Physiological and biochemical changes in naturally ... - IngentaConnect

NATURAL AND ARTIFICIAL AGING OF COTTON SEEDS

Freitas, R.A., Dias, D.C.F.S., Oliveira, M.G.A., Dias, L.A.S. and José, I.C. (2006), Seed Sci. & Technol., 34, 253-264

Physiological and biochemical changes in naturally and artificially aged cotton seeds R.A. FREITAS, D.C.F.S. DIAS*, M.G.A. OLIVEIRA, L.A.S. DIAS AND I.C. JOSÉ Department of Fitotecnia, University Federal of Viçosa, 36571-000, Viçosa, MG, Brazil (E-mail: [email protected])

(Accepted September 2005)

Summary This study was done to determine the physiological and biochemical changes occurring in cotton seed during natural and artificial aging. Cotton seeds cv. IAC-20 RR were stored for 12 months under environmental condition and in a cold room (10 ± 2°C). The artificial aging was induced by storing seeds for 0, 24, 48, 72, 96 and 120 hours, in an incubator at 42°C and 100% RH. Physiological quality of seeds (germination, accelerated aging and cool germination tests) and biochemical changes (lipoxygenase and acid phosphatase activity and lipid content) were determined after each artificial aging period and at two month interval for stored seeds. The viability, vigour, lipoxygenase and acid phosphatase activity and lipid content of cotton seeds decreased with the increasing artificial or natural aging, especially in seeds stored under environmental condition. Cool germination test, lipid content, acid phosphatase and lipoxygenase activities were good parameters for detecting deterioration of naturally or artificially aged seed lots.

Introduction Seed deterioration is a natural phenomenon that occurs in all seeds and leading to gradual decline of seed viability during storage. The process is most pronounced in oil-rich seeds, such as cotton, because of high susceptibility to peroxidation of polyunsaturated fatty acids present in these seeds (Priestley and Leopold, 1983). Accelerated aging test has been recognized as a good predictor of seed storability. Therefore, the mechanisms of seed deterioration have been mostly studied by manipulating the aging rate by exposing the seeds to high temperature and high relative humidity, as normally used in the accelerated aging test (Priestley et al., 1980; Sun and Leopold, 1995, Sung, 1996, Machado Neto et al., 2001 and Basra et al., 2003). Although, in several studies this test has been used to elucidate the events leading to deterioration, there is still no agreement as to whether the alterations caused by accelerated aging are similar to those of natural aging. According to Priestley and Leopold (1983), accelerated aging, although reduces seed vigour, it also induces alterations other than those observed in natural aging. Powell and * Author for correspondence

