IJOEAR-APR-2017-26

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 12 Improvement of Crop Production by Means of a Storage Effect Gustáv Murín 1 Karol Mičieta 2 Department of Botany Comenius University Révová 39 811 01 Bratislava Slovakia Abstract — This study summarizes the results of 30 years of our experiments with Vicia faba L seeds. Our long-term practical observations of different Vicia faba L. cultivars points out the method useful for the higher yield of seeds in terms of their viability and thus higher crop production. Our experiments led to the following important findings regarding of seed viability: 1. Individual and group variability of seeds 2. Storage condition before germination and 3. The condition of their germination. All these three influential conditions is possible to optimalize by method of storage effect described in this our report resulting in the improvement of crop production. This is especially important in case of seeds that are rare and/or expensive i.e. seeds that are genetically modified or with rearranged karyotypes. Keywords — seed color higher germination improvement of viability higher crop production. I. INTRODUCTION Seeds have been studied for more than hundred years cf. Murín 2001 Murín and Mičieta 2009. The first reports uncovered the relationship between the decline of their vitality and their storage conditions Navaschin 1933 Cartledge and Blakeslee 1934 1935 Stube 1935 Nichols 1942 D’Amato 1951 Murín 1961 Avanzi et al. 1969. Since respiration is the most marked manifestation of metabolism in stored seeds it should also be considered. Rieger and Michaelis 1959 found that V. faba seeds are susceptible to the action of ethanol or other “automutagens” which can accumulate during the respiration of seeds stored over long period. Bewley and Black 1982 1994 explored the relationship between the color of the testa and the dormancy and germination of wheat as affected by the level of inhibitors catechinins and their derivates occurring in the testa. Floris and Anguillesi 1974 made a major contribution to the understanding of this external manifestation of the internal state of broad bean seeds when they reported on several biochemical and functional changes in aging seeds. Over the course of long storage enzymes like catalase peroxidase cytochrome oxidase and decarboxylase display diminished activity while the protein-synthesizing capacity of older seeds is lost in the process of germination. Furthermore membrane permeability increases resulting in reduced sugars and other metabolic products. According Roos 1980 four factors must be considered in seed storage – time temperature relative humidity seed moisture content and a level of oxygen. With the exception of recalcitrant species two factors – time and oxygen level have very little effect on storability if the optimum seed moisture content and storage temperatures are observed. For example Roberts and Ellis 1977 predicted the 95 survival of pea Pisum sativum L. seeds after 1090 years of storage at -20°C and 5 seed moisture content. If the storage temperature is reduced further the viability may be extended indefinitely. Attempts to prolong life of seeds during the storage were focused at the use of liquid nitrogen LN2 as a storage medium with a temperature -196°C. At this temperature presumably all biochemical activity is reduced to essentially zero. Thus the deteriorative changes noted above should be eliminated. According Babasaheb 2004 safe seed storage moisture should be less than 8. In 1981 King et al. reported that the survival of lemon Citrus limon L. lime C. aurantifolia Swing. and sour orange seeds C. aurantium L. was examined under a wide range of constant moisture contents and temperatures. Seed longevity was increased by decreasing the moisture content and temperature of the storage environment. Maximum viability was maintained in a combination of storage conditions including the lowest moisture content 5 and lowest temperature - 20°C. The practicality of the dry storage of citrus seeds for genetic conservation was pointed out.