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Article

In Vitro Screening and Field Performance of EMS-Treated Eggplants for the Selection of Shoot and Fruit Borer-Resistant Plants

by
Md Ashraful Islam
1,
Md Muntasir Bin Mohi Uddin
1,
Md Golam Rasul
1,
Md Ahsanul Haque Swapon
2,
Minhaz Ahmed
3 and
Mehfuz Hasan
1,*
1
Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh
2
Department of Entomology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh
3
Department of Agroforestry and Environment, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(8), 1832; https://doi.org/10.3390/agronomy12081832
Submission received: 30 June 2022 / Revised: 26 July 2022 / Accepted: 29 July 2022 / Published: 2 August 2022
(This article belongs to the Special Issue In Vitro-Based Systems for Horticultural and Floricultural Crop Improvement)
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Versions Notes

Abstract

:
The shoot and fruit borer has asserted itself as a severe pest of eggplant. In vitro mutagenesis is an excellent method for producing mutants resistant to biotic and abiotic stresses. This study aimed to create eggplant mutants that were resistant to shoot and fruit borer infestation. In the Murashige and Skoog (MS) medium, two genotypes, including a landrace, were treated with varying amounts of EMS. Among the treatments, MS medium supplemented with 2% EMS demonstrated the most favorable effect. Explant performance dropped by up to 1.5% with an increase in EMS in the medium. The 2% EMS dose was appropriate for in vitro mutant eggplant development, but the higher dose was extremely damaging. EMS made several mutants sterile. Three landrace mutants were insect-resistant. Total phenols, leaf chlorophylls A and B, antioxidants, and solasodine were abundant in these genotypes. Correlation studies found a link between affected fruits and shoots and total phenols, antioxidants, and solasodine. Solasodine was shown to be related to chlorophylls A and B. The eggplant shoot and fruit borer infestation rate can be reduced by increasing these specific biochemical properties.