253

R.A. FREITAS, D.C.F.S. DIAS, M.G.A. OLIVEIRA, L.A.S. DIAS AND I.C. JOSÉ

Harman (1985) also doubt the belief that physiological events occurring during accelerated aging reflect those happening during natural aging. Artificially aged rice seeds showed higher activity of the acid phosphatase than those aged naturally or not aged (Rajagopal and Sen-Mandi, 1992). During natural and accelerated aging of seeds of several species, Likhatchev et al. (1984) found reduced concentrations of soluble sugars, accompanied by an increase in phytin hydrolysis and changes in the electrophoretic mobility of specific proteins. The authors concluded that the biochemical changes during accelerated aging were similar to those of natural aging, varying only the occurrence rate. Sung (1996) reported that both natural and accelerated aging enhanced lipid peroxidation and reduced seed germination; aging also inhibited the activity of superoxide dismutase, catalase, ascorbate oxidase and peroxidase. Machado Neto et al. (2001), in common bean seeds, found that electrophoretic patterns of total proteins changed after 72 hours of artificial aging, while in naturally aged seeds the alterations were detected only after two-year storage at 15°C and suggested that physiological parameters, such as germination and vigour, were more sensitive to monitor deterioration of naturally aged seeds, while electrophoretic profiles were more suitable for detecting deterioration of artificially aged seeds (41°C/100%RH). Accelerated aging can denature enzymes to different extent or affect their synthesis as reported by Bailly et al. (1996) who found a clear relation between the activity loss of scavenging enzymes, lipid peroxidation and deterioration of sunflower seeds during accelerated aging. Basavarajappa et al. (1991) reported that in aging maize seeds while the total protein content decreased, free amino acid concentration increased, which indicates protein degradation by proteases. There are several reports suggesting that seed polyunsaturated fatty acids are highly susceptible to non-enzymatic peroxidation in the presence of oxygen (Sung and Jeng, 1994; Trawatha et al., 1995). Thus, there is a general belief that lipid peroxidation is the basic cause of seed deterioration and the major changes related to this process are depletion of lipid reserves resulting in production of free fatty acids (Trawatha et al., 1995; Basra et al., 2003), enzyme degradation and inactivation (Sung and Jeng, 1994; Bailly et al., 1996; Hsu et al., 2003) and loss of membrane integrity (Sung, 1996; Basra et al., 2003). On the other hand, lipoxygenase, present in many unimbibed seeds, is also capable of catalyzing lipid peroxidation by using membrane phospholipids components as substrates (Wang et al., 1990). Deterioration process also affects the activity of several peroxide-scavenging enzymes such as catalase, superoxide dismutase, peroxidase and acid phosphatase (Sung and Jeng, 1994; Sung, 1996; Bailly et al., 1996; Spinola et al., 2000; Hsu et al., 2003). Acid phosphatase catalyzes monoesters phosphate hydrolysis (Aoyana et al., 2001) and can be related to lipid peroxidation (Spinola et al., 2000). According to these authors, estimation of this enzyme was more sensitive than vigour tests to monitor maize seed deterioration during accelerated aging. Oleaginous seeds, such as cotton seeds, have greater propensity to deleterious changes in the deterioration process, but the studies with this species are rare. Reduced germination of cotton seeds accompanied by increased solute leaching, free fatty acid content, lipid peroxidation and increased mean time for seedling emergence after exposure to different periods of accelerated aging has been reported (Basra et al., 2003). 254


The objective of this study is to compare biochemical and physiological alterations in cotton seeds after natural aging due to storage and artificial aging induced by the accelerated aging process. Materials and methods The study was done in the Laboratories of Seeds and Enzymology of the Federal University of Viçosa, Brazil. For natural aging, the cotton seeds (Gossypium hirsutum L.) cv IAC-20 RR were stored in paper bags for 12 months in a cold room at 10 ± 2°C and 70 ± 5% relative humidity or in environmental laboratory conditions. The physiological quality and biochemical parameters were assayed every two months of storage. The artificial aging was induced in a climatic chamber in conditions recommended for accelerated aging test (AOSA, 1983). The seeds were distributed in a single layer onto a wire mesh screen and suspended over 40 ml of water inside a plastic germination box (11 × 11 × 4 cm) and incubated at 42 ± 0.5°C and 100% RH. Evaluations for physiological and biochemical parameters were done every 24 h for a total of 120 h of ageing. Standard germination: Eight sub-samples of 25 seeds each were planted in rolled paper towels moistened with amount of water equivalent to 2.5× the weight of the dry paper and allowed to germinated at 25 ± 2°C. The germinated seeds were counted four and seven days after seeding, following criteria established by the Rules for Seed Testing (Brasil, 1992). The results were expressed as mean percentage of normal seedling. Accelerated aging: The seeds were placed on a wire mesh screen and suspended over 40 ml of water inside a plastic germination box (AOSA, 1983). The boxes were held in an incubator at 42 ± 0.5°C. After 72 h, four sub-samples of 50 seeds each were tested for germination as previously described. The evaluation was made four days after seeding and the mean normal seedling percentage was calculated. Cool germination: The method recommended by AOSA (1983) was adopted. Four subsamples of 50 seeds were seeded in rolled paper towels as described for the standard germination test. The rolls with seeds were placed in polyethylene bags and incubated for eight days at 18°C in dark and the percentage of normal seedlings with total length of 4 cm or longer was determined. Lipoxygenase activity: The seeds were ground in sodium phosphate buffer (50 mM, pH 6.5), in proportion of 1:10 (w/v) using a chilled mortar and pestle and the suspension was centrifuged at 17,200 × g for 30 min, at 4°C. The supernatant was collected to determine lipoxygenase activity and protein concentration. The protein concentration was determined by the bicinchoninic acid method (Smith et al., 1985) and the lipoxygenase activity was measured spectrophotometrically using linoleic acid as substrate (Axelrod et al., 1981). The absorbance at 234 nm was measured at 30-s intervals for a period of 120 seconds. The reaction mixture without seed extract was used as blank. The results were expressed as mM.s-1/mg of protein. 255