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 13 Bonner 1990 offered classification of stored seeds into four classes of storage characteristics: „‘true orthodox’ seeds can be stored for long periods at seed moisture contents of 5–10 and sub-freezing temperatures ‘sub-orthodox’ seeds can be stored under the same conditions but for shorter periods due to high lipid content or thin seed coats ‘temperate recalcitrant’ seeds cannot be dried at all but can be stored for 3–5 years at near-freezing temperatures and ‘tropical recalcitrant’ seeds also cannot be dried and they are killed by temperatures below 10–15°C.“ Grilli et al. 1995 described the level of Poly A Polymerase as a significant marker of the viability of seeds during their long term storage. Also during imbibition the production of the major organic volatiles ethanol and acetaldehyde depends greatly on the long term storage of the seeds Górecki et al. 1992. Murthy et al. 2002 identified two primary biochemical reactions responsible for deterioration of seed vigour during long term storage – lipid peroxidation and non-enzymatic protein glycosylation reducing sugars. The PCR analysis of Chwedorzewska et al. 2002 led the authors to the conclusion that long term storage of seeds causing the loss of their viability also generates heritable changes in the preserved germplasm. On the other hand antioxidant activity in stored seeds under different conditions temperature and w.c. is not related to seed viability Merritt et al. 2003. However Andreev et al. 2004 found that the loss of germination during the storage of rye seeds was accompanied by a decreased excision of chromatin loop domains. As Patrick and Stoddard 2010 stated “the large seed size of the faba bean has enabled this species to be a model for studies of the molecular physiology of seed development.” The darkening of the testa of aging V. faba L. seeds and its manifestation has been a practical part of our work since 1988 Murín 1988 a b. Today we know that the color of the testa indicates the viability as well as the age of the seeds. Our goal was to study the relationship between the different storage conditions the color of the testa of seeds and the viability of the seed samples. II. MATERIALS AND METHODS 2.1 Seed samples For our experiments we used sets of V. faba L. cv. Inovec seeds of the standard karyotype harvested in 1974 and also for each year from 1976 to 1982. Non-standard rearranged ACB karyotype Michaelis and Rieger 1971 were harvested in 1975 and 1982. The oldest cultivar Přerovský was from 1971. The colors of the seeds were classified from A to U according to Fisher-Sallers scale in order to determine their individual variability. Originally designed for hair color this scale was used for the first time by us because it registers a wide spectrum of brown hues. In the first experiment a total of 1419 broad bean seeds were examined in this way. The effect of storage time and conditions on seed coat color was also reported by Yousif et al. 2003 in their study of Australian adzuki beans. 2.2 Soaking and germination The conditions for standard soaking and germination were altered during our experiments according to the knowledge we obtained in each experimental stage. Our optimal set involved the soaking of seeds in plastic jars that allowed their continual air-bubbling of distilled water. The seeds were then germinated in wet sawdust. The last six experiments were germinated and grown in intact material Perlite. As V. faba is sensitive to hypoxia we made the following arrangements to prevent higher sensitivity affecting the results of our experiments: a better air circulation in the desiccators which were not kept at 25 °C but at laboratory room temperature b during soaking we used 5 chloramine B to prevent microbe contamination of the seedlings which were treated for 30 min and then washed with distilled water and c the seeds were germinated in wet sawdust instead of wet cotton wool which does not permit the satisfactory respiration of seeds. 2.3 Storage 2.3.1 Storage conditions All of the harvested V. faba seeds from our supply were stored at room temperature. Rearranged ACB karyotype was stored at 4 °C.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 14 Twenty-two seed samples from eight countries nineteen cultivars and nine harvests from 1972 to1984 from the Seed Bank in Gatersleben Germany were divided into two groups A and B. Groups A and B were stored in the seed bank at temperatures of -10 to -17 °C and +14 to +20 °C respectively. The seeds were then stored for 0 or 8 days at 25 o C above 600-mL sterile water at room temperature in the desiccators. Following treatment washing and re-drying half of the seed samples were allowed to germinate immediately and the root- tips were cut and put into fixation solution after two recovery times 48 h and 72 h. The other half of the seed samples were allowed to germinate after 8 days of storage. 