Application of nanohydroxyapatite in alleviating abiotic stress of microplastics on plants

Abstract

The present invention discloses the application of nanohydroxyapatite in alleviating the abiotic stress of microplastics on plants. Nanohydroxyapatite is added to a sodium citrate solution to form a suspension, ultrasonically treated, centrifuged, and dried to obtain sodium citrate-modified nanohydroxyapatite. In soil containing microplastics, after plants are sown and seedlings are emerged or transplanted, adding sodium citrate-modified nanohydroxyapatite to the soil can alleviate the abiotic stress of microplastics on plants. The present invention uses sodium citrate to modify nanohydroxyapatite to alleviate the abiotic stress of MP generated after the degradation of PBAT mulch on plants. Not only can the slow release and efficient utilization of P be achieved, but the soluble sugar content, soluble protein content, and VC content of peppers can also be increased under MP stress, the flowering and fruiting periods of peppers can be advanced, the number of flowers and fruits of peppers and the plant height can be increased, and the malondialdehyde content of peppers can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of plant stress resistance technology, and in particular to the application of nano-hydroxyapatite in alleviating abiotic stress of microplastics on plants. Background Art

[0002] In 2004, Richard C. Thompson coined the term "microplastics," referring to particles typically present as fragments, fibers, or films, typically less than 5 mm in size. As an emerging pollutant, the environmental impacts of microplastics have become a hot topic of research. Once introduced into soil, microplastics not only alter soil physical and chemical properties (such as pH, bulk density, water storage capacity, and nutrient availability) but also reduce the activity and functional diversity of soil microorganisms. These effects can directly impact plant health and performance by altering root characteristics, nutrient uptake, and growth, leading to severe white pollution. Terrestrial ecosystems, particularly agricultural soils, serve as a long-term reservoir for microplastics (MPs). MPs infiltrate agricultural soils from a variety of sources, including landfills, sewage irrigation, the application of biosolids and compost, polymer-based fertilizers and pesticides, and atmospheric deposition. Agricultural mulch films are also a type of application that releases significant amounts of micro- and nanoplastics (MNPs). To reduce and prevent residual pollution from mulch films, biodegradable mulch films have emerged with the development of biodegradable materials. These materials are gradually being used in agricultural production as an ideal solution to the global problem of "white pollution." For example, polybutylene terephthalate (PBAT), due to its excellent softness and ductility, is widely used as a raw material for biodegradable mulch films and is one of the most common and widely used biodegradable materials on the market. Current studies have shown that the impact of biodegradable plastics on plants may be much more severe than that of low-density polyethylene (LDPE). Wang (Wang F, Zhang X, Zhang S, et al. Interactions of microplastics and cadmium on plant growth and arbuscular mycorrhizal fungal communities in an agricultural soil [J]. Chemosphere, 2020, 254: 126791) reported that 10% polylactic acid (PLA) significantly inhibited photosynthesis and reduced corn biomass. Furthermore, 10% 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) caused wheat plant death after 25 days.Qi et al. found (Qi Y, Yang X, Pelaez AM, et al. Macro-and micro-plastics in soil-plant system: effects of plastic mulch film residues onwheat (Triticum aestivum) growth[J]. Science of the Total Environment, 2018,645: 1048-1056) that starch-based biodegradable microplastics have stronger negative effects on wheat than low-density polyethylene microplastics, including damage to leaf area, inhibition of plant height, delayed tillering, reduced fruit set rate, and reduced biomass and yield; Lian Yuhang et al. found (Lian Yuhang, Liu Weitao, Shi Ruiying, et al. Effects of polyethylene and polylactic acid microplastics on soybean growth, physiology, biochemistry and metabolism[J]. Chinese Environmental Science, 2022, 42(6): 2894-2903.) PLA-MPs can significantly inhibit soybean root length and affect the antioxidant system of soybean leaves. In addition, it can also lead to the downregulation of organic acid metabolism pathways and carbohydrate metabolism pathways in leaves; Boots et al. ( Boots B, Russell CW, Green D S. Effects ofmicroplastics in soil ecosystems: above and below ground[J]. Environmentalscience&technology, 2019, 53(19): 11496-11506) showed that PLA-MPs significantly inhibited the germination rate and stem length of ryegrass, while high-density polyethylene microplastics had little effect on it; deSouza Machado et al. ( de SouzaMachado AA, Lau CW, Kloas W, et al. Microplastics can change soilproperties and affect plant performance[J]. Environmental science&technology,2019, 53(10): 6044-6052) Studies have found that MPs significantly affect the biomass, root characteristics, elemental composition of tissues, and soil microbial activity of green onions. Biodegradable plastics, once introduced into the soil, can completely decompose in around 100 days, leading to the production and accumulation of MPs. This can also lead to the short-term production of more biodegradable microplastics, posing a threat to plant and soil health.

