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J.B. ZONTA, E.F. ARAJO, R.F. ARAJO, M.S. REIS AND J.

DA SILVA LIMA

Zonta, J.B., Arajo, E.F., Arajo, R.F., Reis, M.S. and da Silva Lima, J. (2009), Seed Sci. & Technol., 37, 140-146

LERCAF test for the assessment of coffee seed quality during storage
J.B. ZONTA1, E.F. ARAJO1, R.F. ARAJO2, M.S. REIS1 AND J. DA SILVA LIMA1
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Universidade Federal de Viosa, Avenida Peter Henry Rolfs, s/n Campus Universitrio 36570-000 Viosa, MG (E-mail: jobazonta@hotmail.com; efaraujo@ufv.br; msreis@ufv.br; julienslima@yahoo.com.br) 2 EPAMIG, Vila Gianneti, n47 Campus Universitrio 36570-000 Viosa, MG (E-mail: rfaraujo@ufv.br)

(Accepted October 2008)

Summary
The objective of the present work was to evaluate the use of the LERCAF test for estimating germination and characterizing deterioration of coffee seeds during storage. The experiment was conducted at the Seed Research Laboratory of the Federal University of Viosa. Seeds of Arabic coffee, with 33, 28, 23, 18 and 13% moisture (wet basis) were stored in waterproof bags at 203C. Seeds were assessed by the germination and LERCAF tests. The tests were carried out at 4 storage times: zero, two, four and six months. There was decrease in germination during the storage for all the studied moisture contents. The LERCAF test had larger percentages of seeds with clear endosperm when evaluated before storage (high germinative capacity) and larger percentages of seeds with dark endosperm (brown or black) at the six months of storage (low germinative capacity). Germination estimates by the LERCAFE test had high correlation with the results of the germination test. The LERCAF test was shown to be efcient to estimate germination of coffee seeds during storage, as well as to characterize deterioration caused by the natural aging of seeds.

Introduction Coffee is most commonly propagated from seed (Afonso Junior et al., 2006). Coffee seeds have, however, a number of problems, such as slow and non-uniform germination. The assessment of coffee seed quality using the germination test requires at least 30 days for the nal count (Brasil, 1992), which slows research and trade. Moreover, it is a laborious test to perform because seed parchment must be removed manually. The slow and non-uniform seed germination is also a great obstacle to production of quality seedlings, making it difcult the establishment of the crop in the eld (Sguarezi et al., 2001). Another problem is the possible occurrence of situations in which the germination test, being too long, may generate results conicting with the actual physiological quality of seeds at the time of presenting the results (Dias and Silva, 1986). Coffee seeds are physiologically characterized as having fast viability losses during storage (Silva and Dias, 1985). Studies showed that coffee seeds, with 15 and 22% moisture content (wet basis) in burlap bags (Vasconcelos et al., 1992) after four months of storage, and with approximately 51% moisture (wet basis), in muslin bags at the fth month (Gentil et al., 2001) lost their germinative capacity.
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LERCAF TEST FOR COFFEE SEED QUALITY DURING STORAGE

The development of fast and non-expensive tests of easy implementation and evaluation is fundamental, since fast quality assessments of seed batches favour not only the seed industry but also nurseries and coffee farmers, as the early release of a seed batch to the market enables nurseries to produce seedlings at a better time and consequently farmers will have more vigorous seedlings available and produce plants with higher eld establishment capacity. This demand has generated new tests, such as tetrazolium test (Dias and Silva, 1998), individual electrical conductivity test (Costa and Carvalho, 2006), visual assessment of exudates (Sera et al., 2006) and visual assessment of shape and colour of embryo (Sera and Miglioranza, 2003). But even being fast, these tests are laborious, expensive and demand highly specialized workers. In view of these problems, the LERCAF test was developed (Reis, 2004) with major advantages as reduction in time and labour for assessing coffee seeds, enabling a fast evaluation of seed quality, as well as low cost and low demand for labour force. The LERCAF test consists of using sodium hypochlorite (NaClO) for a fast assessment of coffee seed quality, in addition to new technologies for agricultural sciences, particularly in the eld of seed technology. Fast assessment is possible because sodium hypochlorite, in determined concentrations, reacts at dead or injured areas of the coffee seed endosperm, giving the tissue, in these conditions, a green colour. If this occurs in a large area of the endosperm or a region on and/or around the embryo, seeds will be considered non-germinable (Reis, 2004). The aim of this article is therefore to evaluate the LERCAF test for estimating germination and characterizing deterioration of coffee seeds during storage.