1. Introduction

In terms of global vegetable crop production, eggplant (Solanum melongena L.), often known as brinjal, ranks fifth overall [1]. This crop is of particular importance because it contains several bioactive chemicals, particularly phenolics, which have been shown to have many health benefits for humans [2,3,4]. It has been consumed as a vitamin-rich meal [5] and is traditionally employed as a therapeutic agent [6,7]. Over the course of more than 1500 years of eggplant cultivation in Asia, there has amassed an extensive body of documentation, evaluation, and conservation efforts surrounding its genetic resources and collections [8]. By digesting eggplants, one can obtain minerals, fiber, calories, protein, as well as bioactive compounds such as steroidal glycoalkaloids (SGAs). They are commonly consumed as a crucial component of low-fat diets [9,10].
Furthermore, many plant tissues, including fruits, tubers, leaves, roots, and sprouts, produce glycoalkaloids. SGAs, the primary ingredient in imparting a unique flavor to the fruits of the Solanaceae family, such as eggplant, are thought to serve crucial roles in the plants’ defensive systems against pests and phytopathogens. Recently, various anticancer properties have been widely observed [9,11,12], suggesting that SGAs may have antioxidant capabilities.
Glycoalkaloid accumulation, such as that of solasodine, may be regulated by a number of factors throughout growth, maturity, harvesting, and post-harvest processing. Moreover, the different genotypes investigated may have a substantial effect, as SGA magnitudes may vary between cultivars. Various environmental factors, including in vitro regeneration, have also been reported to influence SGA production in the Solanaceae family [13,14,15].
In vitro organogenesis and somatic embryogenesis of cultured explants could regenerate eggplant with relative ease. Adding hormones to the medium for organogenesis and somatic embryogenesis has allowed eggplant to regenerate [16,17]. Advances in laboratory techniques, especially in the regeneration of different explants, have made it possible to create and use many powerful biotechnological strategies for managing and improving genetic resources.
The introduction of new biotechnology techniques has created new opportunities for eggplant micropropagation and genetic enhancement. Regenerated plants have a high frequency of variation as a result of plant cell culture. Due to this variation, the offspring may not exhibit the same characteristics as those of the parent plant. Molecular, biochemical, and morphological methods can be used to describe these somaclonal variants. The changes generated in an in vitro environment can be used to develop stress-resistant plants [18,19].
In the quest to boost crop yields, mutation can be seen as a viable alternative to traditional breeding methods. Mutation initiation is a promising new way to improve crops that could compensate for traditional breeding methods’ problems when developing cultivars with desirable traits [20,21]. Because it may be applied on a small or big scale and is effective for all plant species, mutagenesis has gained popularity in recent decades. Saturation can be quickly reached, and the frequency of mutations induced can be matched by altering the mutagen dose. Mutations caused by a wide range of mutagens have increased genetic variability, which has helped plant breeders improve their crops.
Along with recombinant and transgenic breeding, it has been crucial to creating improved plant varieties, proving the monetary worth of mutant breeding technology. Mutagenesis refers to the modification of DNA by any means, whether chemical, physical, or biological. Ethyl methane sulfonate (EMS), one of the most widely used compounds for inducing chemical mutagenesis, is notable for its ability to induce both a high rate of genetic alterations and a low rate of chromosome abnormalities in plants. The number of plants, their ages, and where they are in their developmental stages when they are used as starting material all impact the mutation rate (explant). Increasing the mutation frequency by employing seed or callus tissue as the primary source is possible. It has been demonstrated that the somaclonal variation of plant material considerably increases the likelihood of high-yielding, stress-resistant cultivars being developed in the future [22,23].
Insect pests, including the lepidopteran eggplant shoot and fruit borer (ESFB), are a key limiting factor in tropical and subtropical eggplant output. High levels of ESFB damage can result in a 65 percent yield reduction or more. Third- and fourth-instar ESFB larvae are most responsible for the damage to eggplant, according to reports [24,25]. The larvae of the eggplant shoot and fruit borer eat the vulnerable shoots of infected plants, causing them to wilt and die and ultimately reducing the plants’ ability to yield fruit. The fruits become unsellable because the larvae eat the insides. Because the larvae hide in fruits and shoots, insecticides used to get rid of them are typically unsuccessful. Insects have developed resistance because farmers use insecticides too liberally, driving up production costs and posing potential risks to human health and the environment. The lack of any natural source of resistance in any cultivated or traditionally cross-compatible species of the Solanaceae family has been a severe obstacle in generating cultivars resistant to ESFB. Therefore, getting plants resistant to insects by in vitro mutagenesis is a viable option.
This study aimed to develop eggplants resistant to the shoot and fruit borer pests by employing ethyl methanesulfonate (EMS) in in vitro regeneration and correlating the phenotypic and biochemical characteristics of the regenerated plants under field conditions.

2. Materials and Methods

2.1. Plant Material and In Vitro Mutagenesis

The study utilized healthy, disease-free seeds of two eggplant genotypes collected from the Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh. Genotype 1 is a landrace, whereas Genotype 2 is a cultivar derived from a landrace and a modern variety.
The seeds were washed for three to five minutes with detergent and running water. During the washing process, floating seeds were eliminated. The seeds were soaked in 70% ethanol for 20 s before being washed with distilled water. After removing the ethanol, the seeds were rinsed in a shaker with a solution containing 0.1% mercuric chloride (HgCl2) for twenty minutes, and then they were washed five times with sterile distilled water. After that, the seeds were spread out on a Petri dish lined with sterile filter paper so that the extra water could be absorbed. The surface-sterilized seeds were then cultivated on an MS medium supplemented with 2 mg/L 2,4-D, 30 g/L of sucrose, and six concentrations of EMS (0% as a control, 0.5%, 1%, 1.5%, 2%, and 2.5%) at a pH of 5.7.
For plant regeneration, calli of at least 2 mm in diameter were transferred to MS regeneration media supplemented with 1 mg/L NAA and BAP and cultured in a temperature-controlled growing environment at 25 ± 2 °C with a 12 h light photoperiod and 2000–3000 lux light intensity. Similar concentrations of EMS were added to the regeneration and callus induction mediums.
The plantlets with an adequate root system were removed from the culture pots, and their roots were cleaned with flowing tap water. The plantlets were transplanted into small plastic pots with a 2:1:1 mixture of sterilized ground soil, leaf smash, and cow dung. Then, translucent perforated polythene bags were used to cover the containers. The interior of these bags was sprayed with water to keep them from drying out. The containers were stored in a growth room for two weeks, with polythene covers for the first week and without covers for the second. These seedlings were exposed to the natural environment for two to eight hours every day before being returned to the growth room for another week. When the regenerated plants were fully established in the small pots three weeks following transplantation, they were transferred to the field for further development and performance under natural conditions.