2.3.2 Specific water content w.c. After treatment and washing the seeds were re-dried at 50 by heating for 2 h at 37 °C in a thermostat with a fan to obtain a specific w.c. Half of the seed samples were allowed to germinate immediately and the root-tips were fixed after various recovery-times. The other half of the seed samples were stored for 8 days and then allowed to germinate. 2.3.3 Control of water content For control of w.c. in seeds during the experiment an extra sample of ten seeds was weighed before and after special drying 8 h at 105 °C and calculated according to the formula 100 – Yx100/X w.c. where X weight before drying and Y weight after drying. 2.4 Mutagen treatment In one experiment of this series Vicia faba seeds were first treated for 5 h with a dose of 2mm of methyl methanesulphonate MMS Merck in distilled water at pH 4.8. After the mutagen treatment the seeds were washed for 2 h in tap water to eliminate the mutagen residue. 2.5 Tests of vitality The vitality and length of the roots from sets of 35 seeds were measured for precise time periods: 36h 48 h 72 h 80 h and 96 h 120 h 144 h and 168 h if necessary. Both groups of seeds from the Seed Bank in Gatersleben were tested periodically from 1991 until 1999 to record the viability and frequency of aberrant ana-telophases. 2.6 Cytological evaluations For cytological evaluation we chose ana-telophases in accordance with other authors Bezrukov and Lazarenko 2002. These mitotic figures are simpler to evaluate and thus allowed us to experiment with a large number of samples under different mutagen doses and recovery times. The mutagen-treated roots of seed samples were fixed in ethanol 1N: acetic acid 1N in rate 3:1 squashed and stained by aceto-orcein. On average 200 ana-telophases 50 in control per recovery time were evaluated on the occurrence of fragments F bridges B or both F+B. 2.7 Statistical methods We used a standard Students t-test to evaluate the SEM. All evaluations were conducted under blind conditions. III. RESULTS AND DISCUSSION 3.1 Individual and Group Variability V. faba L. seeds have been used for decades as an experimental model in cytological laboratories and the biological characteristics of this genus are widely known. However insufficient attention was paid to the darkening of seeds during their storage. A closer examination of this phenomenon bore striking implications for our research. Although the seeds seem to be the same they express their individual and group variability by the colors of their testa. In our first experiment we studied the seeds’ light and dark colors in relation to the years when they were harvested. Only seeds showing a definite color were evaluated those of intermediate colors were excluded from our final evaluation. Table 1 and Fig. 1 show the gradual darkening of the seeds over an 11-year period.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 15 TABLE 1 PROPORTION OF DARK AND LIGHT SEEDS IN THE COURSE OF AGING Harvest year and cultivar Proportion and color type of seeds No. of seeds examined No. and color type of seeds excluded light dark 1982 ACB 100 ACB 0.00 32 0 1981 Inovec 68.78 A-B 12.14 G-H 173 33 E F 1980 Inovec 25.80 B-C 20.00 Q R S 155 84 F G H 1979 Inovec 17.05 B-C 65.88 Q R S 129 22 G H 1978 Inovec 11.46 C-E 71.97 Q R S 157 26 L M 1977 Inovec 1976 Inovec 1975 ACB 1971 Přerovský 5.48 A B C 5.71 C-E 7.05 A-C 0.00 70.54 R S T 86.28 S T 85.47 R S T 100 U 146 175 241 211 35 E F G 14 F G 17 K-L 0 FIGURE 1: PROPORTION ORDINATE OF LIGHT AND DARK SEEDS IN DIFFERENT HARVESTS ABSCISSA In a set of seven-year-old seeds harvested in 1975 of the rearranged ACB karyotype that were stored in 4 o C instead of room temperature we observed a small deviation from the gradual darkening tendency. The higher the proportion of viable seeds and thus the better germination rate of this seed set confirmed the relationship between seed viability and storage conditions that was also stated by Michailov and Korytova 1971. We noted with interest the complete loss of viability of 9- year-old cv. Přerovský seeds and their uniformly dark color among the darkest on the color scale – U. In addition to confirming the irreversible tendency of seeds to darken in the course of their storage these experiments suggested a possible difference in the viability and germinating capacity of seeds harvested in the same year but differentiated by external darkening. Three differently arranged independent experiments Tables 2 – 4 revealed a clear difference between dark and light seeds. It shows the importance of individual or inter-individual variability among seeds of the same age externally manifested by darkening. 0 10 20 30 40 50 60 70 80 90 100 1982 1981 1980 1979 1978 1977 1976 1975 1971 Light seeds Dark seeds