[0003] To mitigate the toxic effects of microplastics on crops, Professor Yao Xiaoqin's team (Geng Z, Zhao B, Duan Y, et al. Phosphorus mitigates the adverse effects of microplastics pollution on wheat and maize: Impacts on growth, photosynthesis, and antioxidant defense [J]. Environmental and Experimental Botany, 2024, 228: 105993.) explored the potential role of superphosphate in improving crop tolerance to microplastic pollution. Experiments showed that superphosphate application mitigated the effects of microplastic pollution by improving photosynthetic pigments, peroxidase (POD), and polyphenol oxidase (PPO) activities in wheat and maize. It also improved the AsA-GSH cycle by increasing antioxidant enzyme activity in wheat and non-enzymatic antioxidant content in maize, thereby mitigating the effects of microplastic pollution. However, the phosphate fertilizer used in this study lacks a slow-release mechanism, limiting its effectiveness in mitigating abiotic stress caused by microplastics. Furthermore, after entering the soil, some of this phosphate fertilizer is oxidized and fixed by iron and aluminum in the soil, preventing efficient phosphorus utilization. Therefore, a more effective method is needed to alleviate the abiotic stress of plants caused by mulch-derived microplastics. Summary of the Invention

[0004] In response to the above-mentioned prior art, the present invention aims to provide the application of nanohydroxyapatite to alleviate the abiotic stress posed by microplastics to plants. This invention uses sodium citrate to modify nanohydroxyapatite to alleviate the abiotic stress posed by MP produced by the degradation of PBAT mulch. This method not only achieves sustained release and efficient utilization of MP, but also increases the soluble sugar, soluble protein, and vitamin C content of peppers under MP stress, accelerates the flowering and fruiting periods of peppers, increases the number of flowers and fruits, and increases plant height, while reducing malondialdehyde content in peppers.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of nano-hydroxyapatite in alleviating the abiotic stress of microplastics on plants. In soil containing microplastics, after plants are sown, seedlings are emerged or transplanted, nano-hydroxyapatite is added to the soil to alleviate the abiotic stress of microplastics on plants.

[0007] Preferably, the microplastics are microplastics derived from ground film; the ground film source comes from biodegradable plastic ground film.

[0008] More preferably, the material of the biodegradable plastic mulch is PBAT.

[0009] Preferably, the nano-hydroxyapatite is added to the 5-10 cm soil layer.

[0010] Preferably, the nano-hydroxyapatite is sodium citrate-modified nano-hydroxyapatite; the preparation method of the sodium citrate-modified nano-hydroxyapatite is:

[0011] Nano-hydroxyapatite is added into a sodium citrate solution to form a suspension, which is then ultrasonically treated, centrifuged and dried to obtain sodium citrate-modified nano-hydroxyapatite.

[0012] More preferably, the mass ratio of sodium citrate to nano-hydroxyapatite is 1-2:5; the ultrasound is performed at room temperature for 12 hours, and the frequency of the ultrasound is 40 kHz.

[0013] Preferably, the plant is pepper.

[0014] Preferably, the amount of the nano-hydroxyapatite added to the soil is 20-100 mg P / kg in terms of phosphorus application.

[0015] More preferably, the amount of the nano-hydroxyapatite added to the soil is 50 mg P / kg in terms of phosphorus application.