Materials and methods The experiment was conducted at the Seed Research Laboratory of the Plant Science Department of the Federal University of Viosa (UFV), Viosa, MG, 2006. We used coffee (Coffea arabica L.) seeds, cultivar IAC Catua 44, from the Experimental Farm Vale do Piranga / Agricultural Research Company of Minas Gerais (EPAMIG). Fruits were handpicked at cherry stage. After harvesting, fruits were depulped and fermented in water for 24 hours and washed for mucilage removal. The seeds were washed in tap water and spread on a screen in the shade to remove excess moisture. Seeds were then dried in 10 15 cm muslin bags containing approximately 1.3 kilograms each, in the shade, to 33, 28, 23, 18 and 13% moisture levels (wet basis). After drying, withered, injured and CBBdamaged seeds were discarded using the same procedures to obtain commercial lots. Seeds of all treatments were stored in waterproof packaging, at 20 3C, in laboratory, and the following evaluations were carried out after zero, two, four and six months of storage. LERCAF Four repetitions of 50 seeds per treatment, with parchment manually removed. Using gerboxes with plastic screen, for complete soaking of seeds with sodium hypochlorite solution, 2.5% active chlorine, using 100 mL of the solution per 50 seeds, or corresponding volume. The gerboxes were placed in a BOD incubator, at 25C, for 3 hours. Seeds were
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J.B. ZONTA, E.F. ARAJO, R.F. ARAJO, M.S. REIS AND J. DA SILVA LIMA

then washed in tap water for 90 seconds, soaked in distilled water for 40 minutes, and nally placed on a bench for evaluation. Following the visual evaluation, seeds were photographed and the percentages of germinable and non-germinable seeds were calculated as a function of endosperm colour and presence of embryo, according to methodology described by Reis (2004). Germination test Two hundred seeds with parchment manually removed were sown on germitest towel paper (3 layers) moistened with distilled water in quantities equivalent to 2.5 times the dry weight of the paper, with four repetitions. The rolls were kept in the incubator at 30C. Evaluations took place 15 and 30 days after sowing, and the results were expressed in mean percentage of normal seedlings (Brasil, 1992). Moisture content Seed moisture contents were determined by the standard oven method at 105 3C, for a period of 24 hours, using two repetitions of approximately 30 grams of seeds without parchment, as in Brasil (1992), and the results were expressed in percentage of moisture content (wet basis). The experiment was arranged in a complete randomized design, with four repetitions. Data were examined by the analysis of variance and the means, for each studied moisturecontent, were compared by the Tukey test, at 5% probability. Pearsons coefcient of correlation between LERCAF and germination tests was calculated, and the signicance of correlation was determined by the t- test at 1% probability (Gomes, 2000).

Results and discussion The LERCAF test carried out at the four storage periods showed seeds with endosperm with different colours. The seeds were separated into 5 classes in order to determine viability (gure 1). Class-1 seeds were classied as germinable, showing clear endosperm and visible embryo. These seeds, after LERCAF and germination tests, produced normal seedlings. Class-2 seeds showed light brown endosperm, and Class-3 seeds had dark brown endosperm. Class-4 seeds had black endosperm and Class-5 seeds had clear endosperm but without visible embryo. Seeds belonging to Classