The major findings from this study are (i) throughout the re-imbibition durations, the hydroprimed seeds had higher levels of seed moisture, gibberellic acid concentration, speed of germination, germination percentage, and seedling vigour traits than unprimed seeds, (ii) dehydration of imbibed seeds for 12 h has resulted in an increased GA concentration in seeds, and seedling vigour traits compared to other dehydration treatments (3, 6, and 9 h), and (iii) higher GA concentration in seeds has increased the α-amylase activity, which results in improved seed germination.
Experiment 1: Effect of seed imbibition duration on moisture content, gibberellic acid content and vigour of hydroprimed and unprimed seeds
The result of the present study indicates that there were significant (P ≥ 0.05) differences among the treatments (re-imbibition for 0, 3, 6, 9 and 12 h ) for seed moisture content (%), germination percentage, and speed of germination (d) (Fig. 1.). Similarly, there were significant (P ≥ 0.05) differences among the priming treatments for root length (cm), shoot length (cm), total dry matter production (g 10 seedlings− 1), vigour index, and GA concentration (µg g− 1) (Fig. 2).
Among the treatments, the hydroprimed seed recorded seed moisture of > 9% at 3 h of re-imbibition, while the unprimed seeds had the same moisture content (> 9%) only after 12 h of re-imbibition (Fig. 1a). Irrespective of the duration of imbibition (3, 6, 9 and 12 h), the unprimed seeds showed a lower speed of germination and seed germination percentage; in contrast, the hydroprimed seeds recorded the higher speed of germination and seed germination. Hydroprimed seeds showed an increased root length (cm), shoot length (cm), dry matter production (g 10 seedlings− 1) and vigour index compared to unprimed seeds (Figs. 1 and 2).
Similarly, the gibberellic acid concentration (µg g− 1) was higher in hydroprimmed seeds than unprimmed seeds throughout the re-imbibition durations (Fig. 2a-d). Further, the increased synthesis of GA was observed earlier in primed seeds (6 h) compared to unprimed seeds (9 h). The increased moisture content or rapid water imbibition, coupled with increased biosynthesis of GA in hydroprimmed seeds at 3, 6, 9, and 12 h of imbibition compared to respective timing of unprimmed seeds, shows early biosynthesis of GA. Obviously, early or rapid biosynthesis of GA might help in a higher speed of germination and increased seed germination than unprimed seeds through higher cell division in germinating seeds (Bewley and Black, 1994). Similar positive effects of hydropriming were observed in wheat (Muzaar et al. 2019), maize (Mohammadi et al. 2014), soybean (Langeroodi and Noora, 2017), and sunflower (Lekic et al. 2015).
In the process of hydropriming, various pre-germination processes have to be activated without rupture of testa so that radicle does not emerge (Weitbrecht et al. 2011). The whole metabolism has already been initiated so that when the seeds are shown, developmental processes go on more rapidly than in the case of non-primed seeds (Kattimani et al. 1999). The increased speed of germination and synchronized seed germination in hydroprimmed seeds compared to unprimmed seeds may be associated with metabolic changes that occur during phase I and II of imbibition accomplished due to seed priming (Khajeh-Hosseini et al. 2003; Musa et al. 2001; Sadeghian and Yavari, 2004). Previous studies have shown that GA produced in the embryonic axis is involved in the induction of α-amylase enzyme activity (Yamauchi et al. 2004; Seo et al. 2009) and weakening endosperm caps (Groot and Karssen, 1987).
Overall, the hydroprimed seeds showed quicker water imbibition potential, concomitantly higher biochemical potential in terms of higher gibberellic acid synthesis, and increased physiological potential in terms of seed germination percentage, speed of germination, and root length compared to unprimed seeds.
Experiment 2. Influence of differential duration of seed dehydration of imbibed tomato seeds on the efficacy of seed priming process
Our earlier study in tomato has shown that imbibing the seeds in water for 48 h, followed by shade drying for 12 h (hydropriming) has improved the seed germination and speed of germination compared to unprimed seeds. Previous reports have established that during the imbibition phase, the pre-germinative events like the repair of mitochondria and genetic material, biosynthesis of protein, hormones, and metabolites and induction of enzymes might happen (Bewley and Black, 1994). The stimulatory effect observed due to hydropriming may be associated with imbibition or dehydration period. In this study, we hypothesized that the observed positive effects of hydropriming might be related to the dehydration period. To validate this the tomato seeds were imbibed for 48 h, and then dehydrated for 3, 6, 9, and 12 h, and various seed germination traits like seed GA concentration, α-amylase activity and starch content were quantified by re-imbibing the seeds for 3, 6, 9 and 12 h, to initiate the seed germination process.
The main effects of dehydration period and re-imbibition period was significant (P ≥ 0.05) for speed of germination (d), germination percentage, root length (cm), shoot length (cm), dry matter accumulation (g 10 seedlings− 1), vigour index and moisture content (%). However, the interaction of dehydration and re-imbibition period was significant (P ≥ 0.05) for α-amylase enzyme activity (mg of maltose min− 1), starch content (mg g− 1), and gibberellic acid concentration (µg g− 1).
Among the dehydration durations (h), seeds dehydrated for 12 h was found to record increased speed of germination, germination percentage, root length, shoot length, dry matter accumulation, and vigour index (Table 1). However, the lowest seed moisture content was recorded at 12 h of dehydration (Table 1) which was on par with 9 h. Among the re-imbibition period, the increased speed of germination, germination percentage, root length, shoot length, dry matter accumulation and vigour index was observed at 9 h of imbibition (Table 2). There was no statistical difference between 9 and 12 h of re-imbibition for the above-mentioned traits (Table 2). Similarly, there is no variation in seed moisture content from 6 to 12 h of re-imbibition (Table 2).
