which boost plant nutrient absorption, are viable in reducing the nutritional imbalance brought on by Cd toxicity and fostering healthier plant development and growth. A study reported that seed priming of maize plants using S. platensis enhanced quantum yield (Fv/Fm) of Cd-treated plants, and an 0.83 ratio has been addressed as the optimum value for the healthy functioning of Photosystem II (PSII) [55]. S. platensis extracts also significantly increased the carotenoids and chlorophyll content of both Cd-treated Phaseolus vulgaris and salt-stressed Triticum aestivum L. [56,57]. The addition of S. polycystum and S. platensis extracts also positively increased the gas exchange characteristics. Both water and alcohol extracts of S. polycystum were found to induce seed germination and growth of pigeon peas (Cajanus cajan L.) [58]. Better growth in plants is due to high chloroplast and stomata numbers that receive more sunlight, which play a vital role in gas exchange during the photosynthesis process [59].

Plants exposed to Cd stress exhibited drastic alterations in the activity of antioxidant enzymes [11,60]. Multiple studies have shown that excessive Cd exposure is associated with a rise in reactive oxygen species (ROS) generation and accumulation in cells, which include H₂O₂, O²⁻, and OH⁻ [47]. It can actually be observed as ROS interfere with the regular function of particular biomolecules such as proteins, nucleic acids, and membrane lipids [61]. However, some evidence suggests that heavy metal exposure boosts antioxidant activity in plants as a defensive mechanism against oxidative stress [62], including the enzymatic activities of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and peroxidase (POD) [47]. This study found that the activity of antioxidant enzymes has been highly generated and boosted under 100 mg/kg of Cd. Consistent with earlier research. Mung beans [63] and strawberries [64] showed increased activities of GPX and CAT and a decrease in APX under Cd stress. According to Rahman et al. [65], the activities of APX, SOD, and glutathione peroxidase (GPX) all increased in Cd-treated rice seedlings over control, while those of DHAR (dehydroascorbate reductase), GST (glutathione S-transferase), and CAT had been reduced. The current study has identified that algal extracts significantly increased the antioxidant enzyme activity of Cd-treated Pak Choi, with the highest concentration, 100 mL/L, dominating the activity level. In response to Cd stress, the PGR increased the activities of antioxidant enzymes, which in turn reduced oxidative stress in the plants [11]. Previous research has demonstrated that brown algae, Fucus spiralis, and Cystoseira ericoides extracts had a considerable ameliorating impact on the development and physiological and biochemical parameters of tomato plants subjected to metal stress [66]. An elevated level of SOD, CAT, and POD activity could be seen in Hordeum vulgare L. plants that had been treated with Sargassum spp. while being subjected to Cd stress [67]. S. platensis has increased the CAT (18.10%) and APX (18.30%) of common beans treated with Cd [56].

Overproduction of oxidative indicators, including ROS, free radicals, and lipid peroxidation, from Cd-contaminated soils generates oxidative stress in plants, which in turn reduces agricultural yields [68]. Oxidative damage caused by a rise in ROS is a frequent response of plants to biotic or abiotic stress. It often manifests itself in a high accumulation of MDA and proline. Measurements of MDA and proline were performed to see the efficacy of algal extracts in counteracting the oxidative stress brought on by Cd in Pak Choi. The study discovered that MDA and proline content increased in Cd-treated Pak Choi compared to the control. Though both MDA and proline were distinctly dropped in Cd-treated Pak Choi with algal extract application. Extracts of algae have been shown to contain bioactive chemicals, particularly phenolic compounds, which have antioxidant characteristics. Phenolic compounds, including polyphenols, phenolic acids, flavonoids, and phenylpropanoids extracted from Botryococcus braunii, Chaetoceros calcitrans, Chlorella vulgaris, Isochrysis galbana, Isochrysis sp., Neochloris oleoabundans, Odontella sinensis, Phaeodactylum tricornutum, Saccharina japonica, Skeletonema costatum, and Tetraselmis suecica, have been reported to protect the plants from pathogens or other biotic and abiotic stress conditions, including scavenging reactive oxygen species (ROS) generated by Cd stress, preventing oxidative damage to plant cells [52,53,69,70,71,72]. While free fatty acids (saturated and unsaturated) from Anabaena, Chlorella, Dunaliella, Nannochloropsis, Porphyridium, Scenedesmus, and Spirulina showed antioxidant, antibiotic, anticarcinogenic, antifungal, antioxidant, and antiviral activity, reducing the stress in plants [52,54,73,74,75,76].

