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Slow post-hydration drying improves initial quality but reduces longevity of primed bitter gourd seeds
Scientia Horticulturae 106 (2005) 114–124 www.elsevier.com/locate/scihorti
Slow post-hydration drying improves initial quality but reduces longevity of primed bitter gourd seeds R.H. Lin a, K.Y. Chen a, C.L. Chen a, J.J. Chen b, J.M. Sung c,* a
Department of Agronomy, National Chung Hsing University, Taichung 402, Taiwan, ROC b Department of Food Science and Nutrition, Hung Kuang University, 34 Chung-Chie Rd, Sha Lu, Taichung County 433, Taiwan, ROC c Division of Biotechnology, Taiwan Seed Improvement and Propagation Experiment Station, Hsin She, Taichung County 426, Taiwan, ROC Received 3 December 2004; received in revised form 25 February 2005; accepted 25 February 2005
Abstract Many environmental factors are known to affect the success of priming, post-hydration drying being critical. Accumulated evidence shows that priming improves the quality of bitter gourd (Momordica charantia L.) seeds. However, the effect of post-hydration drying on the their longevity remains unclear. This study evaluated the effects of post-hydration drying speed on the emergence performance and antioxidative activities of primed bitter gourd seeds stored for 48 weeks. Results indicated that priming improved emergence percentage, mean emergence time and seedling growth of treated seeds. Primed seeds also showed lower peroxidation, as indicated by the accumulation of malondialdehyde (MDA) and total peroxide, and higher total anti-oxidative activities (TAA) than non-primed seeds. The seeds receiving slow post-hydration drying treatment exhibited faster emergence and better seedling growth than the seeds receiving fast post-hydration drying treatment and the non-primed control. However, the longevity of slow-dried seeds decreased considerably during 48 weeks of storage as compared to that of non-primed and fast-dried seeds. Fast drying treatment maintained the longevity of primed seeds for up to 24 weeks as compared with non-primed control or slow-drying seeds. Improved longevity was due in part to enhanced TAA that minimized the accumulation of MDA and total peroxide during storage. # 2005 Elsevier B.V. All rights reserved. Keywords: Bitter gourd; Momordica charantia; Post-hydration drying; Seed priming; Seedling emergence; Storage; Total anti-oxidative activity * Corresponding author. Present address: Department of Food Science and Nutrition, Hung Kuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 433, Taiwan, ROC. Tel.: +886 4 22873459; fax: +886 4 22860307. E-mail address: [email protected] (J.M. Sung). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.016
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1. Introduction Priming is a common practice for seed enhancement in the seed industry (Taylor et al., 1998). It induces faster and more uniform seed germination over broader temperature ranges in many crop species (McDonald, 1999). During priming, seeds are partially hydrated so that pre-germinative metabolic activities proceed while radicle protrusion is prevented, then are dried back to the original moisture level. The beneficial effects of priming are associated with the repair and build up of nucleic acid, the increased synthesis of protein as well as the repair of membranes (McDonald, 2000). Priming also enhances the activities of anti-oxidation (Wang et al., 2003; Hsu et al., 2003). Many environmental factors are known to affect the success of priming, post-hydration drying being critical (McDonald, 2000). However, there are conflicting data concerning the effects of post-hydration drying speed of primed seeds upon their quality. Primed seeds of tomato (Lycopersicon esculentum Mill.) (Gurusinghe et al., 2002), pepper (Capsicum annuum L.) and pansy (Viola x wittrockiana) (Bruggink et al., 1999) dried back at a slower dehydration rate germinated faster than the seeds dried more quickly. In primed leek (Allium porrum L.) seeds, no difference in germination percentage was observed between fast- and slow-dried seeds but the incidence of abnormal seedlings from fast-dried seeds increased with increase in duration of storage (Maude et al., 1994). In contrast, primed snap bean (Phaseolus vulgaris L.) (Ptasznik and Khan, 1993) seeds dried at a higher dehydration rate had better vigor responses than the seeds dried back slowly. Thus, the effects of drying conditions on the vigor responses of primed seeds seem to be species specific. Bitter gourd (Momordica charantia L.) seeds germinate well between 25 and 28 8C, but poor germination is common at sub-optimal temperatures (Lin and Sung, 2001). Research evidence shows that priming improves the germination of bitter gourd seeds (Lin and Sung, 2001; Chen and Sung, 2001; Hsu et al., 2003). However, while priming enhanced the germination performance, it made the primed bitter gourd seeds more susceptible to deterioration in prolonged storage (Yeh et al., 2005). Decreased longevity of primed bitter gourd seeds was related to the reduced anti-oxidative activity that minimized the peroxidative damages during storage (Yeh et al., 2005). The influences of post-hydration drying on the longevity of primed bitter gourd seeds in storage are still unknown. The present study was undertaken to determine how post-hydration drying speed affected the initial quality and the longevity of matriprimed bitter gourd seeds. The emergence responses of primed seeds were determined during 48 weeks of storage duration. The levels of malondialdehyde and total peroxide accumulation and total antioxidative activity in the dry seeds during storage were also compared between the primed bitter gourd seeds subjected to slow and fast drying treatments.