Table 1
Main effect of dehydration period (h) on seedling traits of tomato seeds
Stages of dehydration (h)
|
Speed of germination (d)
|
Germination percentage
|
Root length (cm)
|
Shoot length (cm)
|
Dry matter production
(g 10 seedlings− 1)
|
Vigour index
|
Moisture content (%)
|
3
|
3.07d
|
64 (53.19)d
|
11.54d
|
5.25d
|
0.018b
|
1075d
|
10.33 (18.74)a
|
6
|
3.39c
|
66 (54.28)c
|
12.25c
|
5.40c
|
0.018b
|
1160c
|
9.68 (18.11)b
|
9
|
4.09b
|
72 (57.95)b
|
13.17b
|
5.63b
|
0.021a
|
1354b
|
9.00 (17.46)c
|
12
|
4.36a
|
74 (59.19)a
|
13.97a
|
5.96a
|
0.021a
|
1470a
|
8.93 (17.37)c
|
LSD (P ≤ 0.05)
|
0.085
|
0.89
|
0.30
|
0.12
|
0.0005
|
24.87
|
0.16
|
LSD = least significant difference; value in parenthesis are arcsine transformed value |
Table 2
Main effect of re-imbibition period (h) on seedling traits of tomato seeds
Stages of
re-imbibition (h)
|
Speed of germination (d)
|
Germination percentage
|
Root length (cm)
|
Shoot length (cm)
|
Dry matter production
(g 10 seedlings− 1)
|
Vigour index
|
Moisture content (%)
|
3
|
3.59b
|
68 (55.80)a
|
12.35c
|
5.46a
|
0.02a
|
1219c
|
9.20 (17.65)b
|
6
|
3.65b
|
69 (56.27)a
|
12.58b
|
5.54a
|
0.02a
|
1256b
|
9.48 (17.91)a
|
9
|
3.80a
|
69 (56.22)a
|
12.81ab
|
5.59a
|
0.02a
|
1274b
|
9.60 (18.03)a
|
12
|
3.87a
|
69 (56.32)a
|
13.19a
|
5.65a
|
0.02a
|
1309a
|
9.65 (18.09)a
|
LSD (P ≤ 0.05)
|
0.085
|
NS
|
0.30
|
NS
|
NS
|
24.8
|
0.16
|
LSD = least significant difference; value in parenthesis are arcsine transformed value |
The GA concentration observed at 3, 6, 9, and 12 h of re-imbibition duration indicated that the seeds which are dehydrated for 12 h had higher levels of GA compared with 3, 6, and 9 h of dehydration (Fig. 3). Similarly, the α- amylase enzyme activity was the highest at 12 h of dehydration and 12 h of re-imbibition (Table 3). In contrast, the starch content in the seed was the lowest at 12 h of dehydration and 12 h of re-imbibition.
Table 3
Effect of re-imbibition period (h) and dehydration period (h) on α- amylase enzyme activity (mg of maltose min− 1) and starch content (mg g− 1) of tomato seeds.
Stages of dehydration – D (h)
|
Stage of re-imbibition - I (h)
|
α- amylase enzyme activity (mg of maltose min− 1)
|
Starch content (mg g− 1)
|
3
|
6
|
9
|
12
|
Mean
|
3
|
6
|
9
|
12
|
Mean
|
3
|
9.74f
|
9.76f
|
10.10f
|
12.10de
|
10.43
|
30.2a
|
28.4b
|
26.9cd
|
21.6f
|
26.7
|
6
|
9.80f
|
12.24de
|
12.38d
|
14.26bc
|
12.17
|
28.7b
|
26.0d
|
22.4f
|
21.6f
|
24.6
|
9
|
11.78e
|
12.28de
|
13.82c
|
14.48b
|
13.09
|
28.2b
|
24.2e
|
20.6g
|
19.8g
|
23.2
|
12
|
11.78e
|
12.56d
|
14.08bc
|
17.70a
|
14.03
|
27.0c
|
23.7e
|
20.5g
|
17.2h
|
22.1
|
Mean
|
10.78
|
11.71
|
12.60
|
14.64
|
|
28.5
|
25.5
|
22.6
|
20.0
|
|
CD (P ≤ 0.05)
|
D
|
|
I
|
|
D x I
|
D
|
|
I
|
|
D x I
|
0.253
|
|
0.25
|
|
0.507
|
0.45
|
|
0.44
|
|
0.91
|
LSD = least significant difference; D = dehydration duration; I = Imbibition duration; D x I = Interaction of dehydration and imbibition duration; value in parenthesis are arcsine transformed value |
The seed germination was lower in 3 h dehydrated seed (64%); subsequently, as the duration of dehydration increased, the germination per cent increased from 66 to 74% (Table 1). Therefore, it is clear that advancement in dehydration increases germination potential. This phenomenon is observed in re-imbibition (Table 2). Complete drying of seeds after the imbibition process of seed priming improves the rate of seed imbibition and GA synthesis in seeds (Table 1; Fig. 3). GA is involved in the upregulation of α-amylase enzyme activity (Gubler et al. 1995), as a consequence, the carbohydrate stored in the endosperm will be broken down into simple sugars and will be transported to the growing embryo (Gupta and Chakrabarty, 2013) to support the respiratory process, resulting in early and uniform seedling emergence and growth.