Acid phosphatase activity: The seeds were ground in potassium acetate buffer (0.1 M, pH 5.0), in the proportion 1:10 (w/v) using a chilled mortar and pestle and the suspension was centrifuged at 24,700 × g for 30 min, at 4°C. The supernatant was collected to determine acid phosphatase activity (Maia et al., 2000). Fifty μl of the supernatant was added to a mixture containing 100 μl of the 0.018 M substrate p-nitrophenyl phosphate and 800 μl of potassium acetate buffer (0.1 M, pH 5.0) in a tube and after five-minute incubation at 30°C one ml of 0.5 M sodium hydroxide was added and the absorbance was measured at 400 nm. The blanks consisted of one ml of the buffer solution and two ml distilled water. Results were expressed as absorbance units at 400 nm. min-1. mg-1 of seed. Lipid content: Total lipids were extracted from three gram of seed powder using petroleum ether as solvent in a Soxhlet extractor, refluxing for 24 hours (Instituto Adolfo Lutz, 1985). The results are expressed as percentage of lipid content of seed. All biochemical determinations were carried out in triplicates. The study was done using completely randomized design and the data were analyzed as a split-plot experiment, with four replications. In natural aging experiment, the storage condition (environmental and cold room) was allocated to plots and the sampling period to subplots and latter was subjected to regression analysis. The data of artificial aging were also subjected to regression analysis. The degree of agreement between the results of physiological quality tests and biochemical assays was calculated for each period of natural and artificial aging using the generalized distances procedure of Mahalanobis (1936), as follows: Considering Xijk as the observation on the jth cultivar in the ith test and in the kth replication, the means [Xij = (Xij/k)], the variance and residual covariance matrix and the deviations (dj) were estimated. The deviations were obtained by, d1= Xi1- Xi'1; d2= Xi2- Xi'2; d4= Xijk/K; Thus, Dii'2= δ' Ψ-1 δ, where, Dii'2 is the Mahalanobis generalized distance between the ith tests results obtained after artificial aging periods and the ith tests results observed after the ith storage periods; δ'= [d1 d2 ]; and Ψ= residual variance and covariance matrix. To calculate Mahalanobis distances among test means, the CANDISC procedure (SAS, 1989) was used. The Fs and probability values associated with each distance value were also performed to test the hypothesis that the pair wise distance values are zero. The value of Mahalanobis distance between tests must be low and not significant to characterize similarity between them. Thus, the less significant the distance, higher is the similarity between results of artificial aging and storage periods. 256


Results and discussion

(a)

% Normal seedlings

The germination percentage of seeds stored in cold room did not change during the 12month period, but declined gradually over time in seeds stored in the environmental conditions (figure 1a). The seed germination also decreased when the seeds were subjected to accelerated aging (figure 1b). Machado Neto et al. (2001) also reported reduced germination and vigour of naturally or artificially aged bean seeds. The increase of artificial aging period had a deleterious effect on cotton seeds germination (Basra et al., 2003). 100 90 80 70

CR (Y=90,96-0,16714nsX) r2=0,4366 Env (Y=92,41-1,86384**X) r2=0,8511

60 50 0

2

4

6

8

10

12

Storage period (months) (b) % Normal seedlings

100 90 80 70 Y=95,52-0,276786**X

60

2

r =0,9120

50 0

24

48

72

96

120

Aging period (hour)

Figure 1. Estimative of normal seedlings (%) obtained in the standard germination test during storage (a) in a cool room (CR) and environmental condition (Env) or after artifical aging (b) of cotton seeds, cv. IAC-20RR.