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 16 TABLE 2 COMPARISON OF GERMINATING CAPACITY OF LIGHT AND DARK SEEDS FROM VARIOUS HARVESTS. Years of storage and color of seeds No. of germinated seeds out of 16 after 96 hrs 168 hrs Germination rate 6 years light 10 4 87.5 dark 0 2 12.5 7 years light 7 6 81.2 ABC dark 0 6 37.5 TABLE 3 COMPARISON OF GERMINATING CAPACITY OF LIGHT AND DARK SEEDS FROM VARIOUS HARVESTS. Years of storage and color of seeds No. of germinated seeds out of 16 after 98 hrs Germination rate 4 years light 14 87.5 dark 3 18.7 7 years light 11 68.7 dark 3 18.7 TABLE 4 COMPARISON OF GERMINATING CAPACITY OF LIGHT AND DARK SEEDS FROM VARIOUS HARVESTS Years of storage and color of seeds No. of germinated seeds after 72 h 96 h 120 h 144 h 168 h Germination rate 3 years light 9 5 0 1 0 93.75 dark 2 3 4 1 2 75.00 6 years light 5 6 2 2 0 93.75 dark 0 0 0 0 2 12.50 The next two experiments aimed at chromosome aberrations of light more viable and dark less viable colored seeds. In the first of these experiments we devoted most of our attention to the 1st-through-2nd mitosis roots 12-20 mm long and 3rd mitosis roots 20-30 mm long. A total of 5 750 anaphases were examined. Table 5 illustrates the differences in the degree of chromosome aberration within a harvest e.g. after 4 years of storage between light 0.75  0.14 and dark 2.30  1.18 seeds the gradual differentiation among harvests having been preserved dark seeds 1979: 1.73  0.7 dark seeds 1976: 6.00  0.7.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 17 TABLE 5 CHROMOSOME ABERRATIONS IN LIGHT AND DARK SEEDS IN THE FIRST MITOSES Years of storage and color of seeds Root length mm Aberration rate No. of anaphases examined Total aberration rate 72 h 94 h 120 h 144 h 4 years light 12-20 2.0 800 1.5 400 0.0 400 1.0 300 1.13  0.4 20-30 0.8 400 1.0 300 0.5 200 n.d. 0.75  0.1 dark 12-20 1.3 300 3.6 300 1.5 300 0.5 200 1.73  0.7 20-30 2.3 300 n.d. n.d. n.d. 2.30  1.2 7 years light 12-20 3.3 700 2.0 200 1.0 200 1.0 150 1.82  0.5 20-30 1.0 250 1.6 300 1.0 150 n.d. 1.20  0.2 dark 12-20 n.d. 7.0 200 5.0 200 n.d. 6.00  0.7 20-30 n.d. n.d. n.d. 5.0 150 5.00  0.0 n. d. not detected Examining seeds stored for 4 and 7 years we found that the differences within a seed set were more marked in older seeds while dark seeds were similarly differentiated in the early mitotic cycles. In an additional experiment we confirmed this tendency in later mitoses as well. After 98 h of germination 30 root-tips containing 3000 anaphases were examined. The root-length varied from 28 to 84 mm depending on the individual variability while in most roots it was about 50 mm. In light seeds of both harvests Table 6 anaphases were evaluated separately at a root-length of 28-34 mm 3rd mitotic cycle and of 44-57 mm 4th mitotic cycle. TABLE 6 CHROMOSOME ABERRATIONS IN LIGHT AND DARK SEEDS IN LATER MITOSES Years of storage and color type of seeds Aberration rate In roots long 28-34 mm 44-57 mm Total 4 years light 0.0 1.2  0.37 0.66  0.3 dark 2.60  0.5 n.d. 2.60  0.5 7 years light 1.5  0.82 0.5  0.82 1.00  0.3 dark 2.00  1.2 n.d. 2.00  1.2 Three hundred anaphases were examined in each test. Table 7 shows some difference in the aberration rate between light and dark seeds of the same harvest while the aberration rate among seeds of the same color harvested in different years was almost the same. This shows a clear manifestation of viability dependent at individual variability of seeds. TABLE 7 COMPARISON OF CHROMOSOME ABERRATION RATE WITH GERMINATION RATE OF SEEDS STORED FOR 4 AND 7 YEARS. Years of storage and color type of seeds Aberration rate Germination rate 4 years light 0.66  0.3 87.5 dark 2.60  0.5 18.7 7 years light 1.00  0.3 68.7 dark 2.00  1.2 18.7 Three hundred anaphases were examined in each test.