Journal of Integrative Plant Biology Formerly Acta Botanica Sinica 2005, 47 (2): 187−193

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Thermal Hardening: A New Seed Vigor Enhancement Tool in Rice Muhammad FAROOQ*, S. M. A. BASRA, Nazir AHMAD and K. HAFEEZ (Department of Crop Physiology, University of Agriculture, Faisalabad-38040, Pakistan)

Abstract: In a laboratory study, indica and japonica rice (Oryza sativa L.) seeds were exposed to thermal hardening (heating followed by chilling followed by heating; chilling followed by heating followed by chilling; heating followed by chilling or chilling followed by heating). In indica rice, heating followed by chilling followed by heating resulted in decreased mean germination time, time to start germination, electrical conductivity of seed leachates, and time to 50% germination, as well as increased germination index, energy of germination, radicle and plumule length, root length, root/shoot ratio, root fresh and dry weight, radicle and plumule growth rate, and shoot fresh weight. In japonica rice, chilling followed by heating followed by chilling performed better than all other treatments, including control. Key words: germination; indica rice; japonica rice; seedling vigor; thermal hardening.

Seed hardening has been used successfully for vigor enhancement in indica (Basra et al. 2003, 2004) and japonica rices (Lee et al. 1998; Lee and Kim 2000). In hardening, seeds are exposed to alternate wetting and drying in distilled or tap water (Pen Aloza and Eira, 1993). This hydration-dehydration cycle may be repeated twice, thrice etc. (Lee et al. 1998; Lee and Kim 2000). Lee and Kim (2000) investigated the effects of osmoconditioning and hardening on the germination of normal and naturally aged seeds by analyzing the total sugars and α-amylase activity. Total sugar content and the α-amylase activity of normal seeds were higher than those of aged seeds. Aged seeds treated with osmoconditioning and hardening increased their total sugar content and α-amylase activity, but hardening was more effective than osmoconditioning. The α-amylase activity was positively correlated with total sugars and the germination rate. Seed hardening for 24 h (two cycles) invigorated the indica rice seeds compared with seed hardening for 18 h, osmoconditioning (–1.1 MPa KNO3), and traditional soaking of indica seeds (Basra et al. 2003, 2004). Presowing chilling treatments are being used Received 7 Sept. 2004 Accepted 12 Nov. 2004 * Author for correspondence. E-mail: .

effectively alone or in combination with other invigoration techniques in order to shorten the time from planting to emergence and to protect seeds from abiotic and biotic stresses during the critical phase of seedling establishment (Basra et al. 2002). In an earlier study (Farooq et al. 2004), japonica and indica rice seeds were exposed to dry heat treatment (namely 40 °C for 72 h and 60 °C for 24 h) and chilling (–19 °C) treatment for 72 h. In indica rice, dry heat treatment at 40 °C for 72 h resulted in increased vigor, whereas in japonica rice none of the treatments resulted in improved germination and seedling vigor. In earlier studies, different presowing seed treatments were successfully integrated for vigor enhancement (Taylor et al. 1998). Most recently, Basra and Farooq (2004) introduced a new technique for rice seed invigoration in which both seed hardening and osmoconditioning were successfully integrated. Seeds of both types of rice were hardened in various salt solutions instead of tap or distilled water. It was concluded that osmohardening in CaCl2 solution (having an osmotic potential of –1.5 MPa) was best for vigor enhancement compared with other salts and simple

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hardening (Basra and Farooq 2004). It was then thought that alternate low and high temperature treatments may be effective for vigor enhancement in both types of rice. Therefore, the aim of the present study was to explore the benefits (if any) associated with presowing alternating treatments of seeds with low and high temperatures (thermal hardening) by exploring the germination, seedling vigor and electrical conductivity of seed leachates of both japonica and indica rice.

