Anvarjon A. Murodov¹, Mirzakamol S. Ayubov*¹, Mukhammadjon Kh Mirzakhmedov², Nurdinjon S. Obidov¹, Bekhzod O. Mamajonov¹, Abdurakhmon N. Yusupov¹, Ziyodulloxon H. Bashirxonov¹, Lola K. Kamalova, Shukhrat O. Kushakov¹, Ilkhomjon E. Bozorov¹, Zabardast T Buriev¹, Ibrokhim Y. Abdurakhmonov¹,³.

Corresponding author *

Email: mirzo.ayubov@gmail.com

Center of Genomics and Bioinformatics, Academy of Sciences of the Republic of Uzbekistan, Tashkent 111 215, Uzbekistan

Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 06466 Seeland, Germany

Ministry of Agriculture of the Republic of Uzbekistan, Tashkent 100000, Uzbekistan.

Tomato (Solanum lycopersicum L) is one of the most widely grown vegetables. In 2024, 192.3 million tons of tomatoes were produced worldwide (FAO, 2024). Tomato fruit is rich in vitamins A and C, minerals, and antioxidants, which are important for maintaining human health 1. Tomato is also important as a model plant for studying the processes that occur during the ripening of climatic fruits. It is also studied by scientists as a model plant, due to the complete genome sequence of several of its variants and the ease of genetic transformation 2.

50 % of the harvested tomatoes in the developing countries lose their quality before they reach consumers 3. The main reason for this is that fruits that have lost their resistance to post-harvest damage lose their skin integrity as a result of external mechanical influences, and as a result, they are more susceptible to bacterial and fungal diseases 4. To overcome this problem, scientists have used various physical, chemical, and genetic engineering methods to increase the shelf life of tomato fruit 5. However, the application of certain physical and chemical preservation methods has been reported to induce significant alterations in the flavor profile and nutritional composition of the fruits 67. Genetic engineering methods are an alternative solution to solve such a problem. Fruit shelf life was significantly increased when the activity of genes involved in fruit ripening, such as 2C protein phosphatases (SlPP2C) 8, aminocyclopropane-1-carboxylate (ACC) 9, and MADS-box protein (SlCMB1) 10 was suppressed by RNAi. However, public opinion remains concerned about plants modified through RNAi, especially those used for consumption 11. One of the main reasons for this is that stable expression RNAi plants are GM 12. In such circumstances, the use of CRISPR/Cas9 technology, a modern method of genome editing, has made it possible to reduce this concern by obtaining transgene-free organisms 13. CRISPR/Cas9 also stands out from other genetic engineering methods in its ease, convenience, and low cost 14.

CRISPR/Cas9 has been widely used to improve fruit storage. In particular, variants with extended shelf life were obtained by knocking out the genes CmACO1 in melon 15, FIS1 and PL in tomato 16, PG2a 17, and FaPG1 in strawberry 18 using CSIPR/Cas9.

More than 22 enzymes have been identified to work together during tomato fruit ripening 19. Among these, direct softening of the fruit skin is observed as a result of the activity of enzymes that break down cell wall components 20. The cell wall is mainly composed of cellulose, hemicellulose, pectin, and proteins. When the activity of the enzymes Polygalacturonase, β-galactosidase, and β-glucanase, which break down cell wall components, was inhibited through genetic engineering, only a slight increase in fruit shelf life was achieved [21–23]. The cell wall also contains N-glycoproteins, and the breakdown products of these substances, free N-glucans, are precursors for the process of glycosylation or glycoprotein proteolysis 24. These free N-glucans are present throughout the entire developing pericarp of tomato fruit and increase during ripening 25.