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 18 This finding is in accordance with already reported changes in the aberration rate of cells in seeds during their long-term storage Avanzi et al. 1969 Sevov et al. 1973 Cebrat 1977 Murín 1988a. The comparison of data presented in Tables 1 – 4 confirms this conclusion in relation to seed viability and chromosome damage in root-tip cells Tables 5 – 7. In the present case the external signs of reduced viability darkening were manifested irrespective of their age with almost corresponding consequences for genetic and physiological damage. The differences observed between whole seed harvests indiscernible among individual seeds could thus be explained by a decreasing proportion of viable seeds and an increasing number of less viable dark seeds in the same set as a result of long-term storage. In V. faba seeds this process could be followed from the 100 proportion of light seeds in the youngest set of one-year-old seeds with the highest germination rate and lowest aberration rate to the complete mortality of a seed set after 9 years of storage when the proportion of dark seeds had reached 100. To confirm these results once again new experiments were designed Tables 8 – 9. Two experiments involved 1-year old seeds selected by their color Table 8 and two experiments involved 5-year old seeds with and without the mutagen treatment Table 9. This time they were not germinating and growing in sawdust but in intact material Perlite. TABLE 8 ONE-YEAR OLD SEEDS SELECTED BY THEIR COLOR TESTED IN VITALITY. GERMINATION AND CHROMOSOMAL ABERRATIONS IN THEIR ROOT TIPS. 72 h 96 h 120 h Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Light seeds 32.97 100.0 1 37.77 100 2 39.92 100 0.5 Dark seeds 22.15 87.5 2 26.12 90 1 27.62 90 1.5 TABLE 9 FIVE-YEAR OLD SEEDS WITHOUT AND AFTER TREATMENT FOR 5 H WITH DOSE OF 2mM OF METHYL METHANESULPHONATE MMS. MERCKSELECTED BY THEIR COLOR TESTED IN VITALITY. GERMINATION AND CHROMOSOMAL ABERRATIONS IN THEIR ROOT TIPS. 72 h 96 h 120 h Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Light seeds 32.67 100 2.5 37.03 100 1.00 38.0 100 0.9 Dark seeds 21.40 87 4.5 23.43 87 5.57 24.8 87 1.0 72 h 96 h 120 h MMS. Merck Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Length of roots mm Germi- nation Chrom. ab. Light seeds 18.47 93.5 12.18 20.87 93.5 25.18 21.47 93.5 14.31 Dark seeds 13.70 66.5 8.50 15.60 70.0 17.79 15.87 70.0 25.04 The summarized results are found in Table 10 and confirm all of the previous findings and shows higher sensitivity of dark colored individuals in vitality germination and chromosomal aberrations in their root tips. However we also found some disturbances in this tendency in comparison with light colored individuals in chromosomal aberrations in recovery times of 72 h and 96 h.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 19 TABLE 10 COMPARISON OF THE PREVIOUS RESULTS FROM TABLES 8-9 SHOWING AN AVERAGE IN ALL TESTED PARAMETERS LENGTH OF ROOTS. GERMINATION AND C.A. WITH SEM BETWEEN 1-YEAR OLD SEEDS LINES 1-2. 5-YEAR OLD SEEDS LINES 3-4 AND 5-YEAR OLD SEEDS TREATED BY MUTAGEN LINES 5-6 72 h 96 h 120 h Length of roots mm Germi-nation Chrom. ab. Length of roots mm Germi-nation Chrom. ab. Length of roots mm Germi-nation Chrom. ab. Light seeds 32.98±0.38 100.0±0.00 1.00±0.00 37.78±0.63 100.0±0.00 2.00±1.00 39.93±0.08 100.0±0.00 0.50±0.50 Dark seeds 22.15±1.80 87.5±2.50 2.00±0.00 26.13±3.23 90.0±0.00 1.00±0.00 27.63±4.48 90.0±0.00 1.50±0.50 Light seeds 32.67±0.20 100.0±0.00 2.50±0.50 37.03±2.70 100.0±0.00 1.00±0.00 38.00±3.47 100.0±0.00 0.90±0.90 Dark seeds 21.40±6.13 87.0±0.00 4.50±0.50 23.44±5.17 87.0±0.00 5.57±1.57 24.80±5.27 87.0±0.00 1.00±0.00 Light seeds 18.47±3.00 93.5±6.50 12.8±5.18 20.87±4.60 93.5±6.50 25.8±11.18 21.47±4.80 93.5±6.50 14.31±1.37 Dark seeds 13.70±5.10 66.5±3.50 8.50±1.50 15.60±5.13 70.0±10.0 17.79±10.79 15.87±5.27 70.0±10.00 25.04±10.90