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further use. 1.4 Germination test Seeds were sown in Petri dishes between layers of moist filter paper at 27 °C in an incubator. Germination was observed daily according to the methods of the Association of Official Seed Analysis (1990). The time to obtain 50% germination (T50) was calculated according to the following formula of Coolbear et al. (1984), as modified by Farooq et al. (2004): T50 = ti +

Material and Methods

1.1 Seed materials Seeds of the japonica rice (Oryza sativa L.) cultivar KS-282 and the indica rice cultivar Super-Basmati were used as the experimental material. Seeds were obtained from the Rice Research Institute (Kala Shah Kakoo, District Sheikhupura, Pakistan). The initial seed moisture content of japonica and indica rice was 7.89% and 8.06%, respectively (on a dry weight basis). 1.2 Seed treatments The following seed treatments (thermal hardening) were integrated for vigor enhancement. 1.2.1 Heating For dry heat treatment, 250 g seeds of each cultivar were incubated at 40 °C for 24 h in an oven (EYLA Forced Air oven, WFO-600 ND; Rikakikai, Tokyo, Japan). Seeds were incubated in glass jars tightly covered with lids. 1.2.2 Chilling Seeds (250 g) of both japonica and indica rice were sealed in polythene bags and placed in a refrigerator (model NR 245 TES; National) at –19 °C for 24 h. The study consisted of the following treatment combinations: (i) H+C+H = heating followed by chilling followed by heating; (ii) C+H+C = chilling followed by heating followed by chilling; (iii) H+C = heating followed by chilling; and (iv) C+H = chilling followed by heating. 1.3 Post-treatment procedures After treatment of seeds with a particular method and for a particular duration, seeds were sealed in polythene bags and stored in a refrigerator at 5 °C until

( N − ni)(tj − ti) 2

(nj − ni)

where N is the final number of germinating seeds and nj and ni are the cumulative number of seeds germinated by adjacent counts at times tj and ti, respectively, when ni < N/2 < nj. Mean germination time (MGT) was calculated according to the equation of Ellis and Roberts (1981): MGT =

Dn n

where n is the number of seeds that had germinated on day D and D is the number of days counted from the beginning of germination. The germination index (GI) was calculated as described by the Association of Official Seed Analysis (1983) using the following formula: GI =

No. of germinated seeds Days of first count

+ ...... +

No. of germinated seeds Days of final count

The energy of germination (GE) was recorded on the 4th day after planting. This is the percentage of germinating seeds 4 d after planting relative to the total number of seeds tested (Ruan et al. 2002). 1.5 Seedling emergence Control and treated seeds were sown in plastic trays (25 seeds in each tray) containing moist sand, replicated four times, and were placed in a growth chamber (Windon Scientific Company, England). Day and night lengths were kept at 15 and 9 h, respectively,

Muhammad FAROOQ et al.: Thermal Hardening: A New Seed Vigor Enhancement Tool in Rice

with temperatures of 30 and 24 °C, respectively. The relative humidity was maintained at 70%. Emergence was recorded daily according to the Listed in Ref. Seedling Evaluation Handbook of the Association of Official Seed Analysis (1990). Mean emergence time was calculated according to the method described earlier by Ellis and Roberts (1981). 1.6 Electrical conductivity of seed leachates After washing in distilled water, 5 g seeds was soaked in 50 mL distilled water at 25 °C. The electrical conductivity (EC) of the soak solution was measured at 0.5, 1.0, 1.5, 2.0, 6.0, 12.0, and 24.0 h of soaking using a conductivity meter (model Twin Cod B-173) and expressed as µs.cm−1.g−1 (dry weight basis; Ashraf et al. 1999).

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Results

2.1 Germination In indica rice, treated seeds germinated earlier compared with untreated (control) seeds; however, seeds subjected to H+C+H germinated earlier than seeds subjected to other treatment combinations. The highest T50 was noted in untreated seeds; all presowing treatments resulted in a lower T50 compared with control (Table 1). Similarly, all treatments resulted in a significantly lower MGT than that of control, with a minimum value observed in seeds subjected to H+C+H that did not Table 1