[0016] Preferably, the application includes: increasing the soluble sugar content of pepper fruits, increasing the soluble protein content of pepper fruits, increasing the VC content of pepper fruits, reducing the malondialdehyde content of pepper leaves, advancing the flowering and fruiting periods of peppers, and increasing the number of flowers, fruiting numbers and plant height of peppers.

[0017] Beneficial effects of the present invention:

[0018] (1) The present invention modifies nanohydroxyapatite with sodium citrate, effectively preventing nanohydroxyapatite from agglomerating and improving its ability to slowly release phosphorus. The phosphorus contained in the nanohydroxyapatite is not easily oxidized and fixed by iron, aluminum, and other substances in the soil. The biocompatibility and low toxicity of nanohydroxyapatite, as well as the natural presence of its degradation products (i.e., phosphate and calcium ions) in the soil, make it an alternative phosphate fertilizer, thereby improving the utilization rate of phosphate fertilizer.

[0019] (2) The present invention utilizes sodium citrate to modify nanohydroxyapatite to effectively alleviate the abiotic stress of MP produced after the degradation of PBAT mulch film on plants, improve the quality of plant fruits, and effectively promote the flowering and fruiting dynamics of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Flowering kinetics of pepper in each treatment group;

[0021] Figure 2 : The result kinetics of pepper in each treatment group;

[0022] Figure 3 : Plant height of pepper in each treatment group;

[0023] Figure 4 : Phosphorus slow release diagram under three treatments. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0025] As described in the background technology section, phosphorus supply improves photosynthetic pigments, peroxidase (POD), and polyphenol oxidase (PPO) activity in wheat and corn. It also improves the AsA-GSH cycle by increasing antioxidant enzyme activity in wheat and non-enzymatic antioxidant content in corn, thereby mitigating the impact of microplastic pollution. However, the addition of conventional phosphate fertilizers such as superphosphate cannot effectively control the release of phosphorus. Furthermore, after entering the soil, these phosphate fertilizers are partially oxidized and fixed by iron, aluminum, and other substances in the soil, preventing efficient phosphorus utilization.

[0026] Based on this, the purpose of the present invention is to provide the application of nanohydroxyapatite in alleviating the abiotic stress of microplastics on plants. The present invention uses sodium citrate to modify nanohydroxyapatite and add it to the soil, which can effectively alleviate the abiotic stress of MP produced by the degradation of PBAT mulch on plants. Sodium citrate has mild acidity and can stimulate the growth of beneficial microorganisms in the soil, promote the activity of soil enzymes, and improve soil texture. Modifying nanohydroxyapatite with sodium citrate can avoid the agglomeration of nanohydroxyapatite and further improve the sustained release of P. However, the amount of sodium citrate added needs to be controlled. The best effect is when the mass ratio of sodium citrate to nanohydroxyapatite is 1~2:5. In addition, the effect of sodium citrate-modified nanohydroxyapatite in alleviating the abiotic stress of MP on plants is not the same as the more it is added. The optimal concentration in the soil is 50mg P / kg.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1

[0030] 0.15 g of sodium citrate was dissolved in 20 mL of distilled water to obtain a sodium citrate solution, and 0.5 g of nanohydroxyapatite was added to the sodium citrate solution to form a nanosuspension; the ultrasonic frequency was adjusted to 40 kHz, and ultrasonication was performed at 25° C. for 12 h; the powder material was centrifuged and dried in a vacuum drying oven at 60° C. for 24 h to obtain sodium citrate-modified nanohydroxyapatite.

[0031] Comparative Example 1

[0032] The difference from Example 1 is that 0.05 g of sodium citrate was added.

[0033] Comparative Example 2

[0034] The difference from Example 1 is that 0.5 g of sodium citrate was added.

[0035] Test Example 1

[0036] The experiment was divided into 10 groups: a normal group, a blank group, a superphosphate group, a nanohydroxyapatite group, a sodium citrate group, an example group (Example Groups 2-4), and a comparative example group (Comparative Example Groups 1-2). Except for the normal group, all other groups added PBAT microplastics to potting soil and transplanted pepper seedlings to simulate abiotic stress caused by MP on peppers.