2. Materials and methods 2.1. Experiment 1 A commercially produced fresh seed lot (100-seed weight 23 g) of bitter gourd (Momordica charantia L. cv. Blue Mountain Giant White), with moisture content of 8.4%
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(dry weight basis), was obtained from a local vendor. For priming, 150 g of seeds and 300 g of vermiculite no. 3 to which 375 ml of 5 mg l 1 sodium selenite solution was added were sealed in a plastic bag, mixed to provide uniform seed-substrate contact, and incubated at 25 8C for 36 h (Wang et al., 2003). The partially hydrated seeds were dried either at 65% relative humidity for 48 h (fast drying) or 95% relative humidity for 120 h (slow drying) to their original moisture level. After drying, samples of primed and non-primed control seeds were used for emergence and physiological measurements. All the remaining seeds were packed separately and used in experiment 2. Laboratory emergence percentage was conducted by planting three replicates of 50 seeds in 50-plug trays filled with a seedling raising mix of 75% peat moss and 25% vermiculite at a depth of 1.5 cm and watered with distilled water as necessary. A total of nine seed trays were sown. The experimental design was a randomized complete block design with three replicates. The seed trays were incubated in controlled chambers with 12 h photoperiod and 300 Wm 2 light intensity at 25 8C. Daily emergence counts of cotyledons visible above the vermiculite surface were taken until no further emergence occurred for four consecutive days. The mean emergence time (MET) was calculated using the formula of Wang et al. (2003). The 14-days-old seedlings (shoot + root) were sampled and oven-dried (60 8C) for seedling dry weight determinations. For malondialdehyde (MDA) and total peroxide determinations, three replicates of five non-primed or primed seeds were hand-homogenized with 5 ml 5% (v/v) trichloroacetic acid at 4 8C in a mortar and pestle and then centrifuged at 14,000 g for 20 min. The supernatants were used for MDA and total peroxide determinations. MDA was determined by adding 0.8 ml of 20% (w/v) trichloroacetic acid (TCA), containing 0.5% (w/v) thiobarbituric acid, to 0.2 ml of supernatant. The reaction was carried out at 95 8C for 30 min and then terminated by soaking in ice-cold water. The reaction solution was monitored at 532 and 600 nm, and the difference in absorbance between 532 and 600 nm was used to calculate the MDA content (Heath and Packer, 1968). Total peroxide was determined by adding 260 ml reaction medium, containing 10 mM ferrous ammonium sulfate, 2.5 mM potassium thiocyanate and 50% (w/v) TCA, to 10 ml of supernatant. The reaction was monitored at 480 nm (Sagisaka, 1976). For total anti-oxidative activity (TAA) determination, three replicates of 0.4 g imbibed seeds each, were ground at 4 8C in a mortar and pestle with 4 ml of 0.1 M potassium phosphate buffer (pH 6.8), followed by centrifugation at 18,000 g for 30 min. The supernatants (0.5 ml) were used for determination of TAA. The ability of antioxidants to scavenge the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical, produced by the ferrylmyoglobin radical generated from metmyoglobin and H2O2 in the presence of the peroxidase, was measured spectrophotometrically at 734 nm using the methods detailed by Pietta et al. (1998). The activity was calculated by plotting the absorbance data against a trolox standard curve. 2.2. Experiment 2 After priming, seven samples each of the slow-dried, fast-dried and control seeds were packed separately in 21 packets using aluminum foil bags coated with polyethylene and stored at 25 8C for 48 weeks. Three packets of seeds (non-primed, fast-dried and slow-
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dried seeds) were removed from storage at 3, 6, 9, 12, 24 and 48 weeks, respectively, and tested for changes in emergence percentage, the levels of MDA and total peroxide accumulation, and the changes in TAA. 2.3. Statistics The experimental design was a randomized complete block design with three replicates. Percentage data were arcsin-transformed before analysis. All data were subjected to an analysis of variance, and a LSD was calculated when a significant (P < 0.05) F ratio occurred for treatment effects. Correlation analysis was also used to characterize the relationships between the percentage of emergence and the levels of MDA and total peroxide accumulation and TAA, and between seedling dry weight and the levels of MDA and total peroxide accumulation and TAA.
3. Results 3.1. Experiment 1 The moisture content of primed bitter gourd seed lots rose to 43% (dry weight basis) at the end of hydration (Fig. 1). The fast drying treatment reduced seed moisture content linearly to below 20% within 6 h (the estimated rate of drying was about 3.83% h 1), while it required more than 56 h to reach the same moisture level under slow drying conditions (the rate of drying was about 0.41% h 1) (Fig. 1). Both seeds had dehydrated to about 8.4% at the end of drying. Non-primed control seeds used in this study had 80% emergence and 7.49 days mean emergence time (MET) (Table 1). Fresh primed bitter gourd seeds (both fast- and slowdried seeds) showed increased emergence percentage and accelerated emergence speed
Fig. 1. Changes in the moisture content (dry weight basis) of bitter gourd (cv. Blue Mountain Giant White) seeds subjected to fast drying and slow drying treatments during priming.
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Table 1 The effects of post-hydration drying methods on percentage of emergence, mean emergence time (MET), malondialdehyde (MDA) content, total peroxide contents, total anti-oxidative activity (TAA) and dry weight of 14-days-old seedling for non-primed control seeds and the primed seeds of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying or slow drying treatments Seed traits
Control
Fast drying
Slow drying
LSD0.05
Emergence (%) MET (days) MDA (nmol g 1 DW) Total peroxide (mmol g 1 DW) TAA (nmol trolox eq g 1 DW) Seedling dry weight (mg)
80 7.49 30.42 6.57 3.05 148
92 6.88 25.02 5.71 3.67 173
97.5 6.31 23.47 5.15 3.86 192
5.38 0.49 3.02 0.59 0.23 16
(Table 1) in comparison with control. They also exhibited more uniform seedling growth (Fig. 2) and produced heavier seedlings than control (Table 1). Primed seeds also had lower levels of malondialdehyde (MDA) and total peroxide accumulation than non-primed control (Table 1). The lower MDA and total peroxide of primed seeds might result from the priming-enhanced total anti-oxidative activities (TAA) (Table 1). Both emergence percentage and MET of primed seeds were affected by drying method, with slow-dried seeds performing better than fast-dried seeds (Table 1). Slow-dried seeds also produced heavier and better seedlings than fast-dried seeds (Table 1 and Fig. 2). Only minor differences in the levels of MDA and total peroxide accumulation and TAA were obtainable between slow-dried and fast-dried seeds, with slow-dried seeds showing greater priming responses than fast-dried seeds (Table 1).
