Effect of Seed Extraction Methods of Tomato on Physiological Quality of Seeds and Seedlings

Tomato (Solanum lycopersicum L.) belongs to the family Solanaceae is one of the most popular and most processed vegetable crops worldwide with a great nutritional contribution to the human diet. Even though its demand is increasing with increasing protability, the production constricted by lack of quality seeds due mainly to lack of proper seed extraction method particularly for large scale seed production. Extensive disease epidemics might also be due to unsatisfactory seed extraction method. Empirical evidences are scanty on instant and latent effects of seed extraction methods of tomato seeds. Thus, this study was initiated to investigate the effect of seed extraction methods on physiological quality of seeds and seedlings of tomato. The experiment was carried under laboratory and eld conditions in a completely randomized design. Results revealed that the highest germination percent (99.33 and 89.76% under laboratory and eld conditions respectively) was obtained at 2% HCl for 60 minutes. Whereas, maximum weight of 1000 seeds (4.277g) was found at 1% of HCl for 30 minutes whereas maximum mycoora load (36%) was observed from 72 hours fermentation. Yet, no mycoora was detected from higher concentration (2% and more HCl) and time length (60 minutes and longer time). Seed quality parameters like seedling length, seedling fresh weight, seedling dry weight, and vigour indices were signicantly higher at extraction method of 2% HCl for 60 minutes. Thus it can be concluded that maximum physiological seed quality and best performance of seedlings of tomato can be obtained from 2% HCl for 60 minutes. experiments) were subjected to analysis of variance (ANOVA) using GenStat statistical software version 15.1. All signicant pairs of treatment means were compared using Fisher’s LSD (Least Signicant Difference Test) at 5% level of signicance. Correlation analysis was also performed to detect the linear relationship among seed physiological quality attributes and seedling characteristics. aforementioned results showed that most important seed physiological quality and seedling characteristic parameters such as rst count germination, germination percent, seed emergence, seedling length, seedling fresh weight, seedling dry weight, vigour index I and vigour index II were signicantly higher at extraction method of 2% HCl for 60 minutes. Furthermore, no mycoora was detected from seeds extracted by dibbing in to 2% HCl for 60 minutes. Thus, it can be concluded that maximum physiological seed quality of tomato can be obtained from a seed extraction method of dipping within 2% HCl concentration for 60 minutes period of time.


