Journal of Asian Horticulture, 2:250-254 (2006)

Influence of Seed Extraction Methods on Seed Quality in Leaf Curl Resistant Tomato Varieties
VISHWANATH, K1., RAJASHEKHAR, B.S.1, KALAPPA V.P1., MUNIYAPPA, V 2 AND NAGARAJAPPA ADIVAYPPAR3
1

Department of Seed Science and Technology, 2 Department of Plant Pathology, 3 Division of Horticulture University of Agricultural Sciences, Bangalore 560065

ABSTRACT: seed extraction method is important step in tomato seed production programme, which involves removal of pulp and gelatinous substance present around the seed. The study was conducted on three leaf curl resistant varieties viz., Sankranthi, Nandi, Vybav and one susceptible variety Arka Vikas with manual extraction, fermentation, fermentation with yeast (1,2 & 4%) and acid extraction with different concentration of HCl for 30 and 60 min. Three treatments (2.5% HCl for 30 min., fermentation for 24h or 48h showed above 90 percent germination. All other treatments (except control, 1.0% HCl for 30min, fermentation with 1% yeast for 18h) showed germination above 80 per cent. Acid extraction (HCl 25ml/kg pulp for 30min) and natural fermentation either for 24h or 48h showed equally better seed quality in terms of germination, seedling vigour index. However seed mycoflora load is low in acid extraction compare to natural fermentation. Varieties did not differ among each other. However, variety Arka Vikas recorded higher E.C. and lower germination after ageing indicating the poor storability.

INTRODUCTION The availability of quality seed is of at most important for increasing production and productivity of tomato. Quality of seed is also found to be influenced by method of seed extraction. Although large-scale seed production is undertaken with natural fermentation, chemical methods proved to be advantageous, as they are easier and faster than natural fermentation (Ghosh and Syamal,1997). Knowledge of correct method of seed extraction is must for obtaining better seed quality. Seed mycoflora load also influenced extraction methods and unsatisfactory method of tomato seed extraction is also one of the reasons of extensive epidemics of Verticillium wilt, which accrued in 1984 in parts of Italy (Cirruli et al.,1985). MATERIALS AND METHOD Field and laboratory studies were carried at Main Research Station, University of Agricultural Sciences, Hebbal, Bangalore during 2002-03. Methods of seed extraction: The seeds were extracted in all the four varieties by using different extraction methods involving manual extraction, natural fermentation, fermentation with yeast and acid treatments. 10 kg fruits were taken from each variety for each treatment. The procedure adopted for each of the extraction methods are detailed below. Manual seed extraction: Red ripe fruits were crushed and squeezed with hand and then the pulp containing the seeds were washed repeatedly with water to separate

the seeds. Here neither fermentation nor acid was used. Later the seeds were dried to moisture content of 8 per cent. Fermentation method: Red ripe fruits were allowed to over ripe for two days by keeping in plastic container. The fruits were crushed manually and crushed pulp along with gelatinous material and the seed was allowed to ferment for a period of 24 hours at room temperature. The pulp and seed mixture was stirred twice daily to keep the rate of fermentation uniform in the containers and to avoid discoloration. At the end seeds were washed repeatedly directing a set of water in to the containers. The good seeds sink, while other debris and light seeds were surfaced and washed off by inclining the vessel. The remaining good seeds were finally poured in to a retaining sieve, later the seeds were placed in nylon mesh bags, washed again and allowed to dry in shade and sun alternatively, till it attained 8 per cent moisture and were preserved in polythene bags. Another set of fruits were crushed and allowed to ferment for 48 hours at room temperature (23 to 25 0C) and seeds were separated and dried to 8 per cent moisture. Fermentation with yeast: Fruit were crushed and treated with 1, 2 and 4% yeast (i.e., at the rate of 10,20 and 40 gm each per kg of crushed pulp) and allowed for fermentation for 18h. Acid extraction: Fruits were crushed and pulp was taken in plastic containers and treated with commercial hydrochloric acid @ 25 ml, 15 ml and 10 ml per kg of crushed pulp by volume/ weight basis. After extraction, seeds were dried to 8% moisture content and evaluated following seed quality parameters. Germination: test was conducted by between paper method as per the procedure outlined by ISTA (Anon.,1996). Vigour index: was calculated by multiplying per cent germination and seedling length (Abdul-Baki and Anderson, 1973). Germination After Accelerated Ageing (GAA): 100 seeds in three replication from each of the treatment were subjected to artificial ageing (100% RH & 400C) in desiccators, with saturated KNO3 solution at 400C for five days. Then removed and set out for germination (Anon., 1996) and GAA was recorded on normal seedling basis. Electrical conductivity (EC) of seed leachates: Fifty seeds in three replication from each of the treatment were soaked in 25 ml distilled water for 24 hours and seed leachate was measured with the EC bridge and was expressed as dS/m at 25 oC ( Brandock and Mathews, 1968). Seed mycoflora: Detection and identification of storage fungi in seeds was done by blotter method as recommended by ISTA (Anon., 1996). Twenty-five seeds in three replication and were plated equidistantly on two layers of moistened blotters in the petri plates and were incubated at room temperature. After 7 days, the number of infected seeds (fungal colonies) were counted and expressed as percentage. Data collected were statistically analyzed by the analysis of variance techniques by adopting Completely Randomized Design (CRD)(Panse and Sukhatme,1967). Arcsine square root method was applied for transformation of data, which was applied on those tables in which the values were less than ten or in percentage.