2.2. Biochemical Analysis

Total phenol, antioxidants, leaf and fruit chlorophyll, solasodine, total sugar, Na, Mg, Ca, Mn, Fe, Cu, and K content of the fruits were measured.

2.2.1. Total Phenol Determination

Standard solutions of gallic acid were generated at concentrations of 0, 0.0125, 0.025, 0.050, 0.100, and 0.150 mg/mL. After being appropriately diluted, the test extracts (0.1 mL) or the gallic acid standards (0.1 mL) were poured into test tubes with a capacity of 15 mL. After adding the Folin–Ciocalteu reagent to each test tube, the contents of each tube were mixed using a vortex mixer (0.2 N, 3.0 mL). After waiting one minute, 2.0 mL of Na2CO3 in water with a concentration of 9.0% (w/v) was added, and the solution was allowed to cool to room temperature. The absorbance was measured at 765 nm after it had been there for 2 h. The content of total phenolic compounds was determined by analyzing the absorbance of the extract samples and contrasting it to the absorbance of the gallic acid standard solutions. Each sample was examined three times.

2.2.2. Antioxidant Determination

A total volume of 4.1 mL was achieved by combining 0.1 mL of sample extract with 1 mL of CuCl2 solution, 1 mL of neocuproine alcoholic solution, 1 mL of NH4Ac buffer solution, and 1 mL of water. After waiting thirty minutes, the absorbance at 450 nm was determined and compared to the blank reagent.

2.2.3. Chlorophyll Determination

Fresh sample (0.2 g) was put in a test tube, and 10 mL of 80% acetone was mixed with the sample in a mortar and pestle. The homogenate was filtered through No. 1 Whatman filter paper to obtain the chlorophyll that had been extracted into the acetone solution. Two or three times, 5 mL of 80% acetone was used to wash out the homogenate. With 80% acetone, the final amount was brought up to 25 mL. By measuring the optical density at 663 and 645 nm, the amount of chlorophylls a, b, and total chlorophyll in samples was calculated.

2.2.4. Solasodine Measurement

The fruits were picked when they were still green and dried in the shade at room temperature. Dry powder (25 mg) and 4 mL of 1 N HCl were put in a sealed test tube and heated in a water bath at 100 °C for 2 h. They became more basic when 0.5 mL of 60% (w/v) sodium hydroxide was added to the hydrolysates. After giving the mixture a good shake, it was spun in a centrifuge for 5 min at about 1000× g. An amount of 5 mL of chloroform was added to the supernatant. By shaking hard, chloroform was taken from the hydrolyzed derivatives. The bottom layer of chloroform was taken and filled to 5 mL. This was mixed with 2.5 mL of 2 × 10−4 M bromothymol blue (BT) in a borate buffer (pH 8.0) for 10 s. The bottom layer, which was straw-colored, was pulled out. This was mixed with 1 mL of 0.2 M sodium hydroxide in methanol to make a blue color. This happened because aglycone-BT blue formed.
Using a standard solution consisting of 1 mg/mL of solasodine dissolved in chloroform, working standards of concentrations of 20, 40, 60, 80, and 100 g were formed by diluting appropriate quantities until the total amount attained was 4 mL. These intensities were used to determine the accuracy of the measurements. The remaining steps of the process were precisely the same as those described earlier. The absorbance of each concentration was plotted against the quantity of solasodine to generate the calibration curve. This was done by measuring the absorbance of each concentration at 610 nm.