[0037] Among them, the normal group used soil without PBAT microplastics and did not add any phosphate fertilizer; the blank group used soil containing PBAT microplastics and did not add any phosphate fertilizer; the superphosphate group used soil containing PBAT microplastics and added superphosphate at a concentration of 50 mg P / kg; the nanohydroxyapatite group used soil containing PBAT microplastics and added nanohydroxyapatite at a concentration of 50 mg P / kg; the sodium citrate group used soil containing PBAT microplastics and added sodium citrate at the same concentration as that of the sodium citrate in the Example 3 group; the Example 2 group used soil containing PBAT microplastics and added sodium citrate-modified nanohydroxyapatite prepared in Example 1 at a concentration of 20 mg P / kg; the Example 3 group used soil containing PBAT microplastics and added sodium citrate-modified nanohydroxyapatite prepared in Example 1 at a concentration of 50 mg P / kg; the Example 4 group used soil containing PBAT microplastics and added sodium citrate-modified nanohydroxyapatite prepared in Example 1 at a concentration of 100 mgP / kg; Comparative Example 1 group used soil containing PBAT microplastics, and added sodium citrate-modified nanohydroxyapatite prepared in Comparative Example 1 at a concentration of 50 mg P / kg; Comparative Example 2 group used soil containing PBAT microplastics, and added sodium citrate-modified nanohydroxyapatite prepared in Comparative Example 2 at a concentration of 50 mg P / kg.

[0038] The test soil was screened to ensure that it did not contain microplastics, and 0.8 kg (dry weight) of the test soil was placed in the pots of the normal group. Nine other groups of screened test soil (0.8 kg (dry weight)) were taken and PBAT microplastics (concentration of 1% w / w, particle size 30-65 μm) were added to them. Phosphate fertilizer or sodium citrate was added according to the settings of the above groups. These were then placed in pots and recorded as the blank group, superphosphate group, nanohydroxyapatite group, sodium citrate group, example groups (Example groups 2-4), and comparative example groups (Comparative example groups 1-2).

[0039] Before formal cultivation, pepper seeds (sweet pepper seeds numbered CJ20 provided by Sichuan Agricultural University) were soaked in 55°C hot water for 15 minutes for disinfection and sterilization. They were then taken out and repeatedly rinsed with deionized water. They were then soaked in warm water for 2 hours to allow the seeds to absorb enough water. Finally, the seeds were taken out and neatly placed in seedling pots and placed in a constant temperature incubator at 28°C for seedling cultivation. When the seedlings grew to five leaves and one heart, pepper seedlings with consistent growth were selected and one plant was transplanted to the pot of each treatment. Four replicates were performed for each treatment.

[0040] All 10 groups received nitrogen fertilizer (ammonium sulfate) and potassium fertilizer (potassium sulfate) as base fertilizer, each at a dosage of 40 mg / kg. Fertilization was broadcast, with nitrogen and potassium fertilizers applied as topdressing twice, at the initial flowering and fruiting stages, at a dosage of 35 mg / kg. Soil moisture was maintained at approximately 70% of field capacity during the growing season, and pots were moved every two days during the planting period to ensure weed and pest-free conditions.

[0041] During the planting period, the plant growth was analyzed. When the peppers entered the budding stage, three plants with medium and relatively consistent growth were selected from each treatment. The plant height and leaf SPAD values ​​were measured every 7 days until the last yield measurement. After 60 days, the peppers were mature and harvested. The growth indicators of each group of plants (fresh weight and dry weight of each part of the plant, number of flowers and fruits, root morphology, stem height and stem thickness) were recorded. The soluble sugar, soluble protein, and VC content of the pepper fruits and the malondialdehyde content of the pepper leaves were measured. The results are shown in Figures 1-3 and Tables 2~3.

[0042] Soluble sugar was determined using the anthrone colorimetric method:

[0043] Extraction of soluble sugars from samples:

[0044] Weigh 0.5-1.0 g of the chopped and mixed pepper fruits from each group, place them in a large test tube, add 15 mL of distilled water, boil in a boiling water bath for 20 min, remove and cool, filter into a 100 mL volumetric flask, rinse the residue several times with distilled water, and dilute to the mark.