Fig. 2. The growth responses of 14-days-old bitter gourd seedlings of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying (F) or slow drying (S) treatments.
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3.2. Experiment 2 Non-primed control seeds stored for 12 weeks showed no evident decline in emergence percentage, but marked reductions in emergence percentage were observed for non-primed seeds stored for 24, 36 and 48 weeks (Fig. 3A). Primed seeds (both slow- and fast-dried seeds) also had reduced emergence percentage during 48 weeks of storage (Fig. 3A). Drying method had significant effect on the longevity of primed seeds. Emergence percentage rapidly declined to 35% for slow-dried seeds after 12 weeks storage as compared to 75% in nonprimed seeds, and no seedling was found after 48 weeks storage (Fig. 3A). For fast-dried seeds, the percentage of emergence declined slightly (85%) during 12 weeks storage, but it was still greater than non-primed control. The emergence percentage of fast-dried seeds further declined to 39% after 48 weeks storage, which was lower than control (51%) (Fig. 3A). The differences in storability were not due to the moisture uptake during storage, since no notable changes in seed moisture level were detected (data not shown).
Fig. 3. Changes in emergence percentage (A) and seedling dry weight (B) of bitter gourd (cv. Blue Mountain Giant White) seeds that subjected to fast drying and slow drying treatments during 48 weeks storage. Vertical bars represent the mean and S.E. of three replications. LSD (P < 0.05) for emergence percentage and seedling dry weight, across all the treatments are 8.32 and 15.79, respectively.
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Non-primed seeds stored for up to 9 weeks showed no evident decline in seedling growth, but marked reductions in seedling dry weight were observed for non-primed seeds stored for 24, 36 and 48 weeks (Fig. 3B). Storing the primed seeds receiving slow drying treatment also resulted in significant declines in seedling dry weight after 48 weeks storage, but the rate of decline was greater than non-primed control, especially at the earlier stages of storage (12 weeks of storage) (Fig. 3B). However, fast-dried seeds performed better than non-primed control for up to 36 weeks storage (Fig. 3B). Both slow- and fast-dried seeds initially had lower levels of MDA (Fig. 4A) and total peroxide (Fig. 4B) than non-primed seeds. The seeds receiving slow drying showed higher levels of MDA and total peroxide accumulations than non-primed control with 24 weeks of storage. The levels of MDA and total peroxide for fast-dried seeds also increased following 24 weeks storage, but the extents of accumulation were lower than non-primed seeds. After 48 weeks storage, the levels of MDA and total peroxide accumulation for fast-dried seeds were greater than their respective controls (Fig. 4A and B). Both primed and non-primed seeds exhibited a decrease in TAA, but the rate of decline for slow-dried seeds was faster than fast-dried and control seeds during 24 weeks storage (Fig. 4C). The primed seeds receiving fast drying had greater TAA than control seeds up to 36 weeks storage. However, a lower TAA in fast-dried seeds compared to non-primed control seeds occurred after 48 weeks storage. Correlation analysis showed that, in all the treatments, the levels of MDA and total peroxide were negatively correlated to the percentage of emergence and the seedling dry weight (Table 2). In contrast, TAA correlated positively to the emergence percentage and seedling dry weight (Table 2).
4. Discussion Previous studies in this laboratory (Chen and Sung, 2001; Hsu et al., 2003; Wang et al., 2003) demonstrated that priming the bitter gourd seeds would improve their germination and emergence performances. The improvements were attributable in part to the enhanced anti-oxidative activity. However, in these studies, the anti-oxidative abilities were examined only in the seeds imbibed for 48 h because the ascorbate–glutathione cycle was not completely operative in dry bitter gourd seeds (J.M. Lin, unpublished data). Thus, the enhanced anti-oxidative abilities that were found in the primed bitter gourd seeds might partially result from the priming-induced advancement of germination. The measurement of total anti-oxidative activity (TAA) was initially developed to monitoring the antioxidant status in premature neonates (Miller et al., 1993). Yeh et al. (2005) recently used this technique to examine the anti-oxidative responses of imbibing bitter gourd seeds (seeds were imbibed for 48 h) and found that TAA was positively related to the anti-oxidative abilities of primed seeds. They further demonstrated that TAA was closely related to germination percentage and germination speed. In the present study, we found that the TAA was detectable in dried bitter gourd seeds (Table 1), therefore, this technique can be used to examine the anti-oxidative status of non-imbibed bitter gourd seeds. Primed bitter gourd seeds also showed marked decreases in MDA and total peroxide accumulations (Table 1), which might have resulted from priming-enhanced TAA. Correlation analysis
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Fig. 4. Changes in malondialdehyde (MDA) content (A), total peroxide content (B) and total anti-oxidative activity (TAA) (C) of bitter gourd (cv. Blue Mountain Giant White) seeds that subjected to fast drying and slow drying treatments during 48 weeks storage. Vertical bars represent the mean and S.E. of three replications. LSD (P < 0.05) for MDA, total peroxide and TAA, across all the treatments are 3.47, 1.72 and 0.45, respectively.
confirmed these findings (Table 2). These results, taken in conjunction with increased emergence percentage and improved seedling growth (Table 1 and Fig. 2), indicate that enhanced anti-oxidative ability may be in part the reason for improved emergence performance of primed bitter gourd seeds (Table 2).
