Variability of fruit shelf-life of commercial tomatoes and their hybrids crossing with insensi ti vity ethylene mutant tomato Sletr1 ­ 2

Tomatoes are climacteric fruits that experience a surge in respira ti on rate and ethylene production. This condition affects the fruits’ physiological deterioration, shortens their shelf-life, and degrades the quality of tomatoes. The study aimed to enhance the shelf-life of commercial tomatoes with di ﬀ erent gene ti c backgrounds by u ti lizing the Sletr1 ­ 2 mutant, which has a lower sensitivity to ethylene. A randomized block design (RBD) was applied to achieve this goal, where 12 different plant genotypes were considered as treatments, and each treatment was replicated four times. The results showed that fruit shelf-life was extended by crossing commercial tomatoes with Sletr1 ­ 2 mutant tomatoes. Genetic variability for all shelf-life characteristics were narrow. In contrast, there were broad phenotypic variability for fruit hardness and weight loss characteris ti cs.


INTRODUCTION
Tomatoes are climacteric fruits, meaning that during the ripening process, there is a surge in respiration rate (Paul and Pandey, 2014;Colombié et al., 2017) and ethylene production (Liu et al., 2015;Li et al., 2020;Iqbal et al., 2017).Increased respiration rate can impact physiological damage where aging or decay occurs, decreasing shelf-life and maturation.At the same time, increasing ethylene affects its nutrient content and can accelerate fruit ripening.Both processes also impact tomatoes' quality during storage (Wicaksana, Wijaya, and Soeparjono, 2019).
Additionally, the biochemical and physiological changes during the ripening process will cause changes in the fruit's taste, color, flavor, and texture (Wang et al., 2018;Quinet et al., 2019), thereby reducing its quality.Short shelf-life in open or unpacked conditions is also one of the causes of the low quality of tomatoes.Therefore, proper handling to suppress the rate of respiration and ethylene needs to be done to extend shelf-life and produce quality tomatoes (Deepika and Rex, 2020;Pholoma, 2020).
The Indonesian Vegetable Research Institute has released several high-yielding tomato varieties that have become popular for public consumption, such as Intan, Mirah, Ratna, and Mutiara varieties.Even though these four varieties are superior and widely used by farmers, they have disadvantages because of their short shelf-life.Tomato fruit has a short shelf-life of 1 to 2 weeks after harvest.In some commercially advanced varieties released and circulated in Indonesia, the most extended shelf-life of fruit at room temperature may be up to 20 days, with a mean of about seven days (Wicaksana et al., 2019).Even in a single hybrid variety, the shelf-life is only eight days (Rachma, 2013).The short shelf-life of tomatoes is because, since harvest, tomatoes will continue to undergo physiological processes that result in several important biochemical reactions, such as changes in metabolites, color, texture, and taste (Amin et al., 2012).This reaction results in a decrease in both the quantity and quality of fruit.Due to these issues, it is necessary to improve commercial tomato varieties to prolong their shelf-life and preserve the quality of the fruit, one of which is through breeding programs.
Breeding programs that can be carried out are hybridization techniques to combine the good qualities derived from two or more parents to obtain superior offspring (Sharma et al., 2019).The existence of crosses with genetic backgrounds of different parents can produce diverse offspring.Several researches have been done to generate tomatoes with a long shelf-life resulting from mutation (Casals et al., 2012;Yogendra and Gowda, 2013).However, many studies have also reported that tomato varieties from mutations or mutant tomatoes are generally less tasty than local varieties.An example of a mutant tomato is Sletr12 tomatoes produced from the Micro-Tom library, which can delay the normal ripening process (Okabe et al., 2012) and decrease the susceptibility to ethylene (Mubarok et al., 2019).Sletr11 along with Sletr12, are the novel mutant alleles which are isolated from an ethylene receptor gene, Solanum lycopersicum ETHYLENE RESPONSE 1 (SlETR1), taken from a Micro-Tom Library mutant population (Okabe et al., 2011).
The appearance of a plant is the result from interactions between plant genotypes and the environment in which they grow.Crowder (1993) states that appearance can be different or change due to the interaction between one gene and another.The four genotypes used, namely Intan, Mirah, Ratna, and Mutiara, have different genetic backgrounds.The Intan tomato was resulted from a cross between Nagcarlan and Anahu (introduced by AVRDC Taiwan).The Mirah variety was generated from Malang (tomatoes PB).Ratna was resulted from a cross between Nagcarlan and Anahu (introduction from BPI Philippines).Meanwhile, Mutiara tomato was generated from a cross between Monaibo and Venus.Since genetic background plays a crucial role in determining the appearance of tomatoes, this study aimed to investigate the variability in the formation of commercial tomatoes and their F1 hybrids resulting from crosses with Sletr12 mutant tomatoes.Information on alteration and variability in fruit shelf-life characteristics due to the introduction of the Sletr12 mutant is currently lacking in Indonesia.Meanwhile, this is needed as very valuable and helpful information for future tomato breeding programs.