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 20 3.2 Condition of seed storage before germination Table 11 shows the spectrum of the tested samples of V. faba L. seeds from the Seed Bank in Gatersleben. It is interesting to note that although the temperature used for group A was expected to be in favor of the longer survival of the V. faba L. seeds in the case of A. manglesii and M. tetragona Merritt et al. 2003 we observed the opposite effect of storage at a temperature of -18 o C in comparison with higher storage temperatures contrary to the findings of other authors mentioned before. According Murthy et al. 2002 another extreme temperature for storage is above 40 o C. The darkening of testa was also confirmed for cv. Fiesta Nasar-Abbaset et al. 2009. TABLE 11 SPECTRUM OF THE TESTED SAMPLES OF V. FABA L. SEEDS AND THEIR CONDITIONS – SHOWS ZERO VIABILITY. Year Cultivar Country of origin A B 1972 Féverole du Gers France + - 1975 Maly Italy + - 1977 Parvin Great Britain + + 1978 Milión Diana Kronberger tennis Murat Czechoslovakia Czechoslovakia Germany Ethiopia + + + + + - + + 1979 Féverole du Gers tennis Murat Latvija Mazur No.18 DornburgerAckerb. France Ethiopia USSR Poland Germany Great Britain + + + + + + - + + + + - 1980 Romana Maly Přerovský Italy Italy Czechoslovakia + + + + - + 1981 Dire Dawa Skorospelka Banská Ethiopia USSR USSR + + + + + + 1982 Equina tennis Murat Italy Ethiopia + - + + 1984 Maly Italy + + Both groups were tested periodically over nine years 1991 – 1999. The first evaluated parameter to be checked was seed vitality i.e. their germinating capacity according to their color the difference is especially evident for summarized data obtained for the 1978-1984 harvests Table 12. TABLE 12 GERMINATION OF LIGHT AND DARK SEEDS FROM 22 DIFFERENT SAMPLESOF 17 CULTIVARS FROM 9 COUNTRIES IN CONSEQUENT YEARS. Year of harvest Light seeds Dark seeds 1991 1992 1996 1997 1999 1991 1992 1996 1997 1999 1972 98.0 95.0 27.3 50.0 55.0 n.d. n.d. n.d. n.d. n.d. 1975 82.0 90.0 41.7 70.0 30.0 n.d. n.d. n.d. n.d. n.d. 1977 94.0 70.0 70.0 73.3 45.0 0.0 0.0 0.0 0.0 5.00 1978 100.0 96.3 95.8 97.5 92.8 100.0 27.5 23.3 10.0 18.3 1979 98.5 92.5 81.6 94.6 63.4 97.6 26.0 36.0 16.7 12.5 1980 92.0 83.3 74.5 51.1 32.4 92.0 43.3 30.0 4.4 7.5 1981 90.6 93.3 86.6 71.9 52.5 90.6 40.0 30.0 35.5 20.0 1982 95.0 75.0 73.3 80.0 40.0 95.0 50.0 13.3 20.0 10.0 1984 86.0 90.0 50.0 55.6 15.0 89.0 47.5 10.0 40.0 5.0 Total 1978-84 93.7 +2.13 88.4 +3.23 76.9 +6.39 75.1 +7.89 49.3 +11.03 94.0 +1.74 39.0 +4.14 23.7 +4.19 21.1 +5.74 12.2 +2.43

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 21 When confirming the weaker vitality of darker seeds stored in less favorable conditions we were interested in the aberration rate as expressed by the summarized data in Table 13. TABLE 13 SUMMARY OF ABERRATIONS PER YEAR. Year of harvest Light seeds Dark seeds 1992 1996 1997 1992 1996 1997  1972 0.33 + 0.0 4.81 + 3.2 1.17 + 1.2 n.d. n.d. n.d.  1975 0.66 + 0.0 1.65 + 4.9 1.51 + 1.0 n.d. n.d. n.d.  1977 0.33 + 0.0 1.00 + 0.0 n.d. 0.00 + 0.0 0.00 + 0.0 n.d.  1978 0.49 + 0.6 1.00 + 3.3 0.08 + 0.8 1.22 + 0.2 1.10 + 1.3 0.00 + 0.0  1979 0.39 + 0.4 0.00 + 0.0 0.47 + 1.2 3.26 + 1.8 4.65 + 2.4 0.19 + 1.3  1980 0.44 + 0.6 0.25 + 0.9 1.04 + 1.2 2.44 + 1.2 0.25 + 0.9 1.00 + 1.0  1981 0.33 + 0.6 0.57 + 1.1 0.25 + 1.3 2.22 + 0.8 0.00 + 0.0 0.92 + 1.9  1982 0.00 + 0.0 0.00 + 0.0 2.85 + 0.0 4.66 + 0.0 1.00 + 0.0 0.00 + 0.0  1984 1.00 + 0.0 1.23 + 4.9 1.58 + 0.8 0.00 + 0.0 3.08 + 2.5 n.d. n. d. not detected As noted the aberration frequency was not very high in old seeds for significant results. Therefore the most important tendencies in the evaluations from the years 1991 1992 1996 1997 and 1999 best expressed themselves in the viability measured by the percentage of germination after 96 hours. The summarized results are shown in Table 14. TABLE 14 AVERAGE GERMINATION IN OF LIGHT AND DARK SEEDS FOR ALL EVALUATION TIMES. YEARS OF HARVEST AND CULTIVARS. Light seeds day 1 day 2 day 3  46.4 + 24.6 32.4 + 20.6 49.2 + 21.7 56.8 + 24.8 Dark seeds day 1 day 2 day 3  11.7 + 7.0 7.00 + 2.5 11.7 + 6.5 13.1 + 7.70 The difference is significant although it is affected by a wide range of germination of light seeds which confirms our earlier results from a lesser number of Czechoslovak cultivars except in the case of the parameter of the root growth Table 15.. TABLE 15 AVERAGE ROOT LENGTH OF LIGHT AND DARK SEEDS FOR ALL EVALUATION TIMES. YEARS OF HARVEST AND CULTIVARS IN CM. Light seeds day 1 day 2 day 3 Σ 2.3 + 1.5 cm 0.7 + 0.12 2.2 + 0.59 4.0 + 0.80 Dark seeds day 1 day 2 day 3 Σ 1.9 + 1.5cm 0.5 + 0.08 1.4 + 0.76 2.9 + 1.65