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Table 2 The correlation coefficients (r) between the percentage of seed germination and malondialdehyde (MDA), total peroxide and total anti-oxidative activity (TAA), and between TAA and MDA and total peroxide of non-primed control seeds and the primed seeds of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying or slow drying treatments MDA
Total peroxide
TAA
Emergence percentage Control Fast drying Slow drying
0.915a 0.935a 0.916a
0.927a 0.929a 0.897a
0.912a 0.952a 0.921a
Seedling dry weight Control Fast drying Slow drying
0.921a 0.909a 0.934a
0.934a 0.897a 0.923a
0.954a 0.915a 0.965a
a
Significant at 1% level.
Many environmental factors are known to affect the success of priming, post-hydration drying being critical (McDonald, 2000). Chiu et al. (in press) reported that both free radicals and peroxides are re-generated during post-hydration drying, which would intensify lipid peroxidation. The intensified peroxidation, possibly resulting from dryinginduced oxidative stress, would damage the anti-oxidative mechanisms and impair seed quality (Palma et al., 2002; Chiu et al., in press). In the present study, significant differences in MDA and total peroxide existed between the primed seed lots that receiving fast and slow drying treatments, with the latter accumulating less MDA and total peroxide than the former (Table 1). The present study also revealed the greater increases in TAA by slow drying treatment (Table 2). As a result, the lipid peroxidation would be markedly reduced during seed imbibition. Thus, improvements in emergence percentage and seedling growth were obtainable for slow-dried seeds as compared to fast-dried seeds, provided that the seeds were immediately planted following priming (Table 1 and Fig. 2). While slow drying improved the initial quality of primed seeds, it resulted in a rapid reduction of seed longevity in storage as compared to that of the seeds receiving fast drying treatment (Fig. 3A). The decreased seedling dry weight confirmed the inferior quality of slow-dried seeds subjected to long-term storage (Fig. 3B). The rapid deterioration of slowdried seeds might be related to the peroxidative damages, possibly resulting from increased MDA (Fig. 4A) and total peroxide (Fig. 4B) accumulations and the decreased TAA during dry storage (Fig. 4C) (McDonald, 1999; Yeh et al., 2005). Decreased longevity for slowdried seeds might also be due in part to the advancement of germination reactions to a level not compatible with the drying back of seed required for storage. Lin and Sung (2001) previously reported that priming enlarged the embryos of bitter gourd seeds. In this study, slow-dried seeds did maintain a relatively higher seed moisture content at longer drying periods in comparison with fast-dried seeds (Fig. 1). Under higher seed moisture contents, the embryo might enlarge to such an extent that drying was injurious (Basu, 1994). Moreover, Lanteri et al. (1996) reported that more DNA replication and greater advancement through the cell cycle (i.e., more radicle meristem cells were in the S or G2 phase) generally occurred at higher seed moisture contents. Thus, under higher seed moisture content (Fig. 1), slow-dried seeds might pass the threshold of desiccation
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tolerance, which would damage the seeds beyond the point of recovery, even though they had greater initial quality than fast-dried seeds initially (Table 1). In conclusion, the results described here re-confirm that priming improves the initial quality of bitter gourd seeds. The improved quality appears partly attributable to decreased peroxidation, resulting from increased TAA. The method of drying following hydration also affects the initial quality of primed seeds. Slow-dried seeds initially accumulated less amounts of MDA and total peroxide as compared to fast-dried seeds. These results might partially explain why the primed seeds receiving slow drying treatment have better quality than that of the seeds receiving fast drying. However, more total peroxide were accumulated in slow-dried seeds during 48 weeks storage, and therefore made them more susceptible to deterioration in long-term storage as compared to fast-dried seeds. The longevity of fast-dried seeds could be maintained for 24 weeks when they were stored at 25 8C conditions, because they were under the enhanced protection of anti-oxidative activity.
References Basu, R.N., 1994. An appraisal of research on wet and dry physiological seed treatments and their applicability with special reference to tropical and sub-tropical countries. Seed Sci. Technol. 22, 107–126. Bruggink, G.T., Ooms, J.J.J., Van der Toorn, P., 1999. Induction of longevity in primed seeds. Seed Sci. Res. 9, 49– 53. Chen, C.C., Sung, J.M., 2001. Priming bitter gourd seeds with selenium solution enhances germinability and antioxidative responses under sub-optimal temperature. Physiol. Plant. 111, 9–16. Chiu, K.Y., Chen, C.L., Sung, J.M., in press. Why 10 8C-primed sh-2 sweet corn seeds were of higher quality than 20 8C-primed seeds: some physiological clues. Seed Sci. Technol. Gurusinghe, S., Powell, A.L., Bradford, K.J., 2002. Enhanced expression of BiP is associated with treatments that extend storage longevity of primed tomato seeds. J. Am. Soc. Hort. Sci. 127, 528–534. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I: kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hsu, C.C., Chen, C.L., Chen, J.J., Sung, J.M., 2003. Accelerated aging-enhanced lipid peroxidation in bitter gourd seeds and effects of priming and hot water soaking treatments. Sci. Hort. 98, 201–212. Lanteri, S., Nada, E., Belletti, P., Quagliotti, L., Bino, R.J., 1996. Effects of controlled deterioration and osmoconditioning on germination and nuclear replication in seeds of pepper (Capsicum annuum L.). Ann. Bot. 77, 591–597. Lin, J.M., Sung, J.M., 2001. Pre-sowing treatments for improving emergence of bitter gourd seedlins under optimal and sub-optimal temperatures. Seed Sci. Technol. 29, 39–50. Maude, R.B., Drew, R.L.K., Gray, D., Bujalski, W., Nienow, A.W., 1994. The effects of storage on the germination and seedling abnormalities of leek seeds primed and dried by different methods. Seed Sci. Technol. 22, 299– 311. McDonald, M.B., 1999. Seed deterioration; physiology, repair and assessment. Seed Sci. Technol. 27, 177–237. McDonald, M.B., 2000. Seed priming. In: Black, M., Bewley, J.D. (Eds.), Seed Technology and Its Biological Basis. Sheffield Academic Press, Sheffield, UK, pp. 287–325. Miller, N.J., Rice-Evans, C., Davies, M.J., Gopinathan, V., Miller, A., 1993. A novel method for measuring antiosidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci. 84, 407–412. Palma, J.M., Sandalio, L.M., Corpas, F.J., Romero-Puertas, M.C., McCarthy, I., Del Rı´o, L.A., 2002. Plant protease, protein degradation, and oxidative stress: role of peroxisomes. Plant Physiol. Biochem. 40, 537–538. Pietta, P., Simonetti, P., Mauri, P., 1998. Antioxidant activity of selected medicinal plants. J. Agric. Food Chem. 46, 4487–4490.