2.2.5. Total Sugar Content

Alcohol was used to extract one gram of plant material, and the extract was spun at 3000 rpm for 15 min. With 2 mL of 80% ethanol, the sugars were taken out. The mixture was then spun again, and the supernatant was saved for total sugar analysis. Five milliliters of 1.1% HCl was added, and 10 mL of distilled water was used to dilute it. One milliliter of test solution and five milliliters of anthrone reagent were mixed in a test tube. After letting the sample sit for 45 min to obtain color, the spectrophotometer showed absorbance at 630 nm.

2.2.6. Estimation of Minerals

The amount of sodium, magnesium, calcium, manganese, iron, copper, and potassium, among other minerals, in fruits was also found. Dried samples (0.5 g) were sieved into a 250 mL digestion tube for the eggplant samples. Five milliliters of concentrated HNO3 acid (65%) was added, and the samples were left to sit overnight. Then, they were heated to 60 °C until the brown smoke stopped. Then, 2 mL of HClO4 acid was added to the samples at 150 °C to make them smaller. Samples were put through a Whatman No. 42 paper filter and diluted in a 50 mL volumetric flask until they reached the right level. An Atomic Absorption Spectrophotometer (AAS, Perkin Elmer, the PinAAcleTM 900H, Waltham, MA, USA) was used to determine how much mineral was in the samples that had been diluted.

2.3. Data Collection and Analysis

Observations of the explants in the tissue culture medium were made on different parameters, such as the percentage of callus formation, regeneration of callus, and root and shoot length of the regrown plants one month after the callus was put in the regeneration medium. Statistical software called SPSS 16.0 was used to run Tukey’s HSD post hoc test.
The field performance of plantlets produced from tissue culture was evaluated for eggplant shoot and fruit borer. Data were collected for morphological characteristics such as days to first flowering, days to first fruiting, plant height, number of total branches, percentage of affected shoots, and percentage of affected fruits. The percentage of shoot and fruit infestation was calculated by counting the number of infested shoots and fruits on each mutant and control plant. The infestation rate was determined by dividing the number of infested shoots by the total number of observed shoots. Multiplying the number by 100 yielded the percentage of shoot infestation. Similarly, the fruit infestation was determined by dividing the number of infested fruits by the total number of fruits detected. Multiplying the number by 100 yielded the percentage of fruit infestation.
The plants were separated into their respective grades according to the percentage of infestation found in each plant. The plants were deemed immune if there were no symptoms of infestation in their shoots or fruits, and they were considered to have a high level of resistance if between one and ten percent of these areas were infested. If between 11 and 20 percent of the plants’ shoots or fruits were infested, it was concluded that the plants exhibited a moderate level of resistance. The infection rate in the shoots or fruits of tolerant plants varied between 21 and 30 percent. If the plants had between 31 and 40 percent infection in their shoots or fruits, they were considered susceptible, and if they had more than 40 percent infestation, they were labeled highly susceptible.
The above-mentioned biological characteristics were also measured. The dendrogram was produced with the software Origin Pro 2017 [25]. Using the computer language R, a graphical depiction of the correlation between the tested attributes was generated.

3. Results

Eggplant seeds of two genotypes (Genotype 1 and Genotype 2) were treated in vitro with varying dosages of EMS to regenerate plantlets resistant to shoot and fruit borer (Figure 1).

3.1. Effect of EMS on In Vitro Performance

Different EMS treatments affected the callus induction and regeneration percentage and the length of the tested genotypes’ shoots and roots (Figure 2). The performance of the examined parameters decreased as the medium’s EMS content grew, and the untreated controls performed better compared to the treated ones. MS medium supplemented with 2% EMS fared the best compared to the other treatments.

