Influence of Hydro-Priming on Reserve Utilization of Differentially Aged Chickpea Seeds Kazem Ghassemi-Golezani* and Ayda Hosseinzadeh-Mahootchi ABSTRACT Priming appears to reverse the detrimental effects of seed aging by modulating pre-germination metabolic activity prior to radicle emergence. However, the impact of these metabolic changes on seed reserve utilization is not clear. is study was conducted to evaluate the effects of hydro-priming on reserve utilization of three differentially aged seed lots (100, 98 and 89% normal germination) of chickpea. Results showed that reserve utilization rate of the most aged seed lot significantly decreased, compared with other seed lots. e highest conversion efficiency was obtained for the high quality seed lot and decreased with decreasing seed lot quality. Increasing seed aging led to significant reduction in germination rate and seedling dry weight, mainly due to a poor reserve utilization rate. e highest seed reserve utilization rate was recorded for high vigor and primed seed lots. Increasing seed vigor due to hydro-priming also resulted in increasing germination rate and seedling dry weight. e highest improvement in germination rate due to hydro-priming was observed in poor vigor seed lots. erefore, hydro-priming can improve reserve utilization of differentially aged seeds and thereby enhance their germination rate and seedling size. INTRODUCTION
Chickpea is one of the most important legume crops in sustainable agricultural systems. Because of its short growth period, the crop is cultivated in Iran in the spring season, with minimum energy expenditures. Seed aging is a serious problem in developing countries where seeds are usually stored without proper humidity and temperature control (Barton, 1964). Several biochemical and physiological changes have been observed in seeds during aging, resulting in a progressive decline in seed quality and performance (McDonald, 1999). Membrane disruption is one of the main reasons of seed aging, the major causes of which are increased free fatty acid levels and free radical productivity by lipid peroxidation (Grilli et al., 1995). Free radicals attack membrane lipids, and cause major disruption of their viscosity and permeability (Van Zutphen and Cornwell, 1973; Ferguson et al., 1990; Panobianco and Viera, 2007). e rate of deterioration in orthodox seeds, due to aging, is positively related to ambient temperature, relative humidity and seed moisture content (Ellis and Roberts, 1981). erefore, reducing temperature and seed moisture content down to certain levels can considerably prolong survival of orthodox seeds, such as chickpea (Cicer arietinum L.), in storage. Department of Plant Eco-Physiology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. *Corresponding author (
[email protected]). Received 8 January 2013.
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Seed aging is generally marked by a reduction in vigor (Trawatha et al., 1995; Gupta and Aneja, 2004), viability, rate and uniformity of seed germination and seedling emergence (Chhetri et al., 1993; Arefi and Abdi, 2003; Abdulrahmani et al., 2007; Ghassemi-Golezani et al., 2008), increased solute leakage (Basra et al, 2003; Ghassemi-Golezani et al., 2012), susceptibility to stresses and reduced tolerance for storage under adverse conditions (Duffus and Slaughter, 1980). Priming may reverse the deleterious effects of seed aging by repairing and building-up nucleic acids, increasing synthesis of proteins, as well as repairing membranes (McDonald, 2000). Quality reversal by priming deteriorated seed generally occurs in the meristematic axis or the radicle tip (Fu et al., 1988). Priming is also thought to increase free-radical scavenging enzyme activity, counteract the effects of lipid peroxidation and reduce leakage of metabolites (McDonald, 1999; Hsu et al., 2003; Wang et al., 2003). Consequently, seed germination and seedling vigor can be improved as a result of seed priming (McDonald, 2000; Halmer, 2004; Ghassemi-Golezani et al., 2010; Ghassemi-Golezani et al., 2012). Seed germination is comprised of two distinct metabolic processes, enzymatic hydrolysis of seed storage substances, and formation of new cell structures (Bewley and Black, 1994; Copeland and McDonald, 