Table 1 shows the generalized Mahalanobis distances between germination percentage of seeds after different natural and artificial aging periods. The initial germination of naturally aged seeds was similar to that of seeds without artificial aging or artificially aged for 24 h. The Mahalanobis distance between germination percentage of seeds artificially aged for 48 h and those stored for 2 months under the environmental conditions was not significant, but significant when compared with germination percentage after four and six-month storage. Germination percentage of seeds after 10-month storage under environmental conditions was similar to that of seeds artificially aged for 96 h. The Mahalanobis distances between germination of 0, 24 or 48 h of artificial ageing and all the storage periods in the cold room were nonsignificant, indicating that seed deterioration in cold room was similar to that caused by up to 48 h artificial aging. 257


Table 1. Mahalanobis’s distances between results of standard germination tests performed after different storage periods (environmental condition and cool room) and after different artificial aging periods of cotton seeds, cv. IAC-20RR. Storage conditions

Environmental

Cool room

Storage periods (months)

Artificial aging periods (hours) 0

24

48

72

96

120

0

.0 ns

0.7 ns

4.1*

19.2**

47.6**

2

7.8**

4.0*

2.2 ns

13.0**

22.9**

88.8**

4

3.6**

1.3 ns

1.1 ns

13.4**

29.2**

102.0**

6

4.4*

1.5 ns

0.3 ns

9.3**

24.2**

92.8**

135.0**

8

13.9**

8.2**

3.1*

5.8**

10.9**

64.1**

10

34.7**

25.6**

15.0**

6.2**

1.0 ns

32.8**

12

75.2**

61.3**

44.2**

24.1**

3.1*

8.7**

0

.0 ns

0.7 ns

4.1*

19.2**

47.6**

2

0.8 ns

0.1 ns

2.1 ns

16.4**

39.1**

120.4**

4

0.2 ns

0.5 ns

3.5*

19.3**

45.5**

131.4**

6

0.6 ns

0.5 ns

3.3*

19.6**

43.7**

128.2**

8

ns

0.1 ns

1.8 ns

15.5**

37.9**

118.4**

10

1.2 ns

0.1 ns

1.1 ns

13.53**

34.9**

112.9**

12

0.6 ns

0.1 ns

2.2 ns

16.6**

40.1**

122.1**

0.8

** , * Significant at a probability level of 0.01 and 0.05, respectively by the F test;

ns

135.0**

no significant

The adjusted curves, corresponding to cool germination percentage of seeds stored for different periods and aged artificially (figure 2a and 2b), showed that seed vigour decreased linearly with increasing periods of storage and artificial aging. There was a slighter decline in the vigour of seeds stored in cold room compared to those stored under environmental conditions. The efficiency of cool germination test to evaluate vigour of cotton seeds has been shown by Dias and Alvarenga (1999) and was considered most efficient test for monitoring the vigour of stored seeds (Freitas et al., 2000). The cool germination test best defined the relationship between storage periods under environmental conditions and artificial aging time (table 2). The cool germination of seeds stored for 10 or 12 months correlated with that of seeds artificially aged for 120 h. Cool germination of seeds artificially aged for 48, 72 or 96 hours was similar to that of seeds stored for 4, 6 or 8 months, respectively, under environmental conditions, suggesting that there is correspondence between cool germination percentage of naturally and artificially aged seeds. However, this correspondence was less evident for seeds stored in the cold room, where only significant correspondence occurred between seeds stored for 12 months and artificially aged for 48 h. Seeds stored in cold room had higher lipoxygenase activity than those stored in environmental condition and its activity decreased linearly with time. It should be pointed out that cold room storage was beneficial to seed quality (figures 1a and 2a), therefore, the higher lipoxygenase activity in these seeds could not be associated with the deterioration process, as reported for soybean seed (Trawatha et al., 1995). Lipoxygenase is present 258