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differ significantly from that of seeds subjected to the other treatments (Table 1). Seed treatments did not significantly affect the final germination percentage; however, compared with control, the energy of germination was improved when presowing treatments were used, with the energy of germination being highest in seeds subjected to H+C+H (Table 1). Maximum GI was recorded in seeds subjected to H+C, then followed by the remaining treatments, except control. Radicle length remained unaffected by presowing treatments (Table 1); however, minimum plumule length was noted in seeds subjected to C+H+C, which was not significantly different from that of control, whereas the plumule length of seeds undergoing the remaining treatment protocols was higher (Table 1). In japonica rice, the highest value of the time to start germination was noted in seeds treated by H+C, followed by control. The remaining treatments resulted in an earlier start of germination (Table 1); the earliest germination was observed in seeds subjected to C+H+C. All seed treatments resulted in lower T50 compared with control, except for H+C treatment, which resulted in the same T50 as control. Seeds subjected to C+H+C exhibited the earliest germination (Table 1). Similarly, the highest value of MGT was recorded in untreated seeds; all seed treatments resulted in a lower MGT value, which was lowest in seeds treated by C+H+C (Table

Effect of seed treatments on the germination vigor of japonica and indica rice

MGT FGP Radicle Plumule Time to start T50 GI GE (%) germination (h) (d) (d) (%) length (mm) length (mm) Indica rice Control 80.00 a 4.87 a 5.05 a 83.00 43.02 c 51.32 c 67.66 58.67 c H+C+H 38.00 d 2.01 d 3.00 bc 86.33 65.83 b 84.00 a 66.33 73.00 a C+H+C 49.00 c 2.67 bc 3.10 bc 82.00 65.65 b 72.66 b 62.36 59.16 c H+C 68.00 b 2.76 b 3.43 b 86.25 84.72 a 66.00 b 64.00 65.36 b C+H 64.00 b 2.76 b 3.23 b 87.66 69.44 b 72.33 b 62.05 72.70 a LSD at 0.05 9.25 0.563 0.312 n.s. 7.213 9.35 n.s. 4.53 Japonica Control 75.00 b 4.16 a 5.83 a 89.67 51.49 b 31.00 d 25.23 d 45.67 d rice H+C+H 63.00 c 3.22 b 3.83 c 88.67 57.63 a 53.00 b 45.83 c 54.00 c C+H+C 44.00 d 2.00 c 3.46 d 86.33 59.33 a 69.33 a 68.60 a 59.77 b H+C 88.00 a 4.10 a 4.86 b 82.67 31.33 d 46.33 c 54.97 b 68.17 a C+H 56.00 c 3.26 b 4.83 b 82.00 41.66 c 55.33 b 42.17 c 45.77 d LSD at 0.05 10.00 0.7301 0.756 n.s. 7.34 6.31 6.36 3.94 Figures not sharing the same letters differ significantly at P = 0.05. H+C+H = Heating followed by chilling followed by heating; H+C = Heating followed by chilling; C+H+C = Chilling followed by heating followed by chilling; C+H = Chilling followed by heating. FGP, final germination percentage; GE, energy of germination; MGT, mean germination time. Treatments

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1). The final germination percentage was not significantly affected by seed treatments (Table 1); however, all treatments resulted in a higher energy of germination compared with control. Maximum GE was recorded in seeds subjected to C+H+C. The highest germination index was noted in seeds treated with C+H+C, which did not differ significantly from that observed for seeds treated by H+C+H. Minimum GI was recorded in seeds subjected to H+C (Table 1). All seed treatments resulted in a higher plumule and radicle length. A maximum radicle length was observed in seeds treated with C+H+C, followed by H+C. Maximum plumule length was noted in seeds subjected to H+C, followed by seeds treated by C+H+C (Table 1). 2.2 Seedling vigor In indica rice, all seed treatments resulted in a lower mean emergence time (MET) compared with control. Minimum MET was noted in seeds subjected to H+C, which was not significantly different from that of seeds subjected to C+H+C. An improved emergence percentage was noted for all seed treatments compared with control, being maximum in seeds subjected to C+H+C, which was similar to that of H+C and C+H (Table 2). Maximum root length was noted in seeds treated with H+C+H, followed by C+H+C. Minimum root length was noted in untreated seeds (Table 2). The smallest shoots were observed in seeds treated with H+C, which did not differ from control, and H+C+H (Table 2). The longTable 2