1995). Gibberellic acid (GA) stimulates the synthesis of hydrolytic enzymes such as amylase, ribonuclease, protease, phosphatase and 1,3-glucanase. ese enzymes are responsible for the hydrolysis of stored carbohydrates, lipids, proteins and phosphorous compounds. e hydrolyzed products are then utilized in seedling tissue synthesis (Bewley and Black, 1994). Heterotrophic seedling growth can be quantitatively described as the product of three components (Soltani et al., 2006). e first is initial seed weight, the second is seed depletion ratio, i.e. the fraction of seed reserves mobilized, and the third is the conversion efficiency of mobilized seed reserves to seedling tissues. erefore, seedling growth is affected by changes in seed reserve utilization and efficiency. Since the effects of priming on reserve utilization of differentially aged seeds have not been clearly elucidated, this research aimed to investigate such priming effects in chickpea. MATERIALS AND METHODS Seeds of chickpea (cv. ILC 482) were obtained from the Dryland Agricultural Research Institute of Maragheh, Iran, and divided into three samples. One sample, with a tested germination of 100%, was kept as control (V1). e other two samples, with a final moisture content (MC) of about 20%, were artificially aged at 40 °C for 3 and 5 d, reducing normal germination to 98 and 89% (V2 and V3), respectively. Consequently, three seed lots with different levels of vigor were made available. Each seed lot (sample) was divided into three sub-samples, one of which kept as a control (non-primed, P1) and the other two soaked in distilled water at 15 °C for 12 (P2) and 18 (P3) h and then dried back to initial MC at a room temperature of 20–22 °C for 24 h. To determine seed MC, two replications of 5 g from each lot were weighed, ground and aer drying at 130 ± 1 °C for 1 h weighed again, and seed MC calculated according to ISTA (ISTA, 2010).
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Laboratory tests were carried out as a factorial experiment laid out in a RCB design at the Seed Technology Laboratory of Tabriz University, Iran. Mean comparisons were based on Duncan’s multiple range test. Four replicates of 25 seeds each were weighed and placed between moist filter papers and germinated in an incubator at 20 °C for 7 d (ISTA, 2010). Germinated seeds (protrusion of radicle by 2 mm) were recorded every 24 h for 7 d. Rate of seed germination was calculated according to Ellis and Roberts (1980) as:
where n is the number of seeds germinated on day D, D is the number of days from the beginning of the test and R is the mean germination rate. At the end of germination test, seeds with normal seedlings in each replicate were counted and seedlings were detached from the remaining seed reserves. e initial dry weight of these seeds was calculated using the data for the fresh weight of seeds of each replicate and seed MC. Normal seedlings with the seed remnants were then dried in an oven at 75 °C for 24 h and separately weighed. e weight of utilized seed reserves (SRU) was calculated as: SRU = initial seed dry weight − seed remnant dry weight
Seed reserve utilization rate (SRUR) was then estimated as: SRUR = SRU/germination duration
Conversion efficiency (CE) of mobilized seed reserves into plant tissues was calculated as: CE = seedling dry weight /SRU
e ratio of utilized seed reserves to initial seed dry weight was used to calculate seed reserve depletion percentage. RESULTS Seed aging had significant effects on seed reserve utilization rate, conversion efficiency, germination rate and seedling dry weight (Table 1). However, seed Table 1. Analysis of variance of the effects of seed aging and hydro-priming on seed reserve utilization parameters of chickpea. Seed reserve utilization Source of variation df rate Replication 3 1.018 Aging 2 4.185* Hydro-priming (HP) 2 11.287** Aging × HP 4 0.773 NS Error 24 1.324 CV (%) – 7.56
MS Seed Seedling Conversion reserve Germination dry efficiency depletion rate weight 0.000 4.361 0.001 1.179 0.005** 6.859 NS 0.037** 208.351** 0.001 NS 54.963** 0.032** 152.551** 0.000 NS 5.594 NS 0.005** 21.204* 0.001 NS 6.101 0.001 6.351 5.62 6.57 5.43 4.71
NS, *, **Not significant, significant at p ≤ 0.05, and significant at p ≤ 0.01, respectively.