RESULTS AND DISCUSSION The seed extraction methods significantly affected the seed quality parameters. Among the acid extraction methods 2.5% HCl for 30 minutes (25ml/kg pulp) gave clean separation of seeds with highest germination (95.43%), which was closely followed by natural fermentation for 24 hours (93.83%). Further extending period of fermentation to 48 hours appears to have no added advantage. Extending the period of acid extraction (2.5% HCl) for 60 minutes reduced the germination (89.25%). This might be due to corrosive effect of acid over prolonged period. However, other acid methods at reduced concentration had reduced germination ranging from 79.47 to 84.29 per cent. Similarly several researchers noticed improved germination in acid extraction, as they are less infected by seed mycoflora (Ghosh et al., 1997; Vadivelu and Ramaswamy, 1979; Talukdar, 1988; Javaregowda et al., 1991; Das et al., 1991) Natural fermentation with yeast can enhance the process of fermentation and achieve seed separation with in 18 hours, but the germination reduced drastically (73.45%). This was probably due to higher per cent of mycoflora infection (Das et al., 1991)(Table-3). The reduced germination (67.16%) in manual seed extraction was probably due to partial removal of mucilage and presence of inhibitors in the gelatinous substance adhered to the seeds. Similarly seedling length, seedling vigour index and germination after accelerated ageing (GAA) showed significant difference due to extraction methods. Acid extraction with HCl (2.5% for 30 min) recorded highest seedling length, seedling vigour index and germination after accelerated ageing (GAA). The next best treatments showing higher seed quality parameters were natural fermentation for 24 hours followed by 48 hours. Other extraction methods involving reduced acid to pulp ratio and fermentation involving yeast has showed reduced seedling length and seedling vigour index. Treatment with higher concentration of HCl (2.5%) for 60 minutes also showed reduced seed quality. This in agreement with the findings of Vadivelu and Ramswamy, 1979). The maximum EC was observed in treatment with HCl (2.5%)for 60 minutes (0.72 dS/m). The increased EC in case of HCl (2.5%) for 60 minutes might be due to corrosive nature of acid, which might have damaged the seed coat. The results are in conformity with the findings of Talukdar (1998), who reported that HCl at higher concentration and longer duration of exposure would damage seed. Despite moderately higher EC values were noticed with fermentation for 24 hours and acid extraction (HCl 2.5%) for 30 minutes. Other seed quality parameters were not affected, which indicated that, the level of damage to membrane integrity is not conspicuous. Acid extraction methods with higher concentration (2.5%) for 60 and 30 minutes had lowest percentage of seeds associated with seed mycoflora ranging from

0 to 5.0 per cent respectively. Highest level of seed mycoflora infection was observed in the fermentation treatments. Fermentation with yeast showed maximum infection ranging from 27 to 30 per cent, which was closely followed by control and other fermentation methods. The seeds in these fermentation treatments were slightly discoloured, which may be attributed to high percentage of seed mycoflora infection. Although varieties showed significant differences for the seed quality parameters viz., germination percentage (before and after ageing), seedling length, seedling vigour index, EC of seed leachate, the difference between the treatments was narrow. The check variety Arka Vikas even though recorded slightly higher germination (85.25%), seedling length (14.85 cm) and seedling vigour index (1277), but recorded lower GAA (70.80%) and higher EC (0.62 dS/m), which indicated that Arka Vikas was prone for reduced storability. The varietal differences with respect to seed quality parameters can be attributed to genetic cause (Brar and Hari Shing, 1984; Ghosh and Syamal, 1997; Vishwanath et al., 2004 ). In conclusion, Tomato Seeds extracted by using HCl at the rate of 25 ml for 1 kg of crushed pulp for 30 minutes appears to be ideal for getting clean and high quality seeds especially for export purposes. Tomato seed extraction through natural fermentation method (24h) still hold the key in commercial scale production as it is eco-friendly and maintains better germination and seedling vigour. Acknowledgement: First two authors express our deepest gratitude to the departed soul of Dr. V.P. Kalappa, Professor, Department of Seed Science & Technology, UAS, Bangalore, for his guidelines throughout this study.