MATERIALS AND METHODS
The equipment used in this study includes calipers, ruler, digital scales, erlenmeyer, magnetic stirrer, pH meter, spectrophotometer, penetrometer, refractometer, measuring cup, pipette, test tube, volumetric flask, color chart, and knife.Meanwhile, the materials used in this study were four commercial parents (Intan, Mirah Ratna, and Mutiara) and 8 F1 plants as progeny between commercial tomato varieties as female parents and tomato mutants (Sletr12 and wild-type) as male parents.Other materials used are distilled water, 1% indicator Phenolphthalein (PP), and 0.1N NaOH.
The experimental design used was a randomized block design (RBD) with a factor in genotype (G).The experiment consisted of 12 treatments, each with four replications.The effect of each treatment was analyzed using Analysis of Variance (ANOVA).Data showing significant differents between treatments were then analyzed using the Scott-Knott post-hoc

Table 1. Main fruit characteristics of four commercial tomatoes
Remarks: The data are summarized from variety descriptions (Anas et al., 2022).

Genotype
Fruit Yields from planting were used as testing samples.The fruit was harvested when the maturation stage is Br+6 (Mubarok et al., 2015) to determine the character of the shelf-life of each treatment.Character analysis was carried out at the Physiology of Postharvest Laboratory, the Indonesian Vegetable Research Institute, Lembang.Observations were made by observing and analyzing the character of the storability of tomatoes for a shelf-life of 0 days, a shelf-life of 10 days, and a shelf-life of 20 days.The sampling method for one replication in one genotype was by combining the yields in each replication, and then ten pieces were taken for the needs of the entire quality analysis.
The treatment in one observation was repeated three times.
Determining long shelf-life involved monitoring the fruit from the start of storage until its quality deteriorates, indicated by the emergence of black spots or wrinkles on the fruit surface.Measurement of fruit hardness was done using a penetrometer.The sample used in each genotype is four pieces to get ten stabbing times.The way to do this is to put tomatoes just below the penetrometer piercing needle.The tomatoes were stabbed at three points on their surface (tip, middle, and base), and the duration required to exert maximum pressure on the tomatoes was measured using a stopwatch for 10 seconds.A ballast with a weight of 50 grams was used during the test.The calculation results were the average numbers obtained from the measurements.The units used are millimeters (mm) per 10 seconds with a specific load weight expressed in grams or mm/second/gram.
Weight loss during storage was measured on the ten days post storage (DPS) and 20 DPS during storage by weighing tomatoes.The level of water loss indicates the weight loss from tomatoes during the storage period because only the water can evaporate during the storage process.The formula used is as follows: Remarks: a = weight before storage or initial weight b = weight after being stored

Fruit freshness
The analysis results showed that Intan, Mirah, Ratna, and Mutiara's four commercial parents have a shorter shelf-life than the tomatoes from crossing with the Sletr12 mutant (Figure 1).This result is in accordance with Afifah et al. (2021), comparing some commercial tomato varieties, including Intan and Ratna varieties, with a mutant breeding line, where this line showed a better performance in shelf-life.According to the findings, Intan x Slert12 hybrids have the longest average shelf-life (35 days), while Mirah, Ratna, and Mutiara hybrids have the shortest average shelf lives (12, 14, and 13 days, respectively).The results of crosses between commercial tomatoes and mutant Sletr12 tomatoes differ from the commercial parents.This result shows that the Sletr12 mutant allele influences the shelf-life of the crosses from tomatoes.The effect given by the mutant allele on tomatoes from crosses is a positive influence that can increase the shelf-life of tomatoes.Hiwasa-Tanase (2016) states that Sletr12 can extend tomatoes' fruit shelf-life.Sletr12 can maintain fruit surface intact even 60 days after harvest.The ability of mutant alleles to increase shelf-life is due to the SlETR1 gene mutation, which can inhibit the normal ripening process in the fruit because it can reduce sensitivity to ethylene (Mubarok et al., 2015).

Fruit hardness
Loss of fruit hardness is one of the significant changes during fruit ripening (Ghaiet al., 2016).The hardness of fruit between commercial tomatoes and the results of their crosses is generally in a different average group.The results indicated significant differences between the two groups.Nevertheless, at 0 DPS, Mirah x Sletr12, at 10 DPS and 20 DPS, Intan x Sletr12, and at 20 DPS, Mutiara x Slert12, exhibited fruit hardness levels that were comparable to their commercial parents.However, the hardness values tended to be slightly lower (Table 2).The lower hardness value suggests that the fruit texture is firmer.
In the fruit from the cross between commercial tomatoes and Sletr12 mutants, the fruit hardness is more potent than commercial tomatoes.The fruit firmness of commercial tomatoes is softer than that of tomatoes crossed with the Sletr12 mutant, probably due to a higher respiration rate.It is well known that  respiration can reorganize carbohydrate polymers in cell walls, particularly pectin and cellulose.As a result, the wall strength and bonding cohesion may decrease, softening the texture of the fruit (Wills et al., 1981).
Crosses between commercial tomatoes and Sletr12 mutants in tomatoes can prevent cell wall breakdown.The SlETR1 gene mutation found in these crosses decreases ethylene sensitivity, avoiding the respiration rate that initiates the process.This state is in line with the research of Sholiha (2018), reporting that the treatment of 1-MCP concentrations of 30.625 mg/100 ml showed a maturity level of 1, which describes the textures of the fruit in harsh conditions.