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Table 2. Means of seed reserve utilization parameters of chickpea following different seed aging periods and hydro-priming durations. Treatments Aging V1† V2 V3
Seed reserve utilization rate
Conversion efficiency
Seed reserve depletion
Germination rate
Seedling dry weight
(mg seed−1 day −1)
(mg seed−1)
(%)
(day −1)
(mg)
15.57a § 15.54a 14.53b
0.5271a 0.5025ab 0.4860b
37.54a 38.39a 36.88a
0.5882a 0.5201b 0.4780c
57.67a 53.43b 49.33c
0.4962a 0.5153a 0.5041a
35.23b 39.40a 38.18a
0.4695b 0.5517a 0.5652a
49.43b 56.17a 54.83a
Hydro-priming duration P1‡ 14.13b P2 P3
16.00a 15.52a
† V1, V2 and V3: control and aging for 3 and 5 d at 40 °C, respectively. ‡ P1, P2 and P3: unprimed seeds and hydro-priming for 12 and 18 h, respectively. § Means followed by the same letter, in each column, for each treatment, do not significantly differ (p ≤ 0.05), according to Duncan’s multiple range test.
reserve depletion was not significantly affected by seed aging. V1 and V2 did not significantly differ, but resulted in significantly higher seed reserve utilization rate and conversion efficiency than V3 (Table 2). Seeds of V2 and V3 had significantly lower germination rates and thus germinated later than those of V1. Increasing seed aging also resulted in reductions of seedling dry weight (Table 2). Hydro-priming had no significant effect on conversion efficiency. However, seed reserve utilization rate, reserve depletion percentage, germination rate and seedling dry weight were all significantly affected by hydro-priming (Table 1). ese traits for P2 and P3 seeds (hydro-primed for 12 and 18 h, respectively) were significantly higher than those for P1 (unprimed) seeds, but no significant differences between the two priming durations were found (Table 2). e interaction of aging with hydro-priming duration was significant for germination rate and seedling dry weight (Table 1). Germination rate of aged seed lots was improved by hydro-priming compared to the control, although this improvement was statistically similar for both priming durations (P2 and P3), and increased with increased seed aging (Fig. 1). On the other hand, seedling dry weight of V1 and V3 seed lots was significantly increased by hydropriming, but for seeds aged for 3 d (V2), only hydro-priming for 12 h resulted in a significant increase over the control (Fig. 1). Seed reserve utilization rate, conversion efficiency and depletion percentage had significant and positive correlations with germination rate and seedling dry weight (Table 3). e highest positive correlation with germination rate and seedling dry weight was recorded for seed reserve utilization rate. Correlation between germination rate and seedling dry weight was also highly significant and positive. DISCUSSION Reduced seed reserve utilization rate following seed aging (Table 2) might be due to hydrolytic enzyme reduction, mitochondrial dysfunction and reduced
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Figure 1. Mean germination rate and seedling dry weight of differentially aged chickpea seeds (V1, V2 and V3: control and aging for 3 and 5 d at 40 °C, respectively) following priming treatments (P1, P2 and P3: unprimed seeds and hydropriming for 12 and 18 h, respectively).
