Bell peppers (Capsicum annuum L.) are a highly nutritious food vegetable, rich in vitamins, minerals and antioxidants. In this study, using the nutrient film technique (NFT) system, the effect of zinc oxide nanoparticles on microscopic, histological, physiological and yield of grafted pepper was evaluated. The treatments used were grafted and ungrafted plants, four concentrations (0, 10, 20, 30 mg/L) of nano zinc oxide, and the experience was organized in a completely randomized design. The increase in the grafted plants in terms of weight, number and fruit size at 18.1%, 21.8% and 9.6%, the concentration of 30 mg/L of nanoparticles had a statistical effect on the weight. , number and size 46.9%, 47.7% and 18% compared with the control. Interaction with grafted plants and treatment of 30 mg/L nano zinc oxide increased fruit weight, fruit number and fruit size by 62.60%, 57.69% and 29.17% compared to ungrafted plants and witness. These results indicate that the use of grafts and zinc oxide nanoparticles can be used in bell pepper production to increase yield.
Bell peppers are one of the highest yielding vegetables worldwide (FAOSTAT, 2019), the world production of bell peppers in 2019 is 1,990,926 hectares harvested, of which the yield is 38,027,164 tons, energy The average yield is 19.10 tons/ha (FAO, 2019). In Mexico, 152,772.55 hectares were planted, of which the production was 3,238,244.81 tons, with a yield of 21.65 tons/ha (SIAP, 2019). However, pepper also has great contributions to human health, providing excellent nutritional value, it contains many nutritional properties and biological functions related to phenolic compounds, flavonoids, capsaicinoids, carotenoids, tocopherols, vitamins C, A, E, B, potassium, magnesium, iron, calcium and phosphorus (Ganguly et al., 2017; Vera-Guzmán et al., 2017). It is very important to know the microscopic, histological, physiological and yield processes in bell pepper plants, as it is one of the most consumed chili peppers both fresh, dried and processed. The micromorphology, histology, physiology and yield were positively modified by the transplantation and use of nanoparticles in bell pepper culture, the use of commercial varieties, and good adaptability to the conditions. of the area. There is currently no work documenting the effect of grafting and nanoparticles and their interaction on bell pepper production established and grown in a nutrient film engineering (NFT) system. Grafting is a special type of plant propagation in which a part of a plant (graft) is grafted with another plant (rootstock) so that the two parts grow together and form a new plant (Rouphael et al., 2018). ). By selecting the appropriate rootstock and with this technology, the morphology of the shoots can be adjusted and biotic and abiotic stress can be managed (Kumar et al., 2015; Kumar et al., 2017). ). Nutrient uptake and utilization in horticultural crops is enhanced by the selection of suitable rootstocks, which play an important role in regulating the nutritional status of shoots by directly influencing the uptake of nutrients. absorb and transport ions (Amiri et al., 2014). The use of suitable rootstocks tends to simultaneously improve the environmental, economic and social aspects of agriculture (Martínez-Andújar et al., 2020). NFT is a water-use system that contains dissolved nutrients essential for plant growth and can be an open or closed system in which the circulating nutrient solution is continuously in direct contact with the plant. directly with the root system of plants (Cooper, 1979). This hydroponic growing technique is gaining acceptance as it offers a high return on investment, supporting the growth of the hydroponic market (Love et al., 2015). NFT originated from England in 60s, it is developed to increase the productivity of hydroponic production field, generally it consists of PVC pipe, collection tank, circulating pump for nutrient solution (Triunfo) et al., 2018). It allows reuse of nutrients, for better use of resources, facilitates absorption in the root system, it is considered a closed system because the nutrient solution circulates as a root plate (Brenes and Jimenez, 2016), seeking to reduce water use, and thereby reduce production costs, the use of grafts would be beneficial with higher vigor, improved plant growth and protect against abiotic stress (Huang et al., 2015; Love et al., 2015; Miglietta et al., 2017). With nanotechnology, the efficiency of using micronutrients, macronutrients and pesticides in crops can be increased (Mazzaglia et al., 2017). Nanoparticle is defined as any engineered particle that is between 1 and 100 nanometers in size and has properties that are not shared by nano-sized particles of the same chemical composition (Auffan et al., 2009). ; Hajra and Mondal, 2017). However, little is known about the effect of nano zinc oxide (ZnO NPs) on the microscopic morphology, histology, physiology and yield of grafted bell peppers. The use of grafts gives vitality, improves absorption and transport of water and nutrients, affects the microscopic morphology, histology and physiology of plants (CamposcoMontejo et al., 2018; García-López et al., 2019; Salehi et al., 2010). Application of zinc oxide nanoparticles induces oxidative stress in plants as plant defense mechanisms generating secondary metabolites that influence micromorphology, histology and physiology (Mantoan et al. , 2016; Rossi et al., 2019; Zhu et al., 2020). The hypothesis used in this study is that the micro, histology, physiology and production are positively modified by grafting and using zinc oxide nanoparticles in bell pepper cultivation. Therefore, in this study, we report the effects induced in ‘SVEN RZ F1’ bell pepper grafted and cultivated in an NFT system with foliar application of zinc oxide nanoparticles.