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Ptasznik, W., Khan, A.A., 1993. Retaining the benefits of matriconditioning by controlled drying of snap bean seeds. HortScience 28, 1027–1030. Sagisaka, S., 1976. The occurrence of peroxide in a perennial plant, Populus gelrica. Plant Physiol. 57, 308–309. Taylor, A.G., Allen, P.S., Bennett, M.A., Bradford, K.J., Burris, J.S., Misra, M.K., 1998. Seed enhancement. Seed Sci. Res. 8, 245–256. Wang, H.Y., Chen, C.L., Sung, J.M., 2003. Both warm water soaking and solid priming treatments enhance antioxidation of bitter gourd seeds germinated at sub-optimal temperature. Seed Sci. Technol. 31, 47–56. Yeh, Y.M., Chiu, K.Y., Chen, C.L., Sung, J.M., 2005. Partial vacuum extends the longevity of primed bitter gourd seeds by enhancing their anti-oxidative activities during storage. Sci. Hort. 104, 101–112.
Slow post-hydration drying improves initial quality but reduces longevity of primed bitter gourd seeds R.H. Lin a, K.Y. Chen a, C.L. Chen a, J.J. Chen b, J.M. Sung c,* a
Department of Agronomy, National Chung Hsing University, Taichung 402, Taiwan, ROC b Department of Food Science and Nutrition, Hung Kuang University, 34 Chung-Chie Rd, Sha Lu, Taichung County 433, Taiwan, ROC c Division of Biotechnology, Taiwan Seed Improvement and Propagation Experiment Station, Hsin She, Taichung County 426, Taiwan, ROC Received 3 December 2004; received in revised form 25 February 2005; accepted 25 February 2005
Abstract Many environmental factors are known to affect the success of priming, post-hydration drying being critical. Accumulated evidence shows that priming improves the quality of bitter gourd (Momordica charantia L.) seeds. However, the effect of post-hydration drying on the their longevity remains unclear. This study evaluated the effects of post-hydration drying speed on the emergence performance and antioxidative activities of primed bitter gourd seeds stored for 48 weeks. Results indicated that priming improved emergence percentage, mean emergence time and seedling growth of treated seeds. Primed seeds also showed lower peroxidation, as indicated by the accumulation of malondialdehyde (MDA) and total peroxide, and higher total anti-oxidative activities (TAA) than non-primed seeds. The seeds receiving slow post-hydration drying treatment exhibited faster emergence and better seedling growth than the seeds receiving fast post-hydration drying treatment and the non-primed control. However, the longevity of slow-dried seeds decreased considerably during 48 weeks of storage as compared to that of non-primed and fast-dried seeds. Fast drying treatment maintained the longevity of primed seeds for up to 24 weeks as compared with non-primed control or slow-drying seeds. Improved longevity was due in part to enhanced TAA that minimized the accumulation of MDA and total peroxide during storage. # 2005 Elsevier B.V. All rights reserved. Keywords: Bitter gourd; Momordica charantia; Post-hydration drying; Seed priming; Seedling emergence; Storage; Total anti-oxidative activity * Corresponding author. Present address: Department of Food Science and Nutrition, Hung Kuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 433, Taiwan, ROC. Tel.: +886 4 22873459; fax: +886 4 22860307. E-mail address: [email protected] (J.M. Sung). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.016
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1. Introduction Priming is a common practice for seed enhancement in the seed industry (Taylor et al., 1998). It induces faster and more uniform seed germination over broader temperature ranges in many crop species (McDonald, 1999). During priming, seeds are partially hydrated so that pre-germinative metabolic activities proceed while radicle protrusion is prevented, then are dried back to the original moisture level. The beneficial effects of priming are associated with the repair and build up of nucleic acid, the increased synthesis of protein as well as the repair of membranes (McDonald, 2000). Priming also enhances the activities of anti-oxidation (Wang et al., 2003; Hsu et al., 2003). Many environmental factors are known to affect the success of priming, post-hydration drying being critical (McDonald, 2000). However, there are conflicting data concerning the effects of post-hydration drying speed of primed seeds upon their quality. Primed seeds of tomato (Lycopersicon esculentum Mill.) (Gurusinghe et al., 2002), pepper (Capsicum annuum L.) and pansy (Viola x wittrockiana) (Bruggink et al., 1999) dried back at a slower dehydration rate germinated faster than the seeds dried more quickly. In primed leek (Allium porrum L.) seeds, no difference in germination percentage was observed between fast- and slow-dried seeds but the incidence of abnormal seedlings from fast-dried seeds increased with increase in duration of storage (Maude et al., 1994). In contrast, primed snap bean (Phaseolus vulgaris L.) (Ptasznik and Khan, 1993) seeds dried at a higher dehydration rate had better vigor responses than the seeds dried back slowly. Thus, the effects of drying conditions on the vigor responses of primed seeds seem to be species specific. Bitter gourd (Momordica charantia L.) seeds germinate well between 25 and 28 8C, but poor germination is common at sub-optimal temperatures (Lin and Sung, 2001). Research evidence shows that priming improves the germination of bitter gourd seeds (Lin and Sung, 2001; Chen and Sung, 2001; Hsu et al., 2003). However, while priming enhanced the germination performance, it made the primed bitter gourd seeds more susceptible to deterioration in prolonged storage (Yeh et al., 2005). Decreased longevity of primed bitter gourd seeds was related to the reduced anti-oxidative activity that minimized the peroxidative damages during storage (Yeh et al., 2005). The influences of post-hydration drying on the longevity of primed bitter gourd seeds in storage are still unknown. The present study was undertaken to determine how post-hydration drying speed affected the initial quality and the longevity of matriprimed bitter gourd seeds. The emergence responses of primed seeds were determined during 48 weeks of storage duration. The levels of malondialdehyde and total peroxide accumulation and total antioxidative activity in the dry seeds during storage were also compared between the primed bitter gourd seeds subjected to slow and fast drying treatments.