β-D-N-acetylhexosaminidase (β-Hex; EC 3.2.1.52) is also one of the N-glucan hydrolyzing enzymes, and its function is to hydrolyze the last N-acetyl-D-hexosamine residues that are part of many N-glucans 26. This enzyme is ripening specific, and its biosynthesis has been found to increase during fruit ripening 24. Expression of beta-hex gene is regulated by RIPENING INHIBITOR (RIN), ABSCISIC ACID STRESS RIPENING 1 (SLASR1), and ethylene 27. Post-harvest treatment of strawberry fruits with alginate oligosaccharides (AOS) resulted in a decrease in β-Hex enzyme activity, resulting in delayed fruit softening in the fruits 28. It has been found that this enzyme increases in quantity during the ripening period of tomato fruit 25. It has been observed that the shelf life of fruits is extended when the expression of the β-hex gene encoding this enzyme is reduced using RNAi technology 24. Using site-directed mutagenesis, it was demonstrated that mutations within the cis-acting elements of the tomato β-hex promoter significantly downregulated its transcriptional activity, thereby delaying the fruit softening process and extending shelf life. Our earlier study 5 covered more details on various methods utilized by numerous laboratories to extend the shelf life of tomato fruits. In this research, we knocked out the β-hex gene using CRISPR/Cas9 technology and obtained transgenic free plants with extended shelf life.

The seeds of S. lycopersicum L. (“cherry” cultivar) were obtained from the Research Institute of Plant Genetic Resources, Uzbekistan. The surface of the seeds was sterilized by first soaking in 70% ethanol for 5 minutes, then in 3% sodium hypochlorite for 8 minutes. The seeds were then rinsed in sterile distilled water 3 times for 10 minutes each and stored on sterile filter paper until excess moisture was removed. The seeds were then grown in a growth chamber with a 16/8 h light-dark photoperiod and 70% humidity for 8-10 days on MS (Murashige and Skoog) medium containing 3% sucrose and 0.3% Gelzan (PhytoTech Labs, Inc. 14610 W. 106th St, Lenexa KS 66215). Fully formed 8-10-day-old cotyledons were used for transformation.

sgRNAs of 19 bp in length were designed to target exons 1 and 2 of the β-hex gene using the online tool CRISPR-P 2.0 ( http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR ) 29. sgRNA_1: GTCGCCGCAGATATGTAGG for exon 1 and sgRNA_2: GCCGCTATGATCAGCCACC for exon 2 were selected (S1 Table). Their minimum off-target mismatch is 3 bp and 4 bp and GC content is 55 and 60%, respectively (S2 Table). The secondary structure of gRNAs was determined using the web program RNAfold (“RNAfold Web Server,” n.d.). The construct for the CRISPR/Cas9 tomato transformation was assembled using the CasCade vector system 30.

The assembled vector construct was transformed into tomato plants using 8-10 day old cotyledons and Agrobacterium tumefaciens strain LB4404. The tops and bottoms of the cotyledons were cut off and infected with Agrobacterium (OD600=0.6) containing our vector construct. First, 2 days in the dark at 19 C in co-cultivation medium, then 2 weeks in 2Z selection medium1 at 24 °C ± 2 °C, with a 16-h photoperiod. Next, 1Z Selection medium 1 was maintained under the same nutrient conditions as above. The shoots (2 cm) that reached the top of the Magenta box were cut and maintained in selective rooting medium until their roots were sufficiently formed. Later it was adapted to the soil. We used hygromycin (6 mg/l) as a selective antibiotic. Plant transformation was conducted following the Agrobacterium tumefaciens-mediated method as previously described by Van Eck et al. (2019). 31.

To confirm the presence of the CRISPR/Cas9 construct and identify genomic mutations, genomic DNA was extracted from the young leaves of 8-week-old transplants using the Thermo Scientific™ GeneJET Plant Genomic DNA Purification Kit (K0791). The initial screening of T_0 generation plants was performed via PCR using Cas9-specific primers (S1 Table) to verify the integration of the genetic element. DNA from wild-type (WT) plants served as a negative control. The PCR thermal cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 59°C for 30 sec, and 72°C for 40 sec, with a final extension at 72°C for 5 min. For mutation characterization in T_0 plants, PCR products amplified with gene-specific primers were first cloned into the TOPO™ TA Cloning™ Kit (Thermo Fisher Scientific, USA) due to the potential chimeric or heterozygous nature of the primary transformants. Subsequently, positive clones were sequenced using the SeqStudio Genetic Analyzer. In contrast, for the T_2 and T_3 generations, where mutations reached homozygosity, genotypes were determined through direct Sanger sequencing of the PCR products without intermediate cloning.