Materials and methods
The present work was carried out during the spring-summer 2020 cycle in a greenhouse located in the Department of Horticulture at the Antonio Narro Autonomous Agricultural University, Buenavista, Saltillo, Coahuila, Mexico, at latitude 25°21’23.4 “, longitude 101° 02’10.6” and 1,760 meters above sea level Figure 1.
Plant material and growing conditions
The ‘SVEN RZ F1’ variety from Rijk Zwaan Seeds is a cube-shaped bell pepper characterized by short internodes, very well adapted to greenhouses, very hardy and high fruit setting in sunny conditions. hot. because it is a very precious material. The fruit is beautiful, bright yellow, very good quality, is a tree with tough vitality and tends to grow well. For the rootstock, ‘ULTRON F1’ pepper from the commercial seed company HM CLAUSE was selected, which is a hybrid of indeterminate growth with high vigor and tolerance to salinity, producing yellow mass fruit. Inside the greenhouse, the average irradiance achieved was 4.5 kWh m-2 day-1, the average recorded temperature was a maximum of 36°C and a minimum of 22°C, and the relative humidity was the average inside the structure is 40%.
The cuttings were sown in the greenhouse on February 7, 2020 using a 200-hole polystyrene tray Figure 2A, peat was used as a substrate, 10 days later, on February 17, 2020, the rootstock was sown in a 200-hole tray. , using peat as a substrate and one seed per cavity. The rootstock has stronger vigor and vegetative growth, that is why it is sown later, for this reason it reaches the stem size and width. There is a similarity in the thickness of the stem, which favors the combination of both plant structures.
On March 7, 2020, grafting was performed using the grafting technique (Lee Jung Myung, 1994) when the plants were 30 days after germination. Figure 2B. Jointing is performed when both structures have a base diameter of two mm. Rootstock and scion were cut at 60° from top to bottom respectively, both tree structures joined by a 2.0 mm silicone clamp.
Plant recovery begins immediately after grafting, where they are kept in a chamber with 80 to 85% relative humidity and between 25 and 28°C, for 10 days. On March 17, 2020, the silicone segment was completely removed as the fusion of the graft and the rootstock took place and healed. Plants were supplied daily with water, 25% nutrient solution (Steiner, 1961), and commercial leaf amino acid Metamin Max® (Agroestimulantes® Mexicanos SA de CV., Aguascalientes, Mexico) with a composition of 64.92% glutamic acid, 5.08% thiamine and 30% inert nutrients, dose of 1 g/L sprayed through the leaves by manual sprinkler.
Nutrition Film Technique (NFT)
The NFT system used consisted of 8 PVC pipes 6 inches in diameter, with 16 holes 6 cm wide, and with 30 cm between holes, 68 liter plastic containers used to collect the nutrient solution and submersible 25 watts. Pumps for aquariums with a circulation capacity of 1500 liters per hour are used to circulate the nutrient solution.
Transplant to the NFT . system
Transplantation was performed on April 13 and 14, 2020. Prophylaxis against pathogenic microorganisms. A 3-inch hydroponic plastic basket is used, to adjust the plants inside the basket, a polyurethane sponge is used so that the roots do not come into contact with the nutrient solution.