3.2. Treatment-Wise Genotypic Performance

Figure 3 depicts the performance of Genotype 1. Genotype 1 performed best in the control condition for all parameters evaluated. When the EMS concentration was raised, a declining trend was seen. Genotype 1 in MS medium supplemented with 2% EMS outperformed the other EMS treatments regarding callus induction percentage, plant regeneration percentage, and root length. Genotype 1 performed best for shoot length in MS medium supplemented with 0.5% compared to the other EMS treatments. In the MS medium supplemented with 2.5% EMS, Genotype 1 did not do well.
Figure 4 depicts the performance of Genotype 2. Genotype 2 performed best in the control condition for all parameters evaluated. Genotype 2 in MS medium supplemented with 0.5 percent EMS outperformed the other EMS treatments in terms of callus induction percentage and plant regeneration percentage, as well as root and shoot length. When placed in MS medium supplemented with 2.5 percent EMS, Genotype 2 performed the worst.

3.3. Genotypic Comparison

The genotypes were compared in callus induction percentage, plant regeneration percentage, root length, and shoot length (Figure 5). When callus induction percent was assessed in control, T1, T2, and T4 circumstances, Genotype 1 outperformed Genotype 2. For Genotype 2, the percentage of callus induction was higher in T3 and T5 circumstances. Genotype 1 was superior for plant regeneration in the control, T1, T2, and T4 stages, while Genotype 2 performed better in the T3 and T5 states. In the T5 condition, Genotype 1 performed poorly. Except for the T1 and T5 situations, it was better for root length in all cases. It was on par with the other genotype in the T1 setting. Genotype 1 had the longest shoots in T2, T3, and T4 circumstances. In T0, T1, and T5 cases, Genotype 2 performed better.

3.4. Field Performance of Regenerated Plantlets

After suitable acclimation, the regenerated plantlets were transferred to the field environment. Among the transplanted plants, only 10 treated plants and the control genotypes produced fruit, while the others did not. There was substantial variation among the plants (Figure 6).

3.4.1. Correlation of Phenotypic and Biochemical Traits

A comparative correlation study was performed to determine the relationship between phenotypic and biochemical characteristics (Figure 7). There was a considerable positive correlation between total phenol and solasodine. There was a substantial link between antioxidant levels and total carbohydrate levels. The relationship between chlorophyll A and chlorophyll B in leaves was extremely strong. The same was true for fruit chlorophylls A and B. Solasodine had a moderate relationship with leaf chlorophylls A and B. Mg had a strong correlation with fruit chlorophylls A and B, and a moderate correlation with Fe and K. Ca and Mn had a strong relationship with Fe and Cu. The correlation between affected fruit percentage and affected shoot percentage was substantial. Days to first flowering was negatively linked with affected shoots and fruits. The association between impacted fruits and fruit chlorophylls A and B was substantial but moderate for affected shoots. Total phenols was moderately linked with plant height. Negative associations were found between affected fruits and shoots and total phenols, antioxidants, solasodine, total carbohydrate, Ca, days to first fruiting, and the total number of branches.

3.4.2. Classification of Mutants

Based on morphological and biochemical characteristics, the regenerated plants were divided into two broad categories (Figure 8). Group 2 consisted of a single mutant (number 6). The remaining plants were separated into two distinct groups. Subcluster 1 consisted of G1, G8, G10, and G9, whereas subcluster 2 comprised G2, G4, G5, G11, G12, G3, and G7.
The morphological and biochemical characteristics were utilized to build a heatmap (Figure 9). The characteristics created two separate groupings. A group consisted of leaf chlorophyll A, leaf chlorophyll B, antioxidants, solasodine, and total carbohydrate, whereas another group comprised the remaining characteristics. The mutants G1, G5, and G6 had the lowest eggplant shoot and fruit borer populations. The infestation rate of these three mutants was less than 10%, indicating a high level of resistance (Figure 9, Supplementary file Table S1). Some mutants fared well in percentage of shoot infestation, but poorly in fruit infestation. These mutants had a substantial amount of leaf chlorophylls A and B, antioxidants, solasodine, and total carbohydrate. The control plants lacked resistance to eggplant fruit and shoot borer.

4. Discussion

The coupling of induced mutation and in vitro cell differentiation has been utilized to generate the required variability and reproduce selected mutants in vitro [26,27]. Moreover, the morphological, biochemical, and molecular characterization of mutants is critical during in vitro mutagenesis. Our research centered on the in vitro generation of shoot and fruit borer-resistant eggplants using EMS-induced genetic variation.