(a) % Cool germination

80 70 60 50 40 30

2

CR (Y=70,25-1,10714**X)

r =0,8334

Env (Y=73,12-3,62054**X)

r =0,9663

2

20 0

2

4

6

8

10

12

Storage period (months) 80

% Cool germination

(b)

70 60 50 40 Y=74,49-0,326191**X

2

r =9407

30 20 0

24

48 72 Aging period (hour)

96

120

Figure 2. Estimative of normal seedlings (%) obtained in the cool germination test during storage (a) in a cool room (CR) and environmental condition (Env) or after artificial aging (b) of cotton seeds, cv. IAC-20RR. Table 2. Mahalanobis’s distances between results of cool germination tests performed after different storage periods (environmental condition and cool room) and after different artificial aging periods of cotton seeds, cv. IAC-20RR. Storage conditions

Environmental

Cool room

Storage periods (months)

Artificial aging periods (hours) 0

24

48

72

96

120

0

.0 ns

0.2 ns

3.7*

51.4**

82.7**

147.1**

2

1.2 ns

0.1 ns

1.9 ns

37.8**

65.6**

124.1**

4

6.7**

5.7**

2.4 ns

21.5**

43.7**

93.1**

6

29.9**

26.5**

14.4**

3.0 ns

13.1**

44.4**

ns

8

102.5**

94.9**

68.5**

11.8**

2.6

10

137.7**

130.7**

101.4**

20.9**

7.9**

2.0 ns

5.5**

12

172.8**

163.8**

129.2**

36.8**

16.5**

1.0 ns

0

.0 ns

0.2 ns

3.7*

51.4**

82.7**

147.1**

2

0.1 ns

0.4 ns

3.9*

50.0**

81.3**

145.4**

4

0.7 ns

0.6 ns

1.8 ns

39.9**

68.2**

127.6**

6

9.8**

8.6**

4.0*

17.1**

37.6**

84.1**

8

15.9**

14.3**

7.5**

11.1**

28.5**

70.1**

10

13.8**

11.3**

4.0*

12.4**

29.0**

70.9**

12

12.5**

10.3**

3.6 ns

13.4**

30.9**

73.8**

** , * Significant at a probability level of 0.01 and 0.05, respectively by the F test;

ns

no significant

259


(a)

Lipoxygenases specific activity (mMs -1/mg protein)

in many unimbibed seeds, capable of catalyzing lipid peroxidation by using membrane phospholipids as substrate (Priestley and Leopold, 1983). The physiological role of this enzyme is not well understood and there is contradictory view about its role in the aging process. Some reports suggest that lipoxygenase and its hydroperoxide products might participate in seed aging, whereas other reports argue against such role (Vernooy-Gerritsen et al., 1983). A concomitant decrease in this enzyme activity occurred with increasing of storage and artificial aging (figure 3a and 3b). Lipoxygenase could promote slow lipid peroxidation, which is accompanied by the formation of activated oxygen, especially at levels of hydration that are far below those normally encountered in stored seeds (Priestley and Leopold, 1983). Sung and Jeng (1994) argued that lipoxygenase seems to have no role in peanut seed aging per se, because its activity declined rapidly with the prolonged aging treatment. According to Kalpana et al. (1993) lipid peroxidation is independent of lipoxygenase activity, because the process is self-propagated. Most Mahalanobis distances (data not shown) were not significant for lipoxygenase activity in seeds aged naturally or artificially, indicating similarity between the two aging processes, regardless of aging period, although the precise correspondence between a specific artificial ageing period and natural storage period could not be determined. The seeds stored in the cold room had higher acid phosphatase activity compared to seeds stored in environmental condition (figure 4) where physiological quality declined expressively (figures 1a and 2a). There was a linear decrease of this enzyme activity 14 12 10 8 6

2

CR (Y=12,88-0,384685**X) r =0,9697 2 Env (Y=12,31-0,683722**X) r =0,9881

4 2 0 0

2

4

6

8

10

12

(b)

Lipoxygenases specific activity -1 (mMs /mg protein)

Storage period (months)

16 14 12 10 8 6 4 2 0

Y=12,53-0,043872

0

24

48

2

r =0,9736

72

96

120

Aging period (hour)

Figure 3. Estimative of lipoxygenase activity (mM.s-1/mg protein) determined during storage (a) in a cool room (CR) and environmental condition (Env) or after artificial aging (b) of cotton seeds, cv. IAC-20RR.