2. Materials and methods 2.1. Experiment 1 A commercially produced fresh seed lot (100-seed weight 23 g) of bitter gourd (Momordica charantia L. cv. Blue Mountain Giant White), with moisture content of 8.4%
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(dry weight basis), was obtained from a local vendor. For priming, 150 g of seeds and 300 g of vermiculite no. 3 to which 375 ml of 5 mg l 1 sodium selenite solution was added were sealed in a plastic bag, mixed to provide uniform seed-substrate contact, and incubated at 25 8C for 36 h (Wang et al., 2003). The partially hydrated seeds were dried either at 65% relative humidity for 48 h (fast drying) or 95% relative humidity for 120 h (slow drying) to their original moisture level. After drying, samples of primed and non-primed control seeds were used for emergence and physiological measurements. All the remaining seeds were packed separately and used in experiment 2. Laboratory emergence percentage was conducted by planting three replicates of 50 seeds in 50-plug trays filled with a seedling raising mix of 75% peat moss and 25% vermiculite at a depth of 1.5 cm and watered with distilled water as necessary. A total of nine seed trays were sown. The experimental design was a randomized complete block design with three replicates. The seed trays were incubated in controlled chambers with 12 h photoperiod and 300 Wm 2 light intensity at 25 8C. Daily emergence counts of cotyledons visible above the vermiculite surface were taken until no further emergence occurred for four consecutive days. The mean emergence time (MET) was calculated using the formula of Wang et al. (2003). The 14-days-old seedlings (shoot + root) were sampled and oven-dried (60 8C) for seedling dry weight determinations. For malondialdehyde (MDA) and total peroxide determinations, three replicates of five non-primed or primed seeds were hand-homogenized with 5 ml 5% (v/v) trichloroacetic acid at 4 8C in a mortar and pestle and then centrifuged at 14,000 g for 20 min. The supernatants were used for MDA and total peroxide determinations. MDA was determined by adding 0.8 ml of 20% (w/v) trichloroacetic acid (TCA), containing 0.5% (w/v) thiobarbituric acid, to 0.2 ml of supernatant. The reaction was carried out at 95 8C for 30 min and then terminated by soaking in ice-cold water. The reaction solution was monitored at 532 and 600 nm, and the difference in absorbance between 532 and 600 nm was used to calculate the MDA content (Heath and Packer, 1968). Total peroxide was determined by adding 260 ml reaction medium, containing 10 mM ferrous ammonium sulfate, 2.5 mM potassium thiocyanate and 50% (w/v) TCA, to 10 ml of supernatant. The reaction was monitored at 480 nm (Sagisaka, 1976). For total anti-oxidative activity (TAA) determination, three replicates of 0.4 g imbibed seeds each, were ground at 4 8C in a mortar and pestle with 4 ml of 0.1 M potassium phosphate buffer (pH 6.8), followed by centrifugation at 18,000 g for 30 min. The supernatants (0.5 ml) were used for determination of TAA. The ability of antioxidants to scavenge the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical, produced by the ferrylmyoglobin radical generated from metmyoglobin and H2O2 in the presence of the peroxidase, was measured spectrophotometrically at 734 nm using the methods detailed by Pietta et al. (1998). The activity was calculated by plotting the absorbance data against a trolox standard curve. 2.2. Experiment 2 After priming, seven samples each of the slow-dried, fast-dried and control seeds were packed separately in 21 packets using aluminum foil bags coated with polyethylene and stored at 25 8C for 48 weeks. Three packets of seeds (non-primed, fast-dried and slow-
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dried seeds) were removed from storage at 3, 6, 9, 12, 24 and 48 weeks, respectively, and tested for changes in emergence percentage, the levels of MDA and total peroxide accumulation, and the changes in TAA. 2.3. Statistics The experimental design was a randomized complete block design with three replicates. Percentage data were arcsin-transformed before analysis. All data were subjected to an analysis of variance, and a LSD was calculated when a significant (P < 0.05) F ratio occurred for treatment effects. Correlation analysis was also used to characterize the relationships between the percentage of emergence and the levels of MDA and total peroxide accumulation and TAA, and between seedling dry weight and the levels of MDA and total peroxide accumulation and TAA.