Abstract
Tomato (Solanum lycopersicum L.) belongs to the family Solanaceae is one of the most popular and most processed vegetable crops worldwide with a great nutritional contribution to the human diet. Even though its demand is increasing with increasing pro tability, the production constricted by lack of quality seeds due mainly to lack of proper seed extraction method particularly for large scale seed production. Extensive disease epidemics might also be due to unsatisfactory seed extraction method. Empirical evidences are scanty on instant and latent effects of seed extraction methods of tomato seeds.
Thus, this study was initiated to investigate the effect of seed extraction methods on physiological quality of seeds and seedlings of tomato. The experiment was carried under laboratory and eld conditions in a completely randomized design. Results revealed that the highest germination percent (99.33 and 89.76% under laboratory and eld conditions respectively) was obtained at 2% HCl for 60 minutes. Whereas, maximum weight of 1000 seeds (4.277g) was found at 1% of HCl for 30 minutes whereas maximum myco ora load (36%) was observed from 72 hours fermentation. Yet, no myco ora was detected from higher concentration (2% and more HCl) and time length (60 minutes and longer time). Seed quality parameters like seedling length, seedling fresh weight, seedling dry weight, and vigour indices were signi cantly higher at extraction method of 2% HCl for 60 minutes. Thus it can be concluded that maximum physiological seed quality and best performance of seedlings of tomato can be obtained from 2% HCl for 60 minutes.
Background Tomato (Solanum lycopersicum L.) belongs to the family Solanaceae is one of the most popular and worldwide consumed vegetable crops. It is also the most processed vegetable crop and ranks rst in commercial and nutritional contribution to the human diet (Dandena et al., 2013;Isack and Monica, 2013;Asfaw and Eshetu, 2015;Ankit et al., 2016;Mawardii et al., 2017).
Domestic production and export of tomato in Ethiopia is signi cantly increasing with increasing pro tability providing higher income for producers (EIA, 2012 andDebela et al., 2016) and making a signi cant contribution to the national economy (Baredo et al., 2014). However, the production is constrained by lack of quality seeds especially of local open pollinated varieties which are being replaced by imported hybrids. The success of germination, growth and nal yield of crops largely depend on quality of seeds. Quality of seed refers to the viability, free from damage, healthy, purity and vigour attributes of a seed that enables the emergence and establishment of normal seedlings under a wide range of environments (Kailappan and Karunanithy, 2006;Khan, 2013;and Savageand Bassel, 2016).
Tomato seed quality is affected by factors such as fruit maturity, methods of seed extraction, time length of fermentation, and fermentation temperature (Nemati et al., 2010). A mucilaginous gel substance in tomato seeds has germination inhibitors. Thus, seed extraction includes removal of pulp and the gelatinous substances surrounding the seed (Jeffrey, 2004;and Vishwanath et al., 2006).
The pulp and gel surrounding the seeds can be removed by different extraction methods such as natural fermentation, using chemicals or by mechanical means. Sodium carbonate, sodium hydroxide, ammonium hydroxide, hydrochloric acid, acetic acid, calcium hypochlorite, pectinases and sulfuric acid are among commonly used chemicals for tomato seed extraction (Demir and Samit, 2001;França et al., 2013;and Rival et al., 2016). A speci c concentration of chemicals applied to the eshy fruits together with the pulp and seeds for speci c time period.
Natural fermentation and manual seed extraction methods are commonly used methods but not effective for large scale production. Chemical methods mostly preferred as they are easier and faster for large scale production and obtain disease free seeds (Kailappan and Karunanithy, 2006;Nemati et al., 2010;Ankitet al., 2016;and Vishwanathet al., 2016). However, it is evident that chemical with higher concentration harm the embryo of seeds so as affect their nutritive value, germination percentage and other seed quality parameters.
Acid extraction methods especially 1-3% hydrochloric acid largely used for separating tomato pulp and seeds (Sachan et al., 2009;Nemati et al., 2010;Ankit et al., 2016;and CAFT, 2017). This method is often favored by large commercial producers as it rapidly degrades the gelatinous seed coating resulting in the production of a very bright clean seed sample (Eevera and Vanangamudi, 2006;and Rival et al., 2016). E cient breakdown of the gel surrounding the seed and quick cleaning, avoiding of the low and high temperature problem, eradication of bacterial canker and producing bright looking seed coat are important features of acid extraction method (Desai, 2004). However, it can be deteriorative on seed quality if application period and concentration are not appropriate.
Seed myco ora load also in uenced by extraction methods. Unsatisfactory method of tomato seed extraction is one of the reasons of extensive disease epidemics (Ankit et al., 2016). Empirical evidences are scanty on the instant and latent effects of seed extraction methods and time length of fermentation on seed physiology and seedling quality of tomato. This study was therefore intended to investigate the effects of seed extraction methods and time length of fermentation on physiological quality of seeds and seedlings of tomato.

Materials And Methodologies
The experiment was carried out in the year 2019/20 at Horticulture Department laboratory of College of Agriculture and Environmental Sciences, University of Gondar, Ethiopia located at an altitude of 1906 m.a.s.l.

Treatments and Experimental Design
Fermentation time for 24, 48 and 72 hours and dipping of tomato fruits with 1%, 2%, and 3% HCl acid for 30, 60 and 90 minutes were the treatment combinations. Thus, there were about 12 treatment combinations replicated thrice; 3*12 = 36 experimental units laid out in a CRD.

Experimental Procedures
For each treatment, about 1 kg of uniform size, shape and fully ripened tomato fruits var. Gelila were taken directly from production eld. The fruits crashed and squeezed with in a clean plastic buckets. Then, seeds were extracted with both methods following speci c procedures for each. After extraction process, the quality of seeds was evaluated by seed physiology and seedling characteristics. Seedlings were planted in petri dishes and pots in laboratory and on eld conditions, respectively.
Fermentation extraction method: The fruits were wrinkled manually then pulp along with gelatinous material and the seed was allowed to ferment for a period of 24, 48 and 72 hours at room temperature of18 -33 o C and relative humidity of 42%. The mixture was agitated daily to allow uniform rate of fermentation and elude discoloration. The seeds were repeatedly lapped with tap water. Good seeds and abnormal seeds and other debris were separated by sink and oating on the water surface. Finally, the good seeds were surface dried over rough papers for three days at room temperature and then weighed and packed in plastic bags (ISTA, 2020;Nemati et al., 2010).
HCl acid extraction method: Fully ripened tomato fruits with uniform maturity stage were lanced and broken. HCl acid solution of 1%, 2%, and 3% for each kg of tomato fruits was prepared in a volume/weight basis. The juicy pulps with gelatinous substances were dipped for 30, 60 and 90 minutes. The seeds were extracted, dried and packed with similar procedure for fermentation process (Fig. 1).