Table 3. Correlation coefficients among seed reserve utilization parameters of chickpea. Parameter 1. Reserve utilization rate (mg seed−1 day −1) 2. Conversion efficiency (mg seed−1) 3. Depletion percentage 4. Germination rate (day −1) 5. Seedling dry weight (mg)
1 1 −0.317 0.900** 0.535** 0.617**
2
3
4
5
1 0.330* 0.382* 0.542**
1 0.476** 0.520*
1 0.799**
1
*, **Significant at p ≤ 0.05 and p ≤ 0.01, respectively
ATP production during germination (McDonald, 1999; Basra et al., 2003). Significant reductions in germination rate and seedling dry weight as a result of seed aging were mainly related to poor reserve utilization rate and conversion efficiency of mobilized reserves (Table 3). Free radicals damage the lipid bi-layer, especially of the mitochondrial membrane, leading to reduced energy production (Booth and Bai, 1999), enzymes, proteins and DNA (Wilson and McDonald, 1986), and ultimately limit conversion of mobilized reserves for germination and seedling growth. Decreasing GA3 concentration during germination of aged seeds may also lead to reductions in seed reserve utilization (Mohammadian et al., 2011). Increasing reserve utilization rate, reserve depletion, germination rate and seedling dry weight in hydro-primed seeds (Table 2) could be associated with various biochemical, cellular and molecular events such as early synthesis of RNA and proteins (Bray et al., 1989; Dell’Aquila and Bewley, 1989; Dell’Aquila
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and Tritto, 1991; Bray, 1995; Kalpana and Madhava Rao, 1997), promotion of DNA replication (Sivritepe and Dourado, 1994) and enhancing enzyme activities (Jeng and Sung, 1994; Amanpour-Balaneji and Sedghi, 2012) during the hydration process. Many seed priming treatments have been used to reduce the damage of aging and invigorate seed performance in many crops (Basra et al 2003; Farooq et al., 2006). In our work, hydro-priming improved reserve utilization of chickpea seeds, leading to a higher germination rate, particularly of the most aged seeds (Fig. 1). Higher reserve utilization of primed seeds may be related to increased enzyme activities such as amylase, protease and lipase, which play a key role in breakdown of macromolecules for embryo growth and development (Dell’Aquila and Tritto, 1990), ultimately resulting in germination. Moreover, priming restores activities of enzymes involved in cell detoxifying mechanisms in aged seeds, such as super-oxide dismutase, catalase and glutathione reductase (Bailly et al., 1997). Priming has also been shown to increase germination metabolism in aged axes compared to non-aged ones (McDonald, 2000). Rapid germination of seeds could ultimately lead to the production of larger seedlings (Table 2, Fig. 1). is is also reflected in significant and positive correlations of seed reserve utilization rate, reserve depletion and germination rate with seedling dry weight (Table 3). In general, seed aging within a range of acceptable germination may considerably reduce seed reserve utilization rate in chickpea, leading to reductions in germination rate and seedling dry weight. ese deleterious effects may be overcome to some extent by hydro-priming of seeds. REFERENCES Abdulrahmani, B., K. Ghassemi-Golezani, M. Valizadeh and V. Feizi-Asl. 2007. Seed priming and seedling establishment of barley (Hordeum vulgare L.). J. Food Agric. Environ. 5: 179–184. Amanpour-Balaneji, B. and M. Sedghi. 2012. Effect of aging and priming on physiological and biochemical traits of common bean (Phaseolus vulgaris L.). Not. Bot. Hort. Agro. ClujNap. 4: 2067–3264. Arefi, H.M. and N. Abdi. 2003. Study of variation and seed deterioration of Festuca ovina germplasm in natural resources gene bank. J. Rang. Forests Plant Breed. Gen. Res. 11: 105–125. Bailly, C., A. Benamar, F. Corbineau and D. Come. 1997. Changes in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds during accelerated ageing and subsequent priming. p. 665–672. In Basic and applied aspects of seed biology. R.H. Ellis, M. Black and A.J. Murdoch (eds.). Kluwer Academic Publishers, Dordrecht. Barton, L.V. 1964. Seed preservation and longevity. Inter-social publishers, Inc. New York. Basra, S.M.A., I.A. Pannu and I. Afzal. 2003. Evaluation of seedling vigor of hydro and matri-primed wheat (Triticum aestivum L.) seeds. Int. J. Agric. Biol. 2: 121–123. Bewley, J.D. and M. Black. 1994. Seeds: physiology of development and germination. Plenum Press. Booth, D.T. and Y. Bai. 1999. Imbibition temperature effects on seedling vigor in crops and shrubs. J. Rang. Manage. 52: 534–538.
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