260


90

Acid phosphatase activity -1 (A400.min /m gseed)

(a)

80 70 60 50

CR (Y=75-1,35973**X) Env (Y=75-2,07774**X)

2

r =0,9470 2 r =0,9757

40 30

(b)

Acid phosphatase activity -1 (A400.min /mg seed)

0

2

4 6 8 Storage period (months)

10

12

90 80 70 60 50

Y=81,64-0,152738**X

40

2

r =0,9919

30 0

24

48

72

96

120

Aging period (hour)

Figure 4. Estimative of acid phosphatase activity (A400 nm. min-1. mg seed-1) determined during storage (a) in a cool room (CR) and environmental condition (Env) or after artificial aging (b) of cotton seeds, cv. IAC-20RR.

during artificial aging. These results suggest a positive relationship between reduced acid phosphatase activity and seed vigour. This enzyme can promote hydrolysis of esters leading to membrane lipid peroxidation (Spinola et al., 2000). The Mahalanobis distances for acid phosphatase activity (data nor shown) in seeds stored in environmental condition followed the tendencies similar to those of cool germination test. With the increase of storage and artificial aging periods distances remained nonsignificant, suggesting good agreement among results. Association between reduced acid phosphatase activity and reduced seed vigour have been reported for other seeds (Vieira, 1996; Spinola et al., 2000). Total seed lipid content declined with increasing storage and artificial aging periods (figure 5a and 5b). These results may be due to lipid peroxidation, which is considered as one of the major cause of seed deterioration (Priestley and Leopold, 1983). Lipid oxidation produces highly reactive free radicals intermediates, lipid hydroperoxides and a variety of secondary products, which potentially can damage membranes, enzymes and nucleic acids. The Mahalanobis distances between lipid contents after each storage and artificial aging period were nonsignificant, suggesting an association among all the storage and artificial aging periods. However, from these results it was not possible to establish a consistent relationship between changes in the lipid content during artificial and natural aging. The mechanisms of seed aging have been widely studied by manipulating the aging rate by exposing the seeds to conditions similar to accelerated aging (Sun and Leopold, 261


(a)

30

Env (Y=25,1024-0,175235**X) r2=0,4944

% Lipid

28

CR (Y=25,0345-0,155929**X) r2=0,5501 26 24 22 20 0

2

4

6

8

10

12

Storage period (months) (b)

30

% Lipid

28 26 24 2

22

Y=25,20-0,009019**X r =0,8331

20 0

24

48

72

96

120

Aging period (hour)

Figure 5. Estimative of lipid content (%) determined during storage (a) in a cool room (CR) and environmental condition (Env) or after artificial aging (b) of cotton seeds, cv. IAC-20RR.

1995, Sung, 1996, Machado Neto et al., 2001; Basra et al., 2003). However, Priestley and Leopold (1983) found that accelerated seed aging promotes alterations other than those occurring during natural aging. Powell and Harman (1985) also doubt whether the physiological changes induced by accelerated aging are similar to those occurring during natural aging. The data from this study show that in general, activities of lipoxygenase and acid phosphatase and total lipid content (figures 3, 4 and 5, respectively) decrease with increasing natural (storage) and artificial aging period. Lipoxygenase activity in sunflower (Leoni et al., 1985) and soybean (Lanna et al., 1996) seeds was lost during storage at low temperatures. Vieira (1996) reported that electrophoretic variation in the acid phosphatase can be a promising parameter to evaluate cotton seed deterioration level. Spinola et al. (2000) reported alterations in the isoenzymatic profile of acid phosphatase and peroxidase in maize seeds artificially aged for 72 hours, which may be due to deteriorative effect of seed exposure to high temperature and high relative humidity. Physiological seed quality tests did not prove sensitive enough to detect the deteriorative process induced by artificial aging and, which were detected only in longer aging periods. In naturally aged bean seeds, the physiological quality tests were more sensitive than the electrophoretic analyses to monitor seed deterioration, while in the artificially aged seeds electrophoretic profiles were more efficient. These results suggested different protein degradation patterns in artificial and natural aging, probably driven by different 262