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 22 This tendency was also confirmed in the case of the aberrant ana-telophases where in the range of 0.0 – 3.66 SEM was 0.0 – 2.42 thus rendering all results non-significant. There was no provable difference between the light and dark seeds or even between cultivars or years of harvest. All of these results support our previous conclusions and findings Murín et al 2007. In all of the evaluated parameters we have observed that despite the great difference in the two basic storage conditions and the time between the harvest years and the evaluation frequency 17 years in the case of the oldest year germination was the only provable parameter. This confirms the possibility of the practical use of this interesting manifestation of different storage conditions. 3.3 Experimental Storage Effect The basic observations mentioned in the introduction regarding various plant species were later supplemented by attempts to demonstrate a relationship between the aging of seeds and the mode of their storage and sensitivity to the action of chemicals e.g. Avanzi et al. 1969. Interesting results were obtained due to the effects of different moisture on stored seeds in the course of the presumed repair mechanisms in plant cells and the corresponding recovery effects after the action of alkylating agents Gichner and Gaul 1971 Gichner and Velemínský 1973. Following the method of the last mentioned authors in a series of experiments Murín and Mičieta 2001 described in detail in our recent report Murín and Mičieta 2014 we found the experimental “storage effect” to be a universal method for recovering and enhancing seed viability for higher crop production. Seeds stored for a long time and impacted by significant chromosome damage by a chosen mutagen can recover by this method. It means that their experimental storage under the defined conditions for 8-days resulted in 3 to 4 times lower frequency of chromosomal aberration in root tips and a significantly higher viability Tables 16 and 17 Figures 2 and 3. Even 12-year old seeds after experimental storage showed viability comparable with 2-year old seeds without storage. This prolongation of the G-1 phase causes a decrease in damage caused by long term storage and other stresses up to a condition similar to that of young undamaged seeds. According to our experience Murín and Mičieta 1997 the prolongation of the storage period for more than 8 days has no greater effect i.e. the “storage effect” is limited probably due to the limited source of repair enzymes stored in dormant seeds. TABLE 16 GERMINATION AND CHARACTER OF ABERRATIONS F-FRAGMENTS. B-BRIDGES. F+B IN ROOT TIP CELLS OF 2. 6 AND 12-YEAR OLD V. FABA SEEDS AFTER 0 DAYS OF EXPERIMENTAL STORAGE. Years MMS in mM Germination in No. of cells scored 48h/72h No. of aberrant cells 48 h recovery time 72h F B F+B F B F+B 0 77.0 150/120 2 1 0 1 3 0 2 3 90.0 155/140 17 6 3 8 1 1 6 87.0 150/130 24 3 4 11 9 2 0 57.0 210/140 40 7 12 15 6 3 6 3 50.0 250/90 40 11 16 11 3 3 6 77.0 250/150 57 26 18 35 4 4 0 19.4 40/70 7 2 2 11 1 0 12 3 17.4 25/15 1 6 2 4 2 2 6 13.0 60/25 10 16 20 14 2 2

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 23 TABLE 17 GERMINATION AND CHARACTER OF ABERRATIONS F-FRAGMENTS. B-BRIDGES. F+B IN ROOT TIP CELLS OF 2. 6 AND 12-YEAR OLD V. FABA SEEDS AFTER 8 DAYS OF EXPERIMENTAL STORAGE. Years MMS in mM Germination in No. of cells scored 48h/7h No. of aberrant cells 48 h recovery time 72h F B F+B F B F+B 0 89.6 270/150 7 1 0 4 1 0 2 3 94.0 260/150 3 0 0 3 1 0 6 86.0 290/150 7 0 1 0 2 0 0 87.5 300/160 2 1 0 0 0 0 6 3 88.6 300/150 8 1 0 2 0 0 6 82.4 300/150 6 2 0 3 1 0 0 60.0 230/150 18 5 2 4 1 0 12 3 46.0 205/150 8 6 0 11 1 1 6 72.0 180/150 12 4 1 2 2 2 FIGURE 2. FREQUENCY OF CHROMOSOME ABERRATIONS ON DAY 0 OF EXPERIMENTAL STORAGE. FIGURE 3. FREQUENCY OF CHROMOSOME ABERRATIONS ON DAY 8 OF EXPERIMENTAL STORAGE. 0 20 40 60 80 100 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 2 years old 6 years old 12 years old Chromosomal aberation 48h 72h 0 10 20 30 40 50 60 70 80 90 100 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 0 mM 3 mM 6 mM 2 years old 6 years old 12 years old Chromosomal aberation 48h 72h