3. Results 3.1. Experiment 1 The moisture content of primed bitter gourd seed lots rose to 43% (dry weight basis) at the end of hydration (Fig. 1). The fast drying treatment reduced seed moisture content linearly to below 20% within 6 h (the estimated rate of drying was about 3.83% h 1), while it required more than 56 h to reach the same moisture level under slow drying conditions (the rate of drying was about 0.41% h 1) (Fig. 1). Both seeds had dehydrated to about 8.4% at the end of drying. Non-primed control seeds used in this study had 80% emergence and 7.49 days mean emergence time (MET) (Table 1). Fresh primed bitter gourd seeds (both fast- and slowdried seeds) showed increased emergence percentage and accelerated emergence speed
Fig. 1. Changes in the moisture content (dry weight basis) of bitter gourd (cv. Blue Mountain Giant White) seeds subjected to fast drying and slow drying treatments during priming.
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Table 1 The effects of post-hydration drying methods on percentage of emergence, mean emergence time (MET), malondialdehyde (MDA) content, total peroxide contents, total anti-oxidative activity (TAA) and dry weight of 14-days-old seedling for non-primed control seeds and the primed seeds of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying or slow drying treatments Seed traits
Control
Fast drying
Slow drying
LSD0.05
Emergence (%) MET (days) MDA (nmol g 1 DW) Total peroxide (mmol g 1 DW) TAA (nmol trolox eq g 1 DW) Seedling dry weight (mg)
80 7.49 30.42 6.57 3.05 148
92 6.88 25.02 5.71 3.67 173
97.5 6.31 23.47 5.15 3.86 192
5.38 0.49 3.02 0.59 0.23 16
(Table 1) in comparison with control. They also exhibited more uniform seedling growth (Fig. 2) and produced heavier seedlings than control (Table 1). Primed seeds also had lower levels of malondialdehyde (MDA) and total peroxide accumulation than non-primed control (Table 1). The lower MDA and total peroxide of primed seeds might result from the priming-enhanced total anti-oxidative activities (TAA) (Table 1). Both emergence percentage and MET of primed seeds were affected by drying method, with slow-dried seeds performing better than fast-dried seeds (Table 1). Slow-dried seeds also produced heavier and better seedlings than fast-dried seeds (Table 1 and Fig. 2). Only minor differences in the levels of MDA and total peroxide accumulation and TAA were obtainable between slow-dried and fast-dried seeds, with slow-dried seeds showing greater priming responses than fast-dried seeds (Table 1).
Fig. 2. The growth responses of 14-days-old bitter gourd seedlings of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying (F) or slow drying (S) treatments.
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3.2. Experiment 2 Non-primed control seeds stored for 12 weeks showed no evident decline in emergence percentage, but marked reductions in emergence percentage were observed for non-primed seeds stored for 24, 36 and 48 weeks (Fig. 3A). Primed seeds (both slow- and fast-dried seeds) also had reduced emergence percentage during 48 weeks of storage (Fig. 3A). Drying method had significant effect on the longevity of primed seeds. Emergence percentage rapidly declined to 35% for slow-dried seeds after 12 weeks storage as compared to 75% in nonprimed seeds, and no seedling was found after 48 weeks storage (Fig. 3A). For fast-dried seeds, the percentage of emergence declined slightly (85%) during 12 weeks storage, but it was still greater than non-primed control. The emergence percentage of fast-dried seeds further declined to 39% after 48 weeks storage, which was lower than control (51%) (Fig. 3A). The differences in storability were not due to the moisture uptake during storage, since no notable changes in seed moisture level were detected (data not shown).
Fig. 3. Changes in emergence percentage (A) and seedling dry weight (B) of bitter gourd (cv. Blue Mountain Giant White) seeds that subjected to fast drying and slow drying treatments during 48 weeks storage. Vertical bars represent the mean and S.E. of three replications. LSD (P < 0.05) for emergence percentage and seedling dry weight, across all the treatments are 8.32 and 15.79, respectively.
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Non-primed seeds stored for up to 9 weeks showed no evident decline in seedling growth, but marked reductions in seedling dry weight were observed for non-primed seeds stored for 24, 36 and 48 weeks (Fig. 3B). Storing the primed seeds receiving slow drying treatment also resulted in significant declines in seedling dry weight after 48 weeks storage, but the rate of decline was greater than non-primed control, especially at the earlier stages of storage (12 weeks of storage) (Fig. 3B). However, fast-dried seeds performed better than non-primed control for up to 36 weeks storage (Fig. 3B). Both slow- and fast-dried seeds initially had lower levels of MDA (Fig. 4A) and total peroxide (Fig. 4B) than non-primed seeds. The seeds receiving slow drying showed higher levels of MDA and total peroxide accumulations than non-primed control with 24 weeks of storage. The levels of MDA and total peroxide for fast-dried seeds also increased following 24 weeks storage, but the extents of accumulation were lower than non-primed seeds. After 48 weeks storage, the levels of MDA and total peroxide accumulation for fast-dried seeds were greater than their respective controls (Fig. 4A and B). Both primed and non-primed seeds exhibited a decrease in TAA, but the rate of decline for slow-dried seeds was faster than fast-dried and control seeds during 24 weeks storage (Fig. 4C). The primed seeds receiving fast drying had greater TAA than control seeds up to 36 weeks storage. However, a lower TAA in fast-dried seeds compared to non-primed control seeds occurred after 48 weeks storage. Correlation analysis showed that, in all the treatments, the levels of MDA and total peroxide were negatively correlated to the percentage of emergence and the seedling dry weight (Table 2). In contrast, TAA correlated positively to the emergence percentage and seedling dry weight (Table 2).