Data Collection
Weight of 1000 seeds (g): once the seeds extracted, samples of 1000 seeds were randomly collected from each treatment combinations and weighed with sensitive balance to three decimal places expressed in grams (ISTA, 2007;França et al., 2013;Debela et al., 2016).
Seed myco ora: the presence of fungi on seeds was detected by blotter method as recommended by ISTA (2020). 100 seeds were planted on a double layer moistened blotters of petri plates maintained at room temperature in three replications. After 7 days, the number of infected seeds (fungal colonies) were counted according to Vishwanath et al., (2006) and expressed as percentage.
First count (%): a rst count data was taken on the 4 th day after planting based on ISTA (2007). Germination (%):100 seeds representing each treatment were planted in petri dish in which moistened blotter paper was inside and the process triplicated. It will be better to convert it in to Average seedling fresh weightPetri dishes then placed in laboratory at room temperature. The nal count was on 14 th day of germination test for normal seedlings and expressed in percentage based on ISTA (2020).
Seedling emergence (%): randomly selected six seeds were planted on triplicated pots representing each treatment. Adequate moisture was maintained to make as suitable as possible for seed emergence. Seedlings emerged 3 cm above the soil surface was recorded on 7 th to14 th days after planting according to Debela et al., (2016), calculated and expressed as percentage of seed emergence.
Seedling length (cm): Seedling length includes the length of shoot tips to root tips. Ten random selected normal seedlings were considered. On the day of nal count, the length from the collar region to the tip of the primary shoot were measured as shoot length (cm) and from the collar region to the tip of primary root were measured as root length (cm). Then, the seedling length was computed by using the following formula, Seedling length (cm) = Shoot length (cm) + Root length (cm).
Seedling fresh weight (g): On the nal count day, four normal seedlings were uprooted from the growing pots of each treatment and the entire biomass weighed.
Seedling dry weight (g): the seedlings considered for fresh weight were dried in a hot-air oven at 80 o C temperature for 24 hours. The weight of the dried seedlings was recorded and the average weight was calculated and expressed as seedling dry weight in grams.
Vigour indices: The vigour indices were calculated using the procedure suggested by (Abdul-Baki and Anderson, 1973 cited in Ankit et al., 2016) and expressed in whole number.

Data Analysis
Data collected in laboratory and on eld (pot experiments) were subjected to analysis of variance (ANOVA) using GenStat statistical software version 15.1.
All signi cant pairs of treatment means were compared using Fisher's LSD (Least Signi cant Difference Test) at 5% level of signi cance. Correlation analysis was also performed to detect the linear relationship among seed physiological quality attributes and seedling characteristics.

Results And Discussion
Data on selected seed quality parameters and seedling characteristics were recorded during the course of the study. The results of the study are presented and discussed sequentially as follows.
The study results revealed that all seed physiological characteristics were signi cantly in uenced by the extraction methods along with duration of time (  (Table 1). As shown in Fig.3. (A) dominantly higher seed weight in 1% HCl across all time lengths as compared with higher concentrations of HCl. The minimum weight (1.440 g) was obtained from 3% HCl for 90 minutes. Generally, weight of 1000 seeds decreased with increasing concentration of HCl. Maximum weight of 1000 seeds at lower HCl concentrations might probably be due to partial removal of mucilage and presence of gelatinous substance adhered to the seeds (Vishwanath et al., 2006).

Seed myco ora
The percent myco ora detection was signi cantly (P< 0.001) affected by seed extraction methods and time length. The highest percentage of myco ora (36%) observed from fermentation method for 72 hours followed by fermentation for 48 hours (29.33) ( Table 1). On the other hand, myco ora load drastically decreased as HCl concentration as well as time length increased ( Fig. 2. B). No myco ora was detected from higher concentration (2% and more HCl) and time length (60 minutes and longer time). As par with this result Vishwanath et al., (2006) also reported highest myco ora load from fermentation extraction and lowest load from higher concentration of (2.5% HCl) acid extraction methods. It has been evidenced that fusarium, root nematodes and verticillum pathogens resided deep within seed coats and fuzzes usually emerge during germination could be disinfected by acid extraction method (Vishwanath et al., 2006;Dick and Dick, 2014;and Alabi, 2019).