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-5 May- 2017 Page | 24 IV. CONCLUSION In addition to the theoretical ramifications of our experiments our work may also have practical implications in three fields: 1. As pointed out by Čupič et al. 2005 widely used agro-technical plant seeds as in the case of Alfalfa Medicago sativa L. are often stored for years after harvest which influences their germination energy germination rate of abnormal sprouts and dead seeds. This can be easily repaired by the “storage effect” with an interrupted germination period under the described conditions causing the prolongation of the G-1 phase with a significant increase of vitality of seeds treated this way. Consequently it will lead to an improvement of their crop production that is most important in the case of seeds that are genetically modified or rearranged see ACB karyotype seeds used in our experiments. 2. Our findings may be very helpful to seed banks worldwide. The regular checking of the viability of seeds according to germination leads to irreversible losses of stored seeds while a simple visual test based at seed color would provide the same answer with no loss of material. Moreover such a test could be conducted in sealed ampules thus not interfering with the storage conditions in the particular seed bank. By using “storage effect” these seeds can be revitalised or rejuvenalised and stored further with the possibility of long-term survival of the seeds in seed bank. 3. Finally with the above mentioned “storage effect” the amount of yield of viable seeds can be significantly recovered and by this method to prolong of their useful survival in the particular agricultural supply. Just at the example of Vicia faba beans it could cause a significant economical improvement as its seeds are distributed in more than 55 countries when 4.56 million tons of dry grains are produced in the harvested area of 2.56 million ha yearly. ACKNOWLEDGEMENTS Thanks to the opportunity to cooperate with Dr. Joachim Keller and the Seed Bank in Gatersleben Germany we had the chance to conduct a long-term experiment using 22 seed samples from eight countries nineteen cultivars and nine harvests. This study was supported by VEGA grant No. 1/0885/16 and is partially the result of the project implementation of Comenius University in the Bratislava Science Park supported by the Operational Programme Research and Development funded by ERDF Grant No.: ITMS 26240220086. The authors are grateful to Dr. Anna Ligasová and Dr. Jana Chrenová for their kind assistance with the practical parts of some experiments Alena Rendeková for her comments to the manuscript and Zuzana Randáková for her revision of the tables. Conflicts of Interest: The authors declare no conflict of interest. REFERENCES 1 Andreev I.O. Spiridonova E.V. Kunakh V.A. Solov´yan V.T. Aging and loss of germination in rye seeds is accompanied by a decreased fragmentation of nuclear DNA at loop domain boundaries. Russ. J. Plant. Physiol. 2004 512:241–248. 2 Avanzi S. Innocenti A.M. Tagliasacchi A.M. Spontanueous chromosome aberrations in relation to seed dormancy in Triticum durum Desf. Mutation Res. 1969 7:199–203. 3 Babasaheb B.D. Seeds Handbook: Processing And Storage. CRC Press 2004 p 800. 4 Bewley J.D. Black M. Physiology and Biochemistry of Seeds in Relation to Germination. Dormancy and Environmental Control. Springer-Verlag Berlin Heidelberg New York 1982 p 376. 5 Bewley J.D. Black M. Seeds: Physiology of Development and Germination. Second Edition. Plenum Press New York 1994 p 445. 6 Bezrukov V.F. Lazarenko L.M. Environmental impact on age-related dynamics of karyotypical instability in plants. Mutation Res. 2002 520:113–118. 7 Bonner F.T. Storage of seeds: Potential and limitations for germplasm conservation. Forest Ecology and Management Issues 1–2 1990 Vol. 35:35–43 8 Cartledge J.L. Blakeslee A.F. Mutation rate increased by aging seeds as shown by pollen abortion. Proc. Nat. Acad. Sci. USA 1934 20:103–110. 9 Cartledge J.L. Blakeslee A.F. Mutation rate from old Datura seeds. Science 1935 81:492–493. 10 Cebrat J. Investigations on spontaneous chromosome aberrations in the roots of onion seedlings and on their effect upon seedling growth. Genet. Pol. 1977 18:39–49. 11 Čupič T. Popović S. Grljušić S. Tucak M. Andrić L. Šimic B. Effect of storage time on alfalfa seed quality. Journal of Central European Agriculture 2005 61:65–68. 12 D´Amato F. Mutazioni cromosomische spontanee in plantule di Pisum sativum L.. Caryologia 1951 3:285–293. in Italian 13 Floris C. Anguillesi M.C. Aging of isolated embryos and endosperm of durum wheat: An analysis of chromosome damage. Mutation Res. 1974 22:133–138.

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