REFERENCES: Abdul-Baki, A.A. AND Anderson, J.P., 1973, Vigour determination in soyabean by multiple criteria. Crop Science, 13: 630-633.

REFERENCES: Abdul-Baki, A.A. AND Anderson, J.P., 1973, Vigour determination in soyabean by multiple criteria. Crop Science, 13: 630-633. Anonymous (1996). International Rules for Seed Testing. Suppliment to Seed Science and Technology, 24: 1-340. Brandock, W.T. AND Mathews, S. (1968) Assessing field emergence potential of wrinkle seeded peas. Horticultual Research, 10: 50-58. Brar, P.S. and Hari Singh, 1984, Effect of different methods of tomato seed extraction on seed recovery and its germination. Haryana Journal of Hortculture Sciences, 13(3-4): 161-164. Cirruli, M., Ciccarse and S. Frisullo, 1985, Extensive epidemics of Verticillum wilt on tomato in Apulia. Informatore fitopatologico, 35(7-8): 29-32. Das, R.K., Baruah, G.K.S. and Paul, S.R., 1997, Effect of extraction techniques on seed quality of tomato (Lycopersicon esculentum Mill.). Annals of Agricultural Research, 18(2): 220-221. Delouche, J.C., 1965, An accelerated ageing technique for predicting relative storability of Crimson Clover and Falfescne seed lots. Agronomy Abstracts, pp.40. Ghosh, P.K. and Syamal, M.M., 1997, A study of seed extraction methods in tomato. Advances in Plant Science, 10(1): 165-167 Javaregowda, S., Talukdar, K.C. and Ramaiah, H., 1991, Optimization of seed extraction techniques in tomato. Seeds & Farms., 17: 15-17. Panse, V. S. and Sukhatme, P.V., 1967, Statistical Methods for Agricultural Workers. Published by Indian Council of Agricultural Research Publication. New Delhi. pp. 34-37. Talukdar, K.C., 1988, Studies on seed extraction, conditioning and storage in tomato (Lycopersicon esculentum Mill.) seed. M.Sc. (Agri.) Thesis, UAS, Bangalore. Vadivelu, K.K. AND Ramaswamy, K.R., 1979, Influence of seed extraction methods on seed quality in tomato. South Indian Horticulture, 25(3): 106-108. Vishwanath, K., Kalappa, V. P. and Lokesh, K., 2004, Influence of sowing dates on seed yield and quality in French bean varieties. Mysore Journal of Agricultural Sciences. 38(1): 56-59.

Table 1. Effect of seed extraction methods on Germination (%) and seedling length (cm) of tomato varieties
Germination (%) Treatments (T) V1 T1 - Control T2 - 2.5% HCl for 30 min T3 - 2.5% HCl for 60 min T4 - 1.5% HCl for 30 min T5 - 1.5% HCl for 60 min T6 - 1.0% HCl for 30 min T7 - 1.0% HCl for 60 min T8 - Fermentation for 24 h T9 - Fermentation for 48 h T10 - Fermentation with 1% yeast for 18 h T11 - Fermentation with 2% yeast for 18 h T12 - Fermentation with 4% yeast for 18 h Mean 69.66 95.00 88.66 80.16 87.33 81.50 85.75 94.33 93.50 75.25 84.00 84.00 84.76 Varieties (V) V2 70.66 96.55 90.25 84.75 85.75 79.50 87.75 94.00 93.33 71.00 79.35 80.50 84.44 S.Em T V TxV 0.43 0.24 0.86 V3 68.66 94.00 87.33 86.00 86.66 80.16 84.75 92.75 91.25 77.33 78.66 82.00 84.13 V4 71.00 96.25 90.75 86.25 88.00 76.75 85.75 94.25 92.25 72.25 84.50 82.75 85.25 70.00 95.43 89.25 84.29 86.93 79.47 85.75 93.83 92.64 73.45 81.62 83.06 84.64 Mean V1 14.91 15.77 15.60 14.40 15.03 13.20 15.01 15.50 15.62 14.70 13.88 13.50 14.76 Seedling length (cm) Varieties (V) V2 15.47 15.41 15.18 15.33 15.18 14.33 14.51 15.10 15.11 14.16 13.41 13.46 14.72 S.Em T V TxV 0.07 0.04 0.14 V3 14.67 15.23 14.70 14.55 14.14 14.40 14.20 15.24 15.10 13.97 14.40 14.60 14.60 V4 15.13 15.36 14.88 14.68 15.00 14.99 15.01 15.63 15.56 14.56 13.81 13.63 14.85 15.04 15.44 15.09 14.74 14.84 14.23 14.68 15.36 15.35 14.35 13.87 13.80 14.73 Mean