Nano zinc oxide
The ZnO NPNs used in the experiment were synthesized and characterized in the Pilot plant of the Center for Applied Chemistry (CIQA) Saltillo, Coahuila, Mexico Figure 3, their size is 52 nm, shape spherical, clear yellow-white powder, purity 99.8% as determined by X-ray, and coated with palmitic acid.
Applications of nano zinc oxide
Application was carried out by foliar route 10 days after graft healing, on March 27, 2020, different doses of nanoparticles were prepared using concentrations of 0, 10, 20 and 30 mg/l. The method for preparing different dosages is to use a nano zinc oxide-stock solution. Then, four doses of nanoparticles were prepared, using a 1 liter volumetric flask, each concentration of nanoparticles was poured separately and made up with distilled water. The solutions were placed in a manual watering can of 1 liter capacity, and the different nanoparticle treatments were uniformly applied on the bunch, underside of leaves and stems (buds). The first application of nanoparticles was during the vegetative phase, when the seedlings were 10 days after grafting. The second application of the nanoparticles was carried out during the flowering period 80 days after transplantation, the third application was carried out during the fruit-filling phase at 95 days after transplantation, on established plants. in the NFT system. Applications are made in the afternoon when a temperature of 22°C prevails, to avoid any problems associated with high heat and radiation.
Measuring Variables (Micromorphic Variables)
Epidermal sampling: To determine the number and size of stomata and leaf cells, fully-opened young leaves were used Figure 4A. The leaves are cut at the same plant height, have the characteristics of mature, full bloom and the same direction. Impressions were made immediately after leaf cutting, from both sides (top and bottom) ensuring that all were from the center of each leaf according to the method described in the manual (Hernández, 1984).
Materials used to print samples are PVC glue and transparent tape, paint brush and microscope slide. A light layer of glue is placed on the middle of the leaf (top and bottom), let it dry for 30 seconds, then cut a centimeter of tape and place it on top of the glue, let it dry and re-glue. The tape has been peeled off with the glue already attached. It is mounted on slides, three random samples are taken for each treatment (Hernández, 1984).
For the evaluation, a complex microscope (Carl Zeiss) with an integrated camera (Pixera Winder Pro) was used to take pictures of the samples above and below for the quantity, width, and length of the gas. size and number of epidermal cells Figure 4B. Measurement software was used (AxionVision Rel. 4.8; Carl Zeiss)
Stomatal Density (SD)
The stomatal density was assessed as follows:
SD = number of stomata /0.0247604 mm2 (image area), the result is the number of stomata per mm2.
SD = Number of stomata / image area (1)
Stomatal Index (SI)
The stomatal index was calculated according to the formula proposed by (Wilkinson, 1979). A compound microscope (Carl Zeiss) with digital camera (PixeraWinder Pro) and measurement software was used (AxionVision Rel. 4.8; Carl Zeiss).
Express the quotient between the number of stomata and the number of epidermal cells.
SI = stomatal density / epidermal cells + number of stomata * 100 (2)
Histological variables: number of xylem vessels and xylem vessel area
The histological examination procedure is as follows Figures 4C and 4D: Fixed in alcohol-formaldehyde acetic acid (FAA), and dehydrated with 50% and 60% alcohol for 30 min, 70%, 80% and 96% respectively for 3 hours, continue with t-Butyl alcohol I, t-Butyl alcohol II, t-Butyl alcohol plus xylol in a ratio of 3:1, t-Butyl alcohol plus xylol in a ratio of 1:1, t-Butyl alcohol plus with xylol in a ratio of 1: 3 and pure xylol, for 2 hours each solution. The inclusion of paraffin in an oven from 30°C to 55°C. Cuts were made in a manual microtome of 18 µm thickness, and were glued onto glass slides with adhesive and heat, colour, and adhesive. very fast green. Finally, the tissues were sealed with a drop of Canadian balsam and a slide the size of the tissue, and left to dry in an oven at 30 °C for one week (Hernández, 1984).