est shoots were noted in seeds subjected to C+H+C. The maximum root/shoot ratio was for seeds treated with H+C+H, followed by control and H+C; however, a minimum root/shoot ratio was noted for seeds treated with C+H+C (Table 2). Maximum seedling fresh and dry weights were obtained for seeds treated with H+C+H. Minimum seedling fresh and dry weights were noted for untreated seeds, which did not differ from seeds subjected to H+C in both cases and C+H in the case of seedling fresh weight (Table 2). In japonica rice, minimum MET was noted in seeds subjected to C+H+C, whereas maximum MET was noted in seeds subjected to H+C. Maximum emergence was recorded in untreated seeds, which did not differ from seeds subjected to C+H+C and C+H. Minimum FEP was observed in seeds subjected to heating followed by chilling (Table 2). Maximum root length was observed in untreated seeds; all treatments resulted in a lower root length compared with control, which was least for seeds treated with H+C+H (Table 2). The longest shoots were observed in seeds subjected to C+H+C, followed by control, H+C and C+H (Table 2). The highest root/shoot ratio was noted in untreated seeds; all seed treatments resulted in a lower root/shoot ratio than that of control, being minimum in seeds treated with H+C (Table 2). Maximum seedling fresh and dry weights were noted in seeds treated with C+H+C. Mini-

Effect of seed treatments on the seedling vigor of japonica and indica rice

MET FEP Root length Shoot length Root/Shoot Seedling fresh Seedling dry (d) (%) (mm) (mm) ratio weight (mg) weight (mg) Indica rice Control 6.51 a 55.42 c 45.05 d 30.88 c 1.46 b 111.25 c 90.75 c H+C+H 5.69 b 77.00 b 72.58 a 38.30 c 1.89 a 165.50 a 153.25 a C+H+C 3.99 d 88.19 a 63.64 b 75.17 a 0.84 d 145.71 b 127.50 b H+C 3.37 d 90.25 a 52.60 c 36.01 c 1.46 b 117.50 c 100.75 c C+H 4.69 c 89.45 a 56.92 c 62.52 b 0.91 c 121.50 c 127.50 b LSD at 0.05 0.66 9.53 7.31 11.21 0.013 11.23 13.45 Japonica rice Control 6.26 b 89.47 a 85.64 a 64.98 b 1.31 a 162.50 c 101.25 d H+C+H 4.06 d 81.23 a 65.82 b 77.82 a 0.84 c 187.75 a 167.50 a C+H+C 4.06 d 81.23 a 65.82 b 77.82 a 0.84 c 187.75 a 167.50 a H+C 6.34 a 59.24 c 50.15 bc 68.27 b 0.73 d 162.75 c 154.25 b C+H 5.83 c 79.11 a 52.42 b 62.87 b 0.83 c 176.25 b 115.50 c LSD at 0.05 0.07 12.50 14.32 13.64 0.012 5.03 5.42 Figures not sharing the same letters differ significantly at P = 0.05. H+C+H = Heating followed by chilling followed by heating; H+C = Heating followed by chilling; C+H+C = Chilling followed by heating followed by chilling; C+H = Chilling followed by heating. FEP, final emergence percentage; MET, mean emergence time. Treatments

Muhammad FAROOQ et al.: Thermal Hardening: A New Seed Vigor Enhancement Tool in Rice

mum fresh and dry weights were recorded in untreated seeds, which did not differ from H+C and H+C+H in the case of seedling fresh weight (Table 2). 2.3 Electrical conductivity of seed leachates In indica rice, the highest EC of seed leachates was noted in seeds treated with C+H+C, followed by H+C, followed by control. Minimum EC of seed leachates was noted in seeds subjected to H+C+H (Fig. 1). In japonica rice, the highest EC of seed leachates was recorded in untreated seeds. All seed treatments resulted in a lower EC of seed leachates compared with control (Fig. 2); however, the minimum EC of seed

Fig. 1. Effect of seed treatments on the electrical conductivity of seed leachates (µs.cm−1.g−1) in indica rice. Data are the mean ± SE. H+C+H, heating followed by chilling followed by heating; H+C, heating followed by chilling; C+H+C, chilling followed by heating followed by chilling; C+H, chilling followed by heating.