4. Discussion Previous studies in this laboratory (Chen and Sung, 2001; Hsu et al., 2003; Wang et al., 2003) demonstrated that priming the bitter gourd seeds would improve their germination and emergence performances. The improvements were attributable in part to the enhanced anti-oxidative activity. However, in these studies, the anti-oxidative abilities were examined only in the seeds imbibed for 48 h because the ascorbate–glutathione cycle was not completely operative in dry bitter gourd seeds (J.M. Lin, unpublished data). Thus, the enhanced anti-oxidative abilities that were found in the primed bitter gourd seeds might partially result from the priming-induced advancement of germination. The measurement of total anti-oxidative activity (TAA) was initially developed to monitoring the antioxidant status in premature neonates (Miller et al., 1993). Yeh et al. (2005) recently used this technique to examine the anti-oxidative responses of imbibing bitter gourd seeds (seeds were imbibed for 48 h) and found that TAA was positively related to the anti-oxidative abilities of primed seeds. They further demonstrated that TAA was closely related to germination percentage and germination speed. In the present study, we found that the TAA was detectable in dried bitter gourd seeds (Table 1), therefore, this technique can be used to examine the anti-oxidative status of non-imbibed bitter gourd seeds. Primed bitter gourd seeds also showed marked decreases in MDA and total peroxide accumulations (Table 1), which might have resulted from priming-enhanced TAA. Correlation analysis
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Fig. 4. Changes in malondialdehyde (MDA) content (A), total peroxide content (B) and total anti-oxidative activity (TAA) (C) of bitter gourd (cv. Blue Mountain Giant White) seeds that subjected to fast drying and slow drying treatments during 48 weeks storage. Vertical bars represent the mean and S.E. of three replications. LSD (P < 0.05) for MDA, total peroxide and TAA, across all the treatments are 3.47, 1.72 and 0.45, respectively.
confirmed these findings (Table 2). These results, taken in conjunction with increased emergence percentage and improved seedling growth (Table 1 and Fig. 2), indicate that enhanced anti-oxidative ability may be in part the reason for improved emergence performance of primed bitter gourd seeds (Table 2).
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Table 2 The correlation coefficients (r) between the percentage of seed germination and malondialdehyde (MDA), total peroxide and total anti-oxidative activity (TAA), and between TAA and MDA and total peroxide of non-primed control seeds and the primed seeds of bitter gourd (cv. Blue Mountain Giant White) subjected to fast drying or slow drying treatments MDA
Total peroxide
TAA
Emergence percentage Control Fast drying Slow drying
0.915a 0.935a 0.916a
0.927a 0.929a 0.897a
0.912a 0.952a 0.921a
Seedling dry weight Control Fast drying Slow drying
0.921a 0.909a 0.934a
0.934a 0.897a 0.923a
0.954a 0.915a 0.965a
a
Significant at 1% level.
Many environmental factors are known to affect the success of priming, post-hydration drying being critical (McDonald, 2000). Chiu et al. (in press) reported that both free radicals and peroxides are re-generated during post-hydration drying, which would intensify lipid peroxidation. The intensified peroxidation, possibly resulting from dryinginduced oxidative stress, would damage the anti-oxidative mechanisms and impair seed quality (Palma et al., 2002; Chiu et al., in press). In the present study, significant differences in MDA and total peroxide existed between the primed seed lots that receiving fast and slow drying treatments, with the latter accumulating less MDA and total peroxide than the former (Table 1). The present study also revealed the greater increases in TAA by slow drying treatment (Table 2). As a result, the lipid peroxidation would be markedly reduced during seed imbibition. Thus, improvements in emergence percentage and seedling growth were obtainable for slow-dried seeds as compared to fast-dried seeds, provided that the seeds were immediately planted following priming (Table 1 and Fig. 2). While slow drying improved the initial quality of primed seeds, it resulted in a rapid reduction of seed longevity in storage as compared to that of the seeds receiving fast drying treatment (Fig. 3A). The decreased seedling dry weight confirmed the inferior quality of slow-dried seeds subjected to long-term storage (Fig. 3B). The rapid deterioration of slowdried seeds might be related to the peroxidative damages, possibly resulting from increased MDA (Fig. 4A) and total peroxide (Fig. 4B) accumulations and the decreased TAA during dry storage (Fig. 4C) (McDonald, 1999; Yeh et al., 2005). Decreased longevity for slowdried seeds might also be due in part to the advancement of germination reactions to a level not compatible with the drying back of seed required for storage. Lin and Sung (2001) previously reported that priming enlarged the embryos of bitter gourd seeds. In this study, slow-dried seeds did maintain a relatively higher seed moisture content at longer drying periods in comparison with fast-dried seeds (Fig. 1). Under higher seed moisture contents, the embryo might enlarge to such an extent that drying was injurious (Basu, 1994). Moreover, Lanteri et al. (1996) reported that more DNA replication and greater advancement through the cell cycle (i.e., more radicle meristem cells were in the S or G2 phase) generally occurred at higher seed moisture contents. Thus, under higher seed moisture content (Fig. 1), slow-dried seeds might pass the threshold of desiccation
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tolerance, which would damage the seeds beyond the point of recovery, even though they had greater initial quality than fast-dried seeds initially (Table 1). In conclusion, the results described here re-confirm that priming improves the initial quality of bitter gourd seeds. The improved quality appears partly attributable to decreased peroxidation, resulting from increased TAA. The method of drying following hydration also affects the initial quality of primed seeds. Slow-dried seeds initially accumulated less amounts of MDA and total peroxide as compared to fast-dried seeds. These results might partially explain why the primed seeds receiving slow drying treatment have better quality than that of the seeds receiving fast drying. However, more total peroxide were accumulated in slow-dried seeds during 48 weeks storage, and therefore made them more susceptible to deterioration in long-term storage as compared to fast-dried seeds. The longevity of fast-dried seeds could be maintained for 24 weeks when they were stored at 25 8C conditions, because they were under the enhanced protection of anti-oxidative activity.
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