To measure gas exchange, a Li-6,800 cellular photosynthetic system (LI-COR, Inc., Lincoln, NE, USA) was used in the morphological production stage as it is a critical stage. in gas exchange, on 22 July 2020, data were obtained 100 days after implantation, using five vehicles per treatment, the light conditions were: light 379.20 µmol m⁻2 s ⁻1; CO2 550 ppm; temperature 32 ° C; 65% relative humidity, daytime data collection at 12 o’clock at night and with completely clear skies, a single measurement was performed, with the PAR and CO2 variables fixed. fixed and there was no change during the measurement. Figure 5A.
Production variables: fruit weight, number of fruits, pole and equator diameter
For the fruit weight variable, an I-2,000 Superior mini digital hand scale (METER8, Co., Shenzhen, China) was used to weigh 5 fruits per recipe Figure 5B. Number of fruits counted at cutting Figure 5C. For the variables of pole and equatorial diameter, a digital cutter HER-411 (Electrónica Steren S.A. de C.V., Azcapotzalco, Mexico) was used, measuring 5 fruits for each treatment Figure 5D.
The experimental design used for experimental development was completely randomized with factorial arrangement (2 * 4). With the data obtained, an analysis of variance (ANOVA) was performed; To detect statistical differences between treatments, Tukey’s comparison of means test was used (p ≤ 0.05). The factors were: with and without grafting, four concentrations of 0, 10, 20 and 30 mg L-1 of zinc oxide nanoparticles resulting in eight treatments and four replicates, statistical software was used. for information analysis (InfoStat edition 2014 Grupo InfoStat, FCA, Universidad Nacional de Córdoba, Argentina).
Microscopic and histological results
Results of the graft effect on microscopic and histological variables. Grafting affects histological variables with a statistical difference in the grafted trees in the xylem vessel area variable (XVA), which is 37.8% larger than in the ungrafted trees, relative to the xylem vessel variable (NXV). ) in non-grafted plants had 20.1% more vessels than grafted tree Table 1.
Effect ofnano zinc oxide ZnO NPs on microscopic and histological variables.
The application of nanoparticles had a statistical effect on the variation of stomatal index (IE), at a concentration of 30 mg L-1 had an index 3.8% higher than the control, while the length of the stomata. at the bottom (LSB) increased by 12.1%. compared with the concentration of 10 mg L-1 and 15.1% compared with the control. Variables length of upper stomata (LST), width of upper stomata (WST) and width of stomatal base (WSB) did not differ between treatments Table 1.
Results of the graft vs nano zinc oxide interaction in microscopic and histological variables.
Table 1, the combination of coupling effect and application of nanoparticles, in which coupling and concentration of 30 mg L-1 have statistical differences in the variables of stomatal density (SD) and index stomata (SI). Regarding LSB, the concentration of 30 mg L-1 combined with grafted and non-grafted plants gave the highest value. For ungrafted WXV plants with 30 mg L-1 interaction and control, significant differences were found for grafted plants and 10, 20, 30 mg L-1 and control nanoparticle concentrations. The combination of the coupling effect and 10 mg L-1 of the nanoparticles had the highest NXV.
Match results on physiological variables
Effect of grafting on variables of CO assimilation (CA), stomatal conductance (SC), transpiration rate (TR), photosynthetic efficiency (PE) and light capture efficiency ( ELC) no
statistical difference, Table 2.
Results of nano zinc oxide ZnO NPs in physiological variables
The nanoparticle effect had a negative statistical difference on the concentration of 30 mg L-1 compared with the control, the results are described below Table 2. Regarding the CA variable, the concentration of 30 mg L-1 increased by 60.7% compared with the control. , SC increased by 29.8% compared to the control. PE increased by 29.2% compared to the control and ELC increased by 60.7% compared to the control. In the variable TR, there was no statistical difference between concentrations of 30 and 20 mg L-1, but concentrations of 30 mg L-1 were 39.4% higher than concentrations of 10 mg L-1 and 44.8. % in the control table. 2. Results of the graft interaction vs ZnO NPs in physiological variables Physiological variables are affected by the interaction, which increases the values of CA, SC, PE and ELC. In contrast, in the variable TT, the control gave the lowest value in Table 2. The interaction between factors did not cause the difference between grafted and ungrafted plants, but had an effect on other concentrations and the control, affect physiological variables.