physiological mechanisms (Machado Neto et al., 2001). Basavarajappa et al. (1991), Varier and Dadlani (1992) and Vieira (1996) found higher number of bands in the electrophoretic profile of proteins of artificially aged seeds compared to non-aged seeds. In contrast, Spinola et al. (2000) reported that isoenzyme electrophoretical patterns of acid phosphatase and peroxidase were more sensitive than the physiological tests to detect deterioration of maize seeds during artificial aging. The results from study show that physiological quality, lipid content, activities of lipoxygenase and acid phosphatase of cotton seeds decreased with increasing artificial aging and storage time in environmental condition. However, a relationship between changes in lipid content, lipoxygenase activity and seed deterioration during storage and artificial aging could not be established as indicated by Mahalanobis distances. Cool germination test, lipid content, acid phosphatase and lipoxygenase activities were good parameters for detecting deterioration of naturally or artificially aged seed lots. Acknowledgements To the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” for financial support. References Aoyama, H., Cavagis, A.D.M., Taga, E.M. and Ferreira, C.V. (2001). Endogenous lectin as a possible regulator of the hydrolysis of physiological substrates by soybean seed acid phosphatase. Phytochemistry, 58, 221– 225. Association of Official Seed Analysts. (1983). Seed vigour testing handbook. East lasing, AOSA, 88pp. (Contribution, 32). Axelrod, B., Cheesbrough, T.M. and Laasko, S. (1981). Lipoxygenases from soybeans. Methods in Enzymology, 71, 441–451. Bailly, C., Benamar, A., Corbineau, F. and Côme, D. (1996). Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging. Physiologia Plantarum, 97, 104–110. Basavarajappa, B.S., Shetty, H.S. and Prakash, H.S. (1991). Membrane deterioration and other biochemical changes associated with accelerated ageing of maize seeds. Seed Science and Technology, 19, 279–286. Basra, S.M.A., Ahmad, N., Khan, M.M., Iqbal, N. and Cheema, M.A. (2003). Assesment of cotton seed deterioration during accelerated ageing. Seed Science and Technolology, 31, 531–540. BRASIL. (1992). Ministério da Agricultura. Regras para análise de sementes. [Rules for testing seeds]. 365pp, Brasília. Dias, D.C.F.S. and Alvarenga, E.M. (1999). Teste de germinação a baixa temperatura. In Vigour de sementes: conceitos e testes. [Seed vigour: concepts and tests], (eds. Krzyzanowski, F.C., Vieira, R.D., França Neto, J.B.). Londrina: ABRATES. chapter 7. Freitas, R.A, Dias, D.C.F.S., Cecon, P.R. and Reis, M.S. (2000). Qualidade fisiológica e sanitária de sementes de algodão durante o armazenamento. [Physiological and sanitary quality of cotton seeds during storage]. Revista Brasileira de Sementes, 22, 94–101. Hsu, C.C., Chen, C.L., Chen, J.J. and Sung, J.M. (2003). Accelerated aging-enhanced lipid peroxidation in bitter gourd seeds and effects of priming and hot water soaking treatments. Scientia Horticulturae, 98, 201–212. Instituto Adolfo Lutz. (1985). Normas analíticas do Instituto Adolfo Lutz; métodos químicos e físicos para análise de alimentos [Analytical rules of Adolfo Lutz Institut]. Vol.1, 3rd. ed. São Paulo, 533p. Kalpana, R. and Madhava Rao, K.V. (1993). Lowered lipoxygenase activity in seeds of pigeonpea Cajanus cajan L. Millsp. cultivars during accelerated ageing. Seed Science and Technology, 21, 269–272.

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