"Potential off-target sites were identified using the CRISPR-P 2.0 online tool. Among these, site with high off-target score that was located within intron regions were selected for experimental validation. These loci were amplified via PCR and verified through Sanger sequencing to ensure the absence of unintended mutations (S2 Table).

The Expasy Translate program ( https://web.expasy.org/translate ) was used to predict the protein sequences of the mutants and the WT sequence.

Phenotypic parameters such as fruit weight, fruit shape index (FSI), pH, and weight loss were evaluated in mutant and wild type plants. First, all fruits were sterilized in distilled water and ethanol. 10 fruits at the red ripe stage were used to determine fruit weight and fruit shape index (FSI).

To measure fruit shape, the ratio of the polar and equatorial diameters of the fruits was calculated. The pH was measured using a FiveEasy Plus Benchtop FP20 pH/mV Standard Kit (METTLER TOLEDO, Malaysia) in 3 biologicals and 3 technical replicates by diluting 250 mg of pericarp in 15 ml of distilled water (pH 7.0).

Fruit weight loss was also measured every 3 days for 8 fruits at the ripe red stage up to day 18. Weight loss was calculated using the following formula:

$$\frac{W_0―W_{1, W_2,…W_n}}{W_0} \text{ x } 100$$

where W0 is the initial picked fruit weight, W1 is the fruit weight 3 days after harvesting, W2 is the fruit weight 6 days after harvesting, and so on up to n=6.

Also, the color change during the ripening process of the fruits was monitored every 5 days for a total of 15 days in the Breaker (Br) stage fruits.

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation (SD). The significance of differences between the wild-type (WT) and mutant lines was determined using Student’s t-test, with p-values < 0.05 considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant).

For bioinformatic analysis, DNA sequencing chromatograms were visualized and aligned using SnapGene software (GSL Biotech; available at snapgene.com ) and VectorBee ( VectorBee.io ). These tools were utilized to verify the presence of indel mutations, analyze frame-shift events, and ensure the integrity of the CRISPR/Cas9 vector constructs. Sequence translations and protein secondary structure predictions were further processed using the ExPASy Translate tool.

A total of 9 putative plantlets were recovered following Agrobacterium-mediated transformation. Molecular screening via PCR revealed that 3 plants carried the target construct. However, significant phenotypic abnormalities and premature senescence were observed in two transgenic lines, preventing them from reaching the reproductive stage. Similar phenomena regarding the low survival rate of primary plantlets (T0) have been widely reported in plant biotechnology, often attributed to tissue culture-induced stress or insertional mutations. As a result, only one vigorous T0 line was advanced to the T1 generation for segregation analysis (Fig 1C). Analysis of the plant harboring the vector construct using Sanger sequencing revealed a 1 bp insertion mutation in exon 1 and a 5 bp deletion mutation in exon 2. (Fig 1A).

The changes in the β-D-N-acetylhexosaminidase (β-Hex) protein resulting from these mutations were analyzed using the Expasy program. According to the results, it was predicted that a premature stop codon may occur in the sgRNA-located part of exon 1 and cause protein coding disruption. In wild type (WT) plants, the stop codon comes after the Serine amino acid at position 575, while in mutant plants, a premature stop codon is formed after the Alanine amino acid at position 127 at the sgRNA mutation site.

Cas9 free plants were identified among the T_2 generation plants and off-target analyses were performed on these transgenic free plants. For 1-gRNA, 6 off-target cites with the highest off-target scores were identified, 2 of which corresponded to intron parts of the genome with 4 mismatches, and these 2 off-target parts were analyzed using Sanger sequencing. For 2-gRNA, sequencing was not performed because all of the high off-target scores corresponded to intergenic parts of the genome. According to the sequencing results, no mutations were detected in the off-target parts (S3 Fig).