Compound results on production variables
Grafting statistics increased fruit weight (FW) with a 21.8% increase in fruit, an 18.1% increase in fruit number (NF) and a pole diameter (PD) in fruits with a diameter greater than 9.6% compared to trees. do not combine Table 3 .
Results of nano zinc oxide ZnO NPs on yield variables
The application of nanoparticles had a statistical effect on the variables FW, NF, PD and equatorial diameter (ED), with statistical differences between different concentrations. PF is affected when increasing
weight 46.9% compared to the control. For NF, the 30 mg L-1 treatment increased 47.7% compared to the control. PD did not differ between concentrations of 30, 20 and 10 mg L-1, but it was compared with the control, where the difference in diameter was 18%. For ED, concentrations of 30, 20, and 10 mg L-1 showed no difference between them, as did 10 mg L-1 concentrations and control with no statistical difference. On the other hand, the concentration of 20 mg L-1 increased by 16.1% and the concentration of 30 mg L-1 increased by 20.2% in the equatorial diameter compared with the control in Table 3.
Results of the interaction between graft and nano zinc oxide ZnO NPs in yield variables
Combinations of effects on production variables Table 3, where the highest results obtained with grafting and nanoparticle application at 30 mg L-1 concentration compared with the lowest results obtained achieved when combining non-grafted plants and control treatment.
The results obtained in this study, Table 1 has a smaller xylem vessel area and a lower number of vessels, which increases the efficiency of xylem displacement, concurrent with the results obtained by Salehi et al. (2010) when all grafted trees tended to carry higher amounts of xylem sap than ungrafted plants. Albornoz et al. (2020) reported that the grafted plants had a higher number of xylem vessels than the control (non-grafted plants). The results of this trial agree with those of Camposeco et al. (2018) where in bell pepper leaves, the length of the stomata was statistically significant, greater than in the grafted bell pepper, exceeding the ungrafted bell pepper by 8.64 and 11.22%, in the same way, people also reported that the stomatal index and density were increased when rootstock were used, increasing the gas exchange and photosynthetic efficiency of the plants. The results are similar to those reported by Albornoz et al. (2020) where the number of vessels and their diameters, in grafting treatment, were similar in autografted and ungrafted plants. Vascular connectivity through the xylem strongly correlates with the appearance of xylem continuums across the junction (Melnyk et al., 2015). Orsini et al. (two thousand and thirteen); Penella et al. (2017) reported that the use of resistant rootstocks improved the photosynthetic performance of grafted shoots under abiotic stress conditions. Xu et al. (2016); Baron et al. (2018) reported that leaf gas exchange in grafted plants was directly affected by grafting, by altering the viability and yield of grafted species. On the other hand, grafting did not affect the micromorphology of the plants compared with ungrafted plants. On the other hand, a trend was shown in the nanoparticle effect at a concentration of 30 mg L-1, a result that is consistent with that reported by García-López et al. (2019) in habanero pepper where foliar application with ZnO NP at 1000 mg L-1 had a greater impact on plant growth and physiology than conventional Zn salt (ZnSO4), possibly due to its ability greater leaf absorption. Rossi et al. (2019), Raliya et al. (2015) reported that NPs in ZnO have dual roles as an essential nutrient and a cofactor for nutrient-mobilizing enzymes. Zhu et al. (2020) reported that Zn administration increased endogenous hormones (auxin, gibberellins and melatonin) and improved aquaporin activity and antioxidant system, thereby supporting photosynthetic efficiency. Regarding the interaction of factors, a trend was found at a concentration of 30 mg L-1 that was not affected by the graft. This is probably due to the greater assimilation of Zn when applied in the . form
Nano zinc oxide due to their higher foil penetration ability (Rossi et al., 2019). On anabolic variables Mantoan et al. (2016) concluded in the study of Annona emarginata used as a benchmark for Atemoya, it has a balance between perspiration and CO2 assimilation rate to optimize water use efficiency, it gives in gas exchange and photochemical processes. Our results in Table 2, agree with those obtained by Dabirian and Miles (2017), who found that the graft survived longer due to reduced stomatal conductivity and possibly simultaneously. reduce perspiration. The results differ from those obtained by Ayala-Arreola (2010), where graft and nanoparticle interactions affect the increase in sweat rate, CO2 assimilation rate and stomatal conductivity. The use of grafts affects the yield variables Table 3, where fruit weight, fruit number and pole diameter increase, a result that is consistent with the results obtained by Velasco-Alvarado et al. (2019) in which the grafted tree yielded 7.4 kg/plant, the yield was 19% superior to that of the non-grafted plant, 6.2 kg/plant. Fruit weight was affected by rootstock, but not by grafting Ergun and Aktas (2018) reported that the yield of bell pepper grafting was about 12% higher than that of the ungrafted control. Soteriou and Kyriacou (2015) reported a study of watermelons that grafted grafts increased commercial yield by an average of 43% compared with non-grafted controls. Riga (2015) reported grafting effects affecting most of the tomato quality characteristics that were strongly influenced by the sample combination.