[0045] Sample determination:

[0046] Take 1.0 mL of the sample extract and add 5 mL of anthrone reagent (0.2 g anthrone dissolved in 100 mL of concentrated sulfuric acid, prepared and used today). Follow the above procedure to develop color and determine the optical density. Repeat three times.

[0047] Calculation results:

[0048] Soluble sugar content (%) = amount of sugar obtained from the standard curve (μg) × volume of extract (mL) × dilution factor / (volume of sample solution for determination (ml) × sample weight (g) × 10 6 )×100%.

[0049] Soluble protein content was determined by Coomassie Brilliant Blue G-250 staining:

[0050] 1. Reagents and preparation

[0051] Extract (50mmol·L -1 Tris-HCl, pH 7.8, containing 0.5 mmol L -1 MgCl2, 1 mmol·L - 1 EDTA,).

[0052] Preparation of Coomassie Brilliant Blue Solution: Weigh 100 mg of Coomassie Brilliant Blue G-250, add 50 mL of 95% ethanol and 100 mL of 85% phosphoric acid, and then dilute to 1000 mL. 0.15 mol·L -1 NaCl solution.

[0053] Preparation of bovine serum albumin standard solution: use 0.15 mol·L -1 NaCl solution was prepared to 100 μg ml -1 Bovine serum albumin standard solution.

[0054] 2. Preparation of bovine serum albumin standard curve

[0055] Take 6 test tubes, number them, and prepare bovine serum albumin standard solution with a content of 0-100 μg in each tube according to Table 1.

[0056] Table 1

[0057]

[0058] After adding the reagents in Table 1, shake well, use tube 0 as a blank control, and measure its absorbance (A) value at a wavelength of 595 nm.

[0059] Standard curve drawing: draw the standard curve with the bovine serum albumin content as the horizontal axis and the absorbance value as the vertical axis.

[0060] 3. Protein extraction

[0061] Weigh 0.3-0.5 g of pepper fruit obtained from each group, chop it into pieces, add 3 mL of extract solution and a small amount of quartz sand in a frozen mortar, and quickly grind it into a homogenate in an ice bath. Pour the homogenate into a centrifuge tube, and then wash the homogenate in the mortar into the centrifuge tube with 5 mL of extract solution (twice), and then centrifuge it at 10,000 r / min and 4°C for 20 min. The supernatant is the soluble protein extract.

[0062] 4. Determination

[0063] Take 0.1 mL of each of the above supernatants and place them into two test tubes respectively. Add 0.9 mL of Tris buffer to each tube and 1 mL of Tris buffer to the blank control tube. Then add 5 mL of Coomassie Brilliant Blue staining solution to each tube, shake well, and measure the absorbance (A) value at a wavelength of 595 nm.

[0064] 5. Result calculation

[0065] According to the measured absorbance value of the sample solution, the protein content is found from the standard curve, and the soluble protein content is calculated according to the following formula:

[0066] .

[0067] The Vc content was determined by the molybdenum blue colorimetric method (Li Jun, "Food Science", 2000).

[0068] Malondialdehyde content was determined by the following method:

[0069] Experimental potion:

[0070] 1. 10% trichloroacetic acid (TCA);

[0071] 2. 0.5% thiobarbituric acid (dissolved in 10% trichloroacetic acid);

[0072] 3. Quartz sand.

[0073] Experimental methods:

[0074] 1. Take 1-2 chili leaves, wash and dry them, cut them into 0.5 cm long pieces, and mix them well.

[0075] 2. Weigh 0.3 g of the leaf pieces, place them in an ice-bathed mortar, add 2 mL of 10% TCA and a small amount of quartz sand, and grind until homogenous.

[0076] 3. Then centrifuge at 4°C, 12,000 ppm for 15 min and collect the supernatant.

[0077] 4. Take 1.5 mL of the supernatant from the centrifugation (add 1.5 mL of 10% TCA for the control), add the same volume of 0.5% TBA solution, and react the mixture in a boiling water bath for 30 minutes. Cool it quickly and then centrifuge it again.