4.1. Optimization of EMS Treatment Methods

In vitro survival and growth parameters of eggplants were investigated to determine the efficacy of the chemical mutagen (EMS). Among the reported treatment methods for various crops, the dip method and direct injection approach are widely used [26,28]. Explant tissue exposed to the mutagen present in the culture medium for an extended period may sustain DNA damage that cannot be addressed by the cellular receptors, leading to apoptosis, which is why the media enrichment approach is not widely used. The media enrichment strategy may also be species- or genotype-dependent [26,29]. On the other hand, it is a convenient procedure, as explant responses may be easily detected after media enrichment with the particular mutagen and inoculation of the explants in the media. The dose-dependent growth response was seen from various EMS concentrations used in the present investigation. All analyzed explant metrics, including callus induction percentage, plant regeneration percentage, root length, and shoot length, were maximum in the control condition. However, as EMS concentration increased, performance decreased until the medium was enriched with 1.5% EMS. Usually, the medium supplemented with 2% EMS showed the highest beneficial reaction among the treatment doses. Even though some plants regenerated at lower doses, they failed to produce fruit in the field. The 2% EMS dose was optimal for developing mutant eggplants in vitro, but the larger dose was highly harmful. Several crop plants have shown that higher concentrations of mutagens slow down growth in different ways [30,31].
Successful mutant breeding programs require careful consideration of mutagen concentration and growing conditions to maximize the frequency with which desirable mutations are produced. We discovered the best amount of EMS to use in the eggplant tissue culture medium to choose plants that were resistant to shoot and fruit borer.
The application of EMS is thought to be helpful in mutant plant generation. There have been no detrimental effects on human health resulting from the development of more than two thousand crop varieties using mutation techniques, including EMS treatment, worldwide [32]. However, some people are concerned about the safety issue these mutants pose as well as the various protocols employed in the process of generating mutants. Additional research is required to verify the consistency of these techniques.

4.2. Field Performance of Regenerated Plants

Plants were transferred to the field after proper acclimatization to determine if variations occurred that may be used to generate eggplant shoot and fruit borer-resistant plants. Only eight mutants were observed to produce fruit in the field. The field performance of these plants was compared to that of in vitro-regenerated control plants.
Mutations in recessive traits cannot be detected in the M1 generation, while those in dominant traits may be. In addition, the M1 generation exhibits several signs of mutagen potency, such as pollen incompatibility, decreased plant growth, delayed or early blooming, or deformed foliage [28,33]. Physical screenings are the most efficient method for detecting phenotypic mutation at that time. The selection of plants with desirable characteristics, such as resistance to biotic and abiotic stress, early flowering, plant height, fruit color variations, or growth cycle length, can be made mainly through visual screening. Although several plants were sterile, the remaining ten mutants showed a significant variation in fruit size, shape, and color. Among the ten mutants, a single mutant of Genotype 2 gave fruit. The fruit color was white, although the source material was pink. One of the parental components of Genotype 2 was white, and the EMS treatment induced this variation. Several factors, notably, explant type and culture circumstances (such as the presence or absence of a growth hormone in the nutrient media, the length of the culture, and the presence or absence of a mutagenesis agent), contribute to the observed diversity in plant tissue cultures [28,34].
This study suggests that chromosomal aberrations and morphological and physiological modifications mediated by the mutagens may have contributed to pollen infertility in plants developed from in vitro mutagen-treated explants. Previous research that revealed mutagen-induced pollen sterility in various crops validated the results. Some data indicate that the percentage of sterile pollen in the M2 generation is significantly lower than in the M1 generation, suggesting a healing process between generations [33,35].