For ionic and osmotic homeostasis, plants generate proline or other suitable solutes to regulate water balance and protein complex stability [77]. A study by [65] reported a 55.56% increase in proline in rice seedlings after 0.3 mM of Cd exposure. A maximum increase of MDA and H₂O₂ was reported at 20 mg/kg Cd stress on mung bean seedlings [78]. The present study revealed that the utilization of algal extracts positively reduced both MDA and proline content under different concentrations of algal extracts, with 100 mL/L of S. platensis and 50 mL/L of S. platensis + S. polycystum giving the maximum decrement, respectively. Brown algae, especially S. polycystum, were reported to have a high fucoidan level that acts as an antioxidant that might increase ROS scavenging activity [62], thus reducing excessive oxidative damage that causes high levels of MDA and proline. S. platensis and Chlorella vulgaris were revealed to reduce free proline content and MDA levels in the leaves of Phaseolus vulgaris [41].

According to the findings, Cd absorption and translocation occurred more often in the root system than in the shoot system. Similar findings were also observed in mustard (Brassica juncea L.), which was exposed to toxic levels of Cd at 200 mg L⁻¹ and 300 mg L⁻¹ [79], and other metals in Panax notoginseng, Chlorophytum comosum, Calendula offcinalis [80], and Solanum lycopersicum [66]. Cd ions are trapped in roots by selectively binding to molecules or cell sites with high metal affinity. This mechanism involves phytochelatins (PCs), which are the major Cd chelators and generate Cd complexes that are sequestered in vacuoles and hence remain inside the root cells [81]. These complexes lose their toxicity when they are trapped in the cell wall, cytoplasm, or vacuoles, respectively. Furthermore, Cd accumulation in root vacuoles mitigates its toxicity and prevents it from being transported great distances to the shoot. Nevertheless, the translocated Cd in shoots would be sequestered and detoxified in cell walls or the vacuole [82]. The present study determined the potential of S. platensis and S. polycystum extracts in reducing Cd content in the root and shoot of Pak Choi, where Cd ions can be bound or chelated by substances found in algae extracts such as polysaccharides, peptides, and organic acids. This chelation mechanism aids in the sequestration of Cd and decreases its bioavailability, both of which lessen the toxicity of Cd to plants. In line with previous findings, a reduction of Cd from 74% to 91% was observed in common bean plants that had been treated through foliar spraying of S. platensis extract [56]. S. platensis was utilized for Cd and Ni adsorption and Cu ion absorption from aqueous solutions [49]. Four biomass types with different biochemical compositions of Arthrospira (Spirulina) platensis were utilized for the removal of heavy metals [83]. The utilization of Sargassum stolonifolium in reducing Cd content in Brassica chinensis has been reported in previous studies. This is likely because of the presence of functional groups such as thiol, carboxyl, hydroxyl, amine, carbonyl, and other common compounds that are responsible for Cd binding [84].

Our findings revealed that the translocation factor and tolerance index increased with increasing algal extracts. Contrarily, root retention has been greatly reduced with the application of algal extracts. A metal’s ability to move from underneath to the surface is referred to as translocation. Translocation factors (TF) (ratio of metal concentration in roots to those in the shoots) are greater in plants that can take up and distribute the metal throughout their entire system, while TF values are lower in plants that can restrict the movement of metals from the soil to the roots and from the roots to the shoots [85]. Our findings are consistent with what has been discovered previously concerning Cd deposition and tolerance in other crops [86,87].

Root structure of Cd-treated Pak Choi was greatly stunted by the results of WinRHIZO software, as illustrated in Figure 6 and Table 6. Both root elongation and root architecture are influenced by Cd presence in the rhizosphere. Root apoplasm contains higher Cd levels than symplasm, and Cd levels decline from the outer to the interior root tissues [88]. Since the root system is apparently unable to distinguish the cations of the required micronutrients for the plant, it is inevitable that it might absorb free Cd ions into the root system. However, this deleterious change was greatly reduced by the application of S. polycyctum and S. platensis extracts to Cd-treated Pak Choi. It is proven by the demonstrated results (Table 6), where root length, root surface area, root projected area, average root diameter, root volume, root tip, and root fork numbers were positively enhanced after algal extract treatments. Algal extracts may contain micro and macro elements, vitamins, and other essential plant growth regulators (PGR) such as auxin, cytokinin, and gibberellin that help the growth of plants overcome the Cd toxicity effects [89]. In addition, microalgae and macroalgae were reported to effectively bioremediate heavy metals from wastewater and soil, thus reducing heavy metal uptake by roots [90].