Fig. 2. Effect of seed treatments on the electrical conductivity of seed leachates (µs.cm−1.g−1) in japonica rice. Data are the mean ± SE. H+C+H, heating followed by chilling followed by heating; H+C, heating followed by chilling; C+H+C, chilling followed by heating followed by chilling; C+H, chilling followed by heating.

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leachates was noted in seeds subjected to C+H+C.

3

Discussion

Thermal hardening had a significant effect on the germination and seedling vigor of both indica and japonica rice seeds (Tables 1, 2; Figs. 1, 2). The responses of both types of rice to different thermal hardening treatments were not similar (Tables 1, 2). In indica rice, earlier and synchronized germination was observed in treated seeds compared with control seeds, as evidenced by a lower time to start germination, MET, T50, and MGT, and a higher GI, GE, FEP, lengths of plumule, root, and shoot, as well as seedling fresh and dry weight (Tables 1, 2). Germination percentage and radicle length remained unaffected, but higher germination energy was recorded in treated seeds. Ruan et al. (2002) have also reported that seed treatments were not able to invigorate rice seeds, but resulted in a higher energy of germination and germination index compared with untreated seeds. Highest invigoration was observed in seeds subjected to H+C+H. The beneficial aspects of seed hardening are primarily due to pre-enlargement of the embryo (Austin et al. 1969), improvement of the germination rate (Gray and Steckle 1977), and effects that are attributed to alternating wetting and drying processes (Basra et al. 2003). Higher enhancement was noted in seeds subjected to dry heat treatment first compared with seeds subjected to chilling first (Tables 1, 2). Farooq et al. (2004) have reported vigor enhancement in indica rice seeds subjected to dry heat treatment. The highest EC of seed leachates was recorded in seeds subjected to C+H+C (Fig. 1), which may be the result of membrane rupture during the chilling process in indica rice seeds subjected to chilling only (Farooq et al. 2004). Minimum EC of seed leachates was noted in seeds subjected to H+C+H (Fig. 1). Earlier, Farooq et al. (2004) reported that indica rice seeds subjected to dry heat treatment showed a lower EC of seed leachates compared with control. In japonica rice, enhanced vigor was noted in seeds subjected to C+H+C compared with other treatments,

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including control, as shown by lower values of MET, MGT, time to start germination, and T50 and higher values of GI, GE, radicle length FEP, shoot length, and seedling fresh and dry weight (Tables 1, 2). Earlier, Farooq et al. (2004) reported enhanced vigor in japonica seeds subjected to low temperature treatment. The germination percentage remained unaffected; however, higher germination energy was recorded in treated seeds. Ruan et al. (2002) have also reported that seed treatments were not able to invigorate rice seeds, but resulted in a higher energy of germination and germination index compared with untreated seeds. Seeds subjected to H+C exhibited higher values of the time to start germination, even more than that of control, and similar reports have been published by Farooq et al. (2004), who stated that japonica rice seeds exposed to dry heat treatments results in delayed germination. The improved performance of seeds subjected to alternate low and high temperatures may be the result of pre-enlargement of the embryo (Austin et al. 1969) and improvement of the germination rate (Gray and Steckle 1977), which are attributed to alternating wetting and drying processed (Basra et al. 2003). The vigor enhancement as a result of alternating low and high temperatures may also be due to the hardening process, as observed in alternate wetting and drying. The highest EC of seed leachates was recorded for untreated seeds (Fig. 1); all seed treatments resulted in a lower EC of seed leachates compared with control. Minimum EC of seed leachates was noted in seeds subjected to C+H+C (Fig. 2). The reduced EC of seed leachates from treated seeds may be the result of better membrane repair, as was reported by Basra et al. (2002, 2003) in wheat and indica rice and by Rudrapal and Nakamura (1988) in radish and eggplant. Both types of rice responsed differently to different thermal hardening treatments. This indicates a genetic difference between the two rice types, as Lee et al. (2002) also observed that different rice cultivars behaved differently to dry heat treatments. From the present investigation, it may be concluded that, like alternate wetting and drying (seed hardening), alternate low and high temperature treatments may also

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