Screening for Eggplant Shoot and Fruit Borer Resistance

In many Asian nations, the damage caused by the eggplant shoot and fruit borer is the primary barrier preventing the realization of potential eggplant yield. The strong reproductive potential and shorter pest life cycle, together with the evolution of resistance to various pesticides, offer a significant obstacle to successful eggplant farming [36]. Our objective was to establish resistant plants via in vitro mutagenesis by applying EMS in the medium. To achieve this objective, morphological and biochemical characteristics of transplanted regenerated plants were measured. The correlation studies were conducted to identify the traits associated with pest resistance.
The control plants did not show resistance to the eggplant shoot and fruit borer. Three mutants obtained from the landrace were tolerant to the insect pest. These genotypes had a substantial amount of total phenols, leaf chlorophylls A and B, antioxidants, and solasodine. The clustering pattern also indicated a cluster composed of leaf chlorophylls A and B, antioxidants, and solasodine. The correlation studies suggested negative associations between affected fruits and shoots and total phenols, antioxidants, and solasodine. Solasodine was associated with chlorophylls A and B. Obviously, with the increasing amount of these biochemical attributes, the eggplant shoot and fruit borer infestation rate can be minimized.
Vegetables such as eggplants are rich in antioxidants such as phenolics, flavonoids, and ascorbic acid [37]. Phenolics in eggplant have been identified as the key bioactive chemicals crucial for their antioxidant properties [38], and cultivars vary significantly in the amounts of these elements [39]. Landraces typically have higher concentrations of these chemicals than farmed cultivars do [37]. Plants having a higher antioxidant content can be bred from the landraces. Our resistant plants were also mutants of the landraces, with a higher concentration of phenolics and solasodine.
The fruit of eggplants is abundant in bioactive compounds such as solasodine [9]. Solasodine is believed to perform crucial functions in the plant’s defensive mechanisms against pests and diseases [13]. Several studies [40,41,42] have demonstrated the effectiveness of solasodine against pests, including eggplant shoot and fruit borer. Plants with higher levels of solasodine and phenols can tolerate insect invasion [9,13], which is consistent with our findings.

5. Conclusions

On the MS medium supplemented with varying concentrations of EMS, screening was conducted in vitro for eggplant shoot and fruit borer infestation. The optimal dose of EMS for eggplants was determined to be 2% for the mutant generation. Solasodine, phenols, antioxidants, leaf chlorophyll A, and leaf chlorophyll B all had a strong relationship. By choosing genotypes with higher levels of these phytochemicals, resistance to shoot and fruit borer infestation could be increased.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12081832/s1. Table S1. Grading of studied genotypes.

Author Contributions

Conceptualization, M.H., M.G.R. and M.A.H.S.; methodology, M.A.I., M.M.B.M.U., M.A. and M.H.; software, M.H. and M.A.H.S.; formal analysis, M.H. and M.A.H.S.; investigation, M.H., M.G.R. and M.A.H.S.; data curation, M.H., M.G.R. and M.A.H.S.; writing—original draft preparation, M.A.I. and M.H.; writing—review and editing, M.A.I. and M.H.; supervision, M.H., M.G.R., M.A.H.S. and M.A. All authors have read and agreed to the published version of the manuscript.