CD (0.05P) 1.20 0.69 2.41

CD (0.05P) 0.19 0.11 0.39

V1 - Vybav V2 - Sankranthi V3 - Nandi V4 - Arka Vikas

Table 2. Effect of seed extraction methods on seedling vigour index and electrical conductivity (EC) of seed leachate of tomato varieties
Treatments (T) Seedling vigour index Varieties (V) V1 T1 - Control T2 - 2.5% HCl for 30 min T3 - 2.5% HCl for 60 min T4 - 1.5% HCl for 30 min T5 - 1.5% HCl for 60 min T6 - 1.0% HCl for 30 min T7 - 1.0% HCl for 60 min T8 - Fermentation for 24 h T9 - Fermentation for 48 h T10 - Fermentation with 1% yeast for 18 h T11 - Fermentation with 2% yeast for 18 h T12 - Fermentation with 4% yeast for 18 h Mean 1039 1498 1383 1154 1312 1076 1287 1462 1460 1106 1166 1134 1256 V2 1093 1488 1370 1299 1302 1139 1273 1419 1410 1005 1064 1083 1245 V3 1007 1432 1284 1251 1225 1153 1007 1414 1376 1081 1133 1198 1222 S.Em T V TxV 7.99 4.61 15.99 V4 1074 1478 1350 1413 1320 1150 1287 1473 1435 1052 1167 1128 1277 1053 1474 1347 1279 1289 1129 1213 1442 1420 1061 1132 1160 1250 Mean V1 0.42 0.57 0.65 0.50 0.51 0.48 0.49 0.50 0.51 0.51 0.52 0.52 0.52 Electrical conductivity (dS/m ) Varieties (V) V2 0.43 0.63 0.69 0.48 0.51 0.48 0.52 0.53 0.54 0.52 0.53 0.54 0.53 S.Em T V TxV 0.002 0.001 0.004 V3 0.44 0.63 0.69 0.47 0.51 0.48 0.52 0.51 0.54 0.48 0.53 0.54 0.53 V4 0.39 0.80 0.85 0.71 0.81 0.69 0.71 0.50 0.52 0.51 0.49 0.55 0.62 0.42 0.66 0.72 0.54 0.58 0.53 0.56 0.51 0.53 0.51 0.52 0.54 0.55 Mean

CD (0.05P) 22.16 12.80 44.32

CD (0.05P) 0.006 0.003 0.012

V1 - Vybav V2 - Sankranthi V3 - Nandi V4 - Arka Vikas

Table 3. Effect of seed extraction methods on seed mycoflora and germination after accelerated ageing (%) (GAA) infection of tomato varieties
Seed Mycoflora (%) Treatments (T) V1 T1 - Control T2 - 2.5% HCl for 30 min T3 - 2.5% HCl for 60 min T4 - 1.5% HCl for 30 min T5 - 1.5% HCl for 60 min T6 - 1.0% HCl for 30 min T7 - 1.0% HCl for 60 min T8 - Fermentation for 24 h T9 - Fermentation for 48 h T10 - Fermentation with 1% yeast for 18 h T11 - Fermentation with 2% yeast for 18 h T12 - Fermentation with 4% yeast for 18 h Mean 25 3.0 0.0 5.0 3.0 17 16 16 19 26 26 28 15 Varieties (V) V2 26 3.0 0.0 5.0 3.0 17 18 15 18 27 27 30 16 S.Em T V TxV 0.42 0.24 0.85 V3 23 4.0 0.0 4.0 3.0 13 16 14 26 26 28 28 15 V4 26 5.0 0.0 6.0 3.0 16 17 15 19 29 30 33 17 25 4.0 0.0 5.0 3.0 16 17 15 18 27 28 30 16 Mean V1 65.33 88.33 71.66 72.33 71.66 72.00 72.00 85.66 86.00 67.00 67.00 67.33 73.86 GAA (%) Varieties (V) V2 67.00 85.33 69.00 71.33 77.33 71.33 72.00 84.33 83.00 65.33 66.33 69.00 73.44 S.Em T V TxV 0.002 0.001 0.004 V3 72.00 86.66 70.00 73.33 72.33 74.33 70.33 85.33 85.33 68.33 66.00 65.33 74.10 V4 64.33 81.00 67.33 73.00 73.66 70.00 70.33 76.33 75.00 64.00 66.66 68.00 70.80 67.16 85.33 69.50 72.50 73.74 71.91 71.16 82.91 82.33 66.16 66.50 67.41 73.05 Mean

CD (0.05P) 1.19 0.68 2.38

CD (0.05P) 0.006 0.003 0.012

V1 - Vybav V2 - Sankranthi V3 - Nandi V4 - Arka Vikas