[0078] 5. Take the supernatant and measure the absorbance at wavelengths of 532nm, 450nm and 600nm.

[0079] Calculated content:

[0080] The MDA content in the sample was calculated based on the weight of the plant tissue:

[0081] MDA concentration (μmoL / L) = 6.45·(OD 532 -OD 600 )-0.56·OD 450 .

[0082] Table 2 Root morphology

[0083]

[0084] Table 3 Physiological indicators

[0085]

[0086] according to Figures 1-3 It can be seen that under the stress of mulch-derived microplastics (PBAT), compared with conventional phosphate fertilizers, sodium citrate, and nanohydroxyapatite, the application of sodium citrate-modified nanohydroxyapatite can advance the flowering period and increase the number of flowers; it can also advance fruiting and increase the number of fruits; and it can also increase plant height. This shows that sodium citrate-modified nanohydroxyapatite can improve the flowering and fruiting dynamics under microplastics (PBAT) stress.

[0087] It can be seen from Table 2 that the peppers in Example 3 group have more developed root systems, which is beneficial to nutrient absorption and plant growth.

[0088] As shown in Table 3, under PBAT stress, at a phosphorus concentration of 50 P / kg, the pepper plants treated with sodium citrate-modified nanohydroxyapatite had the highest soluble sugar content, soluble protein content, and VC content, while the malondialdehyde content was the lowest. This indicates that sodium citrate-modified nanohydroxyapatite can improve the quality of peppers under microplastic PBAT stress.

[0089] Test Example 2

[0090] The experiment was divided into three groups: a superphosphate group, a nano-hydroxyapatite group, and an Example 3 group, with the specific settings being the same as those of Experimental Example 1.

[0091] The release of phosphate from the superphosphate group, nanohydroxyapatite group and Example 3 group was analyzed in aqueous solution. The three groups of phosphate fertilizers were added to sealed glass bottles containing deionized water to a concentration of 200 ppm P, and placed on a constant temperature oscillator controlled at 25°C. Samples were taken at corresponding times (1, 3, 5, 10, 20, 30, 40, 50, 60, 90, 120, 180 min). The concentration of phosphate was measured by UV-Vis spectrometer (Thermo Scientific, GENESYS50, USA), and the absorbance at 880 nm was recorded using a UV-Vis spectrometer using the molybdenum blue method. The P release of the three groups is shown in Figure 4 .

[0092] according to Figure 4 It can be seen that the superphosphate group released all the P after contacting water. The nanohydroxyapatite group and the Example 3 group both showed a slow release of P, but the P release rate of Example 3 was higher, which could meet the P demand of peppers during growth.

[0093] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of nanohydroxyapatite in alleviating abiotic stress of microplastics on plants, characterized in that: In soil containing microplastics, after plants are sown or transplanted, nanohydroxyapatite is added to the soil to alleviate the abiotic stress of microplastics on plants; The microplastics are microplastics from ground film sources; the ground film sources are biodegradable plastic ground films; The material of the biodegradable plastic mulch is PBAT; The nano-hydroxyapatite is sodium citrate-modified nano-hydroxyapatite; the preparation method of the sodium citrate-modified nano-hydroxyapatite is: Nanohydroxyapatite is added to a sodium citrate solution to form a suspension, subjected to ultrasonic treatment, centrifuged, and dried to obtain sodium citrate-modified nanohydroxyapatite; the mass ratio of sodium citrate to nanohydroxyapatite is 1-2:5; the ultrasonic treatment is performed at room temperature for 12 hours at a frequency of 40 kHz; The plant is pepper; The amount of the nano-hydroxyapatite added to the soil is 50 mg P / kg, calculated as the amount of phosphorus applied.

2. The use according to claim 1, characterized in that The nano-hydroxyapatite is added into the 5-10 cm soil layer of the soil.

3. The use according to claim 1, characterized in that The applications include: increasing the soluble sugar content of pepper fruits, increasing the soluble protein content of pepper fruits, increasing the VC content of pepper fruits, reducing the malondialdehyde content of pepper leaves, advancing the flowering and fruiting periods of peppers, and increasing the number of flowers, fruiting numbers and plant height of peppers.

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