The use of nano zinc oxide ZnO NPs at a concentration of 30 mg L-1 tended to increase because when applied they resulted in higher fruit weight, number of fruits, and pole and equatorial diameters obtained, the results agreeing. with results reported by García-López et al. (2019) in habanero pepper, where similarly, the maximum mean weight of fruit obtained with ZnO NPs was 1000 mg L-1, which exceeded the control treatment by 7%. Du and associates. (2019); Servin et al. (2015) reported that the application of nano zinc oxide ZnO NPs in all treatments increased wheat grain yield, Adhikari et al. (2016) reported that when coating 50 mg of ZnO NP in corn kernels, it promoted dry sprout weight of 22.35 g, compared with the NP-free control of 13.70 g. Elizabeth and associates. (2017) reported that in a study conducted on carrots fertilized with zinc oxide, the yield and plant growth were increased compared with the control treatment. Saadati et al. (2016) reported higher fruit weight when applying Zn with an average of 3.16 kg. Zinc is a phytohormone production promoter promoting an increase in fruit development and production, it obtained a significant difference compared with the control treatment of 3.08 kg (Elizabath et al., 2017) ). The use of Zn increases endogenous hormones (auxin, gibberellins and melatonin) that will be beneficial for fruit growth, development and production (Zhu et al., 2020). Works performed by García-López et al, interaction, coupling and 30 mg L-1 of nanoparticles directly affected the yield with trends in all variables and yielded energy values. highest capacity. (2019) reported maximum mean fruit weight using ZnO NP at 1000 mg L−1, exceeding the control treatment by 7% and the ZnSO4 treatment by 3.6% Penella et al. (2017) reported that grafted tomato plants had increased commercial yield compared with non-grafted plants (44 and 40% higher, respectively). Ergun and Aktas (2018) reported that fruit length was significantly affected by grafting.
The graft does not affect microscopic morphology, but it does affect histology where the number of vessels and xylem vascular area are increased. Physiology is not affected by graft use; on the other hand, yield variables increased, such as fruit weight, number of fruits and pole diameter. The application of zinc oxide nanosheets affects microscopic morphology by increasing stomatal density, stomatal index and length of the stomatal base. The nanoparticles did not affect histology, while they influenced the physiology, where CO assimilation, stomatal conductivity, transpiration rate, photosynthetic efficiency, light capture efficiency, yield for found a favorable effect, which translates into fruit weight, pole diameter and equatorial diameter. Interaction between factors affects microscopic morphology by increasing stomatal density, stomatal index and length of basal stomata, histology is influenced by the interaction between xylem vessel area and number of xylem vessels, physiology affects only the largest application of nanoparticles, increasing CO2 assimilation, stomatal conductivity, transpiration rate, photosynthetic efficiency and light capture efficiency . Yields increased with the interaction, with the best results in terms of fruit weight, number of fruits, pole and equatorial diameters. Due to the results obtained in this study, we recommend the use of zinc oxide nanoparticle grafting and foliar application, which can be used in bell pepper production to increase yield.