The effect of the mutations on protein coding was analyzed using the Expasy program. It was found that the insertion in exon 1 and the deletion in exon 2 resulted in the formation of premature stop codons. (S1 Fig).

A progressive linear increase in cumulative weight loss was observed across all samples over the storage period. This is a characteristic physiological process primarily driven by transpiration and respiratory water loss. According to our data, the weight loss in the beta-hex knockout mutant fruits showed no significant difference compared to the wild type during the initial three days. However, from day 6 through day 12, a significant reduction in weight loss was observed in the mutant lines compared to the wild-type controls (Fig. 2A). These quantitative findings were further corroborated by phenotypic observations; the mutant fruits exhibited superior post-harvest resilience, maintaining their firmness and structural integrity for up to 25 days, whereas the wild-type fruits showed visible shriveling and loss of turgor (Fig. 2B).

Sun X, Jiao C, Schnable PS, Cushman JC, Luo H, Rost TL, et al. Tomato Genomics Reveals Distinct Cumulative Gene Expression Patterns - An Evolutionary Perspective. Plant J. 2011;66: 213–225. doi:10.1111/j.1365-313X.2010.04500.x

Nishimura N, Tsuchiya W, Iwasaki T, Kuromori T, Masuno MN, Morosawa T, et al. Functional implications of protein N-glycosylation in Arabidopsis. Plant Physiol. 2016;171: 1754–1773. doi:10.1104/pp.16.00881

Gao M, Gao X, Zhang Y, Ren Z, Chen Z, Luo H, et al. CRISPR/Cas9-Based Targeted Mutagenesis of SlPL in Tomato. Agronomy. 2023;12: 1647. doi:10.3390/agronomy12071647

Chakraborty S, Roy S. Breaking the Wall: A New Approach to Enhance Shelf Life of Tomato and Other Fruits. In: Chakraborty S, Mitra A, Sharma K, editors. Emerging Technologies in Plant Science. New Delhi: Springer India; 2018. pp. 245–268. doi:10.1007/978-81-322-3774-0_14

El-Sharkawy I, Sherif S, Kumar PP. Deficient moisture-induced stress memory primes redox homeostasis and defense responses in tomato plants. Environ Exp Bot. 22020;171: 103953. doi:10.1016/j.envexpbot.2020.103953

Sun X, Jiao C, Schnable PS, Cushman JC, Luo H, Rost TL, et al. Tomato Genomics Reveals Distinct Cumulative Gene Expression Patterns - An Evolutionary Perspective. Plant J. 2011;66: 213–225. doi:10.1111/j.1365-313X.2010.04500.x

Nishimura N, Tsuchiya W, Iwasaki T, Kuromori T, Masuno MN, Morosawa T, et al. Functional implications of protein N-glycosylation in Arabidopsis. Plant Physiol. 2016;171: 1754–1773. doi:10.1104/pp.16.00881

Gao M, Gao X, Zhang Y, Ren Z, Chen Z, Luo H, et al. CRISPR/Cas9-Based Targeted Mutagenesis of SlPL in Tomato. Agronomy. 2023;12: 1647. doi:10.3390/agronomy12071647

Chakraborty S, Roy S. Breaking the Wall: A New Approach to Enhance Shelf Life of Tomato and Other Fruits. In: Chakraborty S, Mitra A, Sharma K, editors. Emerging Technologies in Plant Science. New Delhi: Springer India; 2018. pp. 245–268. doi:10.1007/978-81-322-3774-0_14

El-Sharkawy I, Sherif S, Kumar PP. Deficient moisture-induced stress memory primes redox homeostasis and defense responses in tomato plants. Environ Exp Bot. 2020;171: 103953. doi:10.1016/j.envexpbot.2020.103953