Funding

Research Management Wing (RMC) of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh, is appreciated for funding the research (37.12.0000.133.34.004.20.26).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All datasets are available on request to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Regeneration of eggplants. (A) Seeds as explants in the petri dish. (B) Shoot regeneration. (C) Regenerated plantlets.
Figure 1. Regeneration of eggplants. (A) Seeds as explants in the petri dish. (B) Shoot regeneration. (C) Regenerated plantlets.
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Figure 2. Impact of different concentrations of EMS on the measured traits grown in vitro. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
Figure 2. Impact of different concentrations of EMS on the measured traits grown in vitro. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
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Figure 3. Performance of Genotype 1 in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
Figure 3. Performance of Genotype 1 in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
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Figure 4. Performance of Genotype 2 in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
Figure 4. Performance of Genotype 2 in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length with yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
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Figure 5. Comparison of genotypes in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length is yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
Figure 5. Comparison of genotypes in the MS medium supplemented with different concentrations of EMS. Callus percentage is indicated with blue color, regeneration percentage with brick color, root length with ash, and shoot length is yellow color. Here, T0 = Control, T1 = MS + 0.5% EMS, T2 = MS + 1% EMS, T3 = MS + 1.5% EMS, T4 = MS + 2% EMS, T5 = MS + 2.5% EMS. Different letters indicate a significant difference at the 5% level.
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Figure 6. Leaf and fruit variations in the regenerated plants. Here, 1 to 10 = EMS-treated mutants, 11 = Genotype 1 without EMS treatment, 12 = Genotype 2 without EMS treatment.
Figure 6. Leaf and fruit variations in the regenerated plants. Here, 1 to 10 = EMS-treated mutants, 11 = Genotype 1 without EMS treatment, 12 = Genotype 2 without EMS treatment.
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Figure 7. Correlation of phenotypic and biochemical traits. Blue represents the positive correlations, while the negative ones are shown in red. The correlation coefficients have a proportional relationship with the size of the square and the color intensity. TP = total phenols, Anox = antioxidants, LCHA = leaf chlorophyll A, LCHB = leaf chlorophyll B, FCHA = fruit chlorophyll A, FCHB = fruit chlorophyll B, Solas = solasodine, TC = total carbohydrate, FFL = days to first flowering, FFR = days to first fruiting, PH = plant height, TBR = number of total branches, AFR = percent affected fruits, ASH = percent affected shoots.
Figure 7. Correlation of phenotypic and biochemical traits. Blue represents the positive correlations, while the negative ones are shown in red. The correlation coefficients have a proportional relationship with the size of the square and the color intensity. TP = total phenols, Anox = antioxidants, LCHA = leaf chlorophyll A, LCHB = leaf chlorophyll B, FCHA = fruit chlorophyll A, FCHB = fruit chlorophyll B, Solas = solasodine, TC = total carbohydrate, FFL = days to first flowering, FFR = days to first fruiting, PH = plant height, TBR = number of total branches, AFR = percent affected fruits, ASH = percent affected shoots.
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Figure 8. Dendrogram of regenerated plants. The numbers 11 and 12 are Genotype 1 and Genotype 2 of untreated plants, while the rest are EMS-treated mutants. Different colors denote the distinction between the genotypes.
Figure 8. Dendrogram of regenerated plants. The numbers 11 and 12 are Genotype 1 and Genotype 2 of untreated plants, while the rest are EMS-treated mutants. Different colors denote the distinction between the genotypes.
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Figure 9. Heatmap of regenerated plants for the phenotypic and biochemical traits. The row indicates genotypes, whereas the column reflects biochemical characteristics. Traits that declined greatly are displayed in green, whereas those that rose significantly are displayed in red. The brightness of each color is proportional to the intensity of the deviation from the mean value.
Figure 9. Heatmap of regenerated plants for the phenotypic and biochemical traits. The row indicates genotypes, whereas the column reflects biochemical characteristics. Traits that declined greatly are displayed in green, whereas those that rose significantly are displayed in red. The brightness of each color is proportional to the intensity of the deviation from the mean value.
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Islam, M.A.; Bin Mohi Uddin, M.M.; Rasul, M.G.; Haque Swapon, M.A.; Ahmed, M.; Hasan, M. In Vitro Screening and Field Performance of EMS-Treated Eggplants for the Selection of Shoot and Fruit Borer-Resistant Plants. Agronomy 2022, 12, 1832. https://doi.org/10.3390/agronomy12081832

AMA Style

Islam MA, Bin Mohi Uddin MM, Rasul MG, Haque Swapon MA, Ahmed M, Hasan M. In Vitro Screening and Field Performance of EMS-Treated Eggplants for the Selection of Shoot and Fruit Borer-Resistant Plants. Agronomy. 2022; 12(8):1832. https://doi.org/10.3390/agronomy12081832

Chicago/Turabian Style

Islam, Md Ashraful, Md Muntasir Bin Mohi Uddin, Md Golam Rasul, Md Ahsanul Haque Swapon, Minhaz Ahmed, and Mehfuz Hasan. 2022. "In Vitro Screening and Field Performance of EMS-Treated Eggplants for the Selection of Shoot and Fruit Borer-Resistant Plants" Agronomy 12, no. 8: 1832. https://doi.org/10.3390/agronomy12081832

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