First count germination
The analysis of variance showed that rst count germination was signi cantly (P< 0.001) affected by treatment effects. The highest rst count germination (93.3%) was recorded at 2% HCl for 60 minutes whereas the lowest (23%) was recorded from seeds extracted with 72 hours fermentation ( Germination percent highly declined when fermentation period prolonged from 24 to 48 then to 72 hours (Fig. 4. B). Darken and swollen (imbibed) seed coat was also observed ( Fig. 1. C). Extended period of fermentation likely imposed seeds to germinate during extraction process and protruded radicle killed during seed drying process. Nemati et al., (2010) also reported analogous ndings. As shown in Fig. 4. (B), the lower germination in lower concentration (1% HCl) and shorter dipping time (less than 60 minutes) might probably be due to presence of inhibitors in the gelatinous substance adhered to the seeds (Vishwanath et al., 2006). Lengthening the time from 60 into 90 minutes or increasing the concentration of HCl from 2% into 3% also resulted signi cantly reduced germination percentage (94.67%) and (88.67%), respectively. This might also be due to corrosive effect of acid over prolonged period (Vishwanath et al., 2006;Nemati et al., 2010;França et al., 2013;and Ankit et al., 2016).

Seeds emergence
Signi cantly maximum (89.76%) seeds emergence was obtained from 2% HCl for 60 minutes followed by 2% HCl for 90 minutes (81.99%). The lowest seeds emergence (33.71%) was found at fermentation for 72 hours ( Table 2).The lowest germination might be either the seeds were damaged by fungal pathogens or seeds were germinated during extended fermentation time thus failed to germinate during germination test. This nding is in conformity with the results of Evera and Vanangamudi (2006) and Nemati et al., (2010) who reported that decreased seed emergence due to fermentation longer than 48 hours. This might be due to premature sprouting and reduced germination from extended fermentation period.

Seedling length
Among all extraction methods, signi cantly (P< 0.01) maximum seedling length (14.00 cm) was recorded at 2% HCl for 60 minutes followed by 1% HCl for 90 minutes (13.50 cm). The minimum seedling length 10.77 cm and 10.83 cm recorded at fermentation for 72 hours and 3% HCl for 90 minutes respectively (Table 3 and Fig. 4. B). Quite the reverse, Ankit et al., (2016) testi ed a maximum seedling length (13.49 cm) from 24 hours fermentation. On the contrary, Nemati et al., (2010) reported no signi cant difference between short-term fermentation and severe plant height reduction with long-term fermentation. In the current study, difference in seedling length might be due to difference in date of germination. Earlier germinated seedlings probably had longer periods for root and shoot growth.

Seedling fresh weight
Seedling fresh weight was signi cantly (P < 0.01) affected by the treatment effects. Maximum fresh weight (558.1g) found at 2% HCl for 30 minutes which is statistically at par with 2% HCl for 60 minutes (556.2g) ( Table 3).

Seedling dry weight
Seedling dry weight was signi cantly (P< 0.01) in uenced by seed extraction methods. The highest seedling dry weight (26.73 g) was observed from 2% HCl for 60 minutes followed 1% HCl for 30 minutes (24.85 g) time length. Minimum dry weight (13.71g) and (15.4 g) was recorded from 72 hours fermentation and 3% HCl for 90 minutes (Table 3).

Vigour indices
Both vigour index I and II were signi cantly affected by extraction method and time length at P < 0.001 and P < 0.01 respectively. The highest vigour index I (1391) and Vigour Index II (2655) were obtained from dibbing of the crashed tomato fruits in 2% HCl for 60 minutes while the lowest vigour index I (329) and Vigour Index II (417) were recorded from fermentation for 72 hours (Table 4).
There was a severe increase in seed vigour indices with increasing HCl concentration and time length up to 2% HCl for 60 minutes then radically decreased beyond that point (Fig. 5. A and B). However, CAFT (2017) recommended 3% HCl for 30 minutes to get the best vigour and seed quality of tomato. But, in this study seed vigour was reduced over 2% HCl. This might be due to differences in pulp thickness. On the other hand, Demir and Samit (2001) reported that best seed vigour can be obtained from 2 and 3% HCl acid extraction. Means different letter within a column are significantly different at 5% level of significance; LSD=Least significant difference; CV=Coefficient of variation.

Conclusion
The aforementioned results showed that most important seed physiological quality and seedling characteristic parameters such as rst count germination, germination percent, seed emergence, seedling length, seedling fresh weight, seedling dry weight, vigour index I and vigour index II were signi cantly higher at extraction method of 2% HCl for 60 minutes. Furthermore, no myco ora was detected from seeds extracted by dibbing in to 2% HCl for 60 minutes. Thus, it can be concluded that maximum physiological seed quality of tomato can be obtained from a seed extraction method of dipping within 2% HCl concentration for 60 minutes period of time.

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Consent for Publication
This piece of manuscript entitled as "Effect of Seed Extraction Methods on Physiological Quality of Seeds and Seedlings of Tomato" is our own original research work and agreed to submit for your journal to be published. Please consider it Effects in 1000 seeds weight (A) and seed myco ora detection (B) due to HCl concentration Changes in vigour index I and II in response to extraction method and time length