Fruit weight loss
Regarding weight loss, commercial tomatoes and their progeny results are generally in the same average group.The findings indicated that the Sletr12 mutant allele on crossed tomatoes had no significant effect.However, there was a significant effect on weight loss   3).The increase in weight loss in tomatoes resulted from crossing with the Sletr12 mutant allele is thought to be because the effect of the mutation on the SlETR1 gene has been lost.As a result, it cannot inhibit ethylene sensitivity.According to Okabe et al. (2012), mutations in SlETR1 (Solanum lycopersicum ETHYLENE RESPONSE 1) produce Sletr11 and Sletr12 mutants that experience inhibition of gene activity as indicated by an unresponsive reaction to exogenous ethylene to a concentration of 10 ppm.The disappearance of the effect of the SlETR1 gene causes the process of respiration and transpiration in the fruit to function normally, so the weight loss that occurs can be equal to or greater than the commercial parent.

Variability of shelf-life appearance
The value of genetic variability for all observed characters is narrow (Table 4).This result indicates that the tested plant population tends to be homogenous.Furthermore, it should be noted that the sampled population is the F1 generation, which does not undergo segregation.This limited genetic variability must be enhanced to succeed in breeding programs.As highlighted by Poehlman and Sleper (1995), plants cannot be improved without genetic variation.Genetic recombination can increase genetic variability, and one approach is crossing with distant relatives.Additionally, Crowder (1993) suggested that plants in the F2 generation exhibit high variability due to segregation under Mendel's law.
The variation in phenotype for fruit freshness is narrow (Table 5), which suggests that only some individuals can be chosen.Conversely, the characteristics of fruit hardness and weight loss display a broad phenotypic variability.Austiet al. ( 2014) indicated that the broad phenotypic variability could be attributed to extensive environmental variations.Furthermore, plant varieties grown under the same conditions may exhibit distinct phenotypic responses.
According to Murti et al. (2002), different genotypes have different abilities in absorbing nutrients even though the environment used is relatively the same, so it can cause a variety of phenotypic appearances from each genotype planted.The high and low variability of the phenotype illustrates the appearance of genotypes tested in the field.

CONCLUSIONS
All hybrid Sletr12 mutants have a more excellent freshness than their commercial parents.Intan x Sletr12 shows the best shelf-life, with a shelf-life of 35 days.All characters show narrow genetic variability.The broad variability of phenotypes is found in the character of fruit hardness and fruit weight loss.
(Lycopersicum esculentum var.servo).ElHayah, 7(2), pp.84-93. Wills, R.H.H., Lee, T.H., Graham, D., McGlasson, W.B., and Hall, E.G. (1981) σ²g) and phenotypic variablity (σ²f) can be obtained based on the analysis of variance.Determination of criteria for genetic and phenotypic variability is done by comparing genetic and phenotypic variance with standard deviation.An observed character has a broad genetic variability value if the value is equal to or twice the genetic standard deviation (σ²g ≥ 2σ σ²g ).The same applies to phenotypic variability.Phenotypic variability is broad if the phenotype values are the same or greater than twice the standard deviation (σ²f ≥ 2σ σ²f ).The standard deviation of genetic variance and phenotype is assumed by the following formulas (Anderson and Bancroft, 1952): Remarks: σ σ²g = standard deviation of genetic variance σ σ²f = standard deviation of the phenotypic variance MSg = genetic mean square MSe = error mean square df g = genetic degrees of freedom r = replication The experiment was carried out using tomatoes planted in the Indonesian Vegetable Research Institute Experimental Field at an altitude of 1,250 m asl with andosol soil types.Planting was done in an area of 100 m².Tomato plants were grown on land with genotype as factor consisting of 12 treatments and four replications.Each replication in one treatment consisted of five plants, resulting in a total of 240 experimental units.

Figure 1 .
Figure 1.The freshness of the fruit of four commercial tomato cultivars and the results of the crossing with tomatoes WT and Sletr12 mutants Wiguna et al.: Variability of fruit shelf-life of commercial tomatoes and their hybrids crossing.....

Table 2 .
The Scott-Knott test results on fruit hardness of 12 tomato genotypes Remarks: Means followed by the same lowercase letters in the same column are not significantly different based on the Scott-Knott test at the level of α=5 %.

Table 3 .
The Scott-Knott test results on fruit weight loss of 12 tomato genotypes

Table 4 .
The genetic variability values of the character of the storability of 12 tomato genotypes

Table 5 .
The phenotypic variability values of the character of the storability of 12 tomato genotypes Sletr12 at 10 DPS and Mirah x Sletr12 at 20 DPS (Table . Postharvest.An introduction to the physiology and handling of fruit and vegetables.Granada.Yogendra, K.N.N., and Ramanjini Gowda, P.H.H. (2013).Phenotypic and molecular characterization of a tomato (Solanum lycopersicum L.) F2 population segregation for improving shelf life.Genetics and Molecular Research, 12(1), pp.506-518.