An amount of 100 mL/L of algal extract mixture (100SS) shows better cell structure and histology. This is based on image analysis using a scanning electron microscope to observe the changes in the cellular structure of the Cd-treated Pak Choi’s leaf when treated with algal extracts. The result shows that treatment with algal extracts has improved stomatal size and opening. More gas and vapor exchange, which is crucial for photosynthesis, may take place through stomata that are bigger and have broader apertures [91], thus, enhancing the photosynthesis activity, growth, and development of the plant. The leaf thickness and vascular bundle (xylem and phloem) were enhanced with 100 mL/L of algal extract exposure. Algal extracts have increased the activity of antioxidant enzymes that are involved in the production of H₂O₂, which is responsible for the prevention of oxidative damage to the cells [92]. It was reported in previous studies that the small open vessel element has thicker tissue walls when compared with unstressed plants [92].

Rubisco (ribulose-1,5-bisphosphate carboxylase) is a carboxylating enzyme that is essential for photosynthesis as it catalyzes the transformation of atmospheric CO₂ into organic molecules, basically carbohydrates, that are needed by plants [91]. This study found that exposure to Cd reduced Rubisco gene expression in Cd-treated Pak Choi leaves. Consistent with our findings of reduced chlorophyll levels, quantum yield, and net photosynthetic rate, plant development and growth were stunted. Leaf photosynthetic rate is strongly influenced by the concentration of Rubisco, which triggers changes in plant activity [92]. S. polycystum and S. platensis extracts, however, reduced the oxidative damage to photosynthetic attributes by increasing the activity of Rubisco gene expression. When compared to 100Cd, which only had a fold change of 0.50, it was discovered that 100SS caused the biggest fold rise, which reached up to 0.99-fold. In conclusion, algal extracts have a great potential for reducing Cd oxidative stress and damage to the cellular structure of Cd-treated Pak Choi, thus increasing the number of fold changes in Rubisco gene expression.

5. Conclusions

In conclusion, this study contributes to the understanding of algal extracts potential for addressing cadmium (Cd) toxicity in Pak Choi plants. The findings demonstrate the efficacy of Sargassum polycystum and Spirulina platensis extracts in reducing Cd-induced stress and improving plant development, biochemical properties, and anatomical abnormalities caused by Cd contamination. Introduction of S. polycystum and S. platensis extracts has lessened the Cd toxicity, improved the growth, photosynthesis, biochemical properties, leaf histology, root structure, and reduced the stress biomarkers and Cd phytotoxicity of Pak Choi. The findings also indicate that the extract treatments of S. polycystum and S. platensis enhanced the expression of the Rubisco gene, which catalyzes the assimilation of CO₂ and increases net photosynthesis in Cd-treated Pak Choi. The great potential of extracts in reducing Cd-induced stress has been demonstrated, particularly when administered in a combination of both algal extracts at 100 mL/L. Extracts of algae have chelating capabilities, antioxidant activity, the ability to induce enzymes involved in detoxification, the ability to boost nutrient absorption, and improvements in plant physiological activities. These processes reduce the stress that Cd induces in the plant by working together to provide a synergistic effect that improves the plant’s capacity to withstand and recover from Cd contamination. Accordingly, this study suggests that extracts of S. polycystum and S. platensis are useful bio-remediation agents for Cd-contaminated soils, and they should be studied further as prospective bio-remediation agents for agricultural systems while reducing the utilization of expensive chemicals and reagents and the limited applicability of physical approaches. Nevertheless, it is essential to take into account the limitations of this method, such as the specific application it requires and the likelihood of variations in its efficiency under a variety of climatic and environmental conditions. More research is needed to learn about the benefits of algal extracts, how to optimize their extraction and application, and what effect they have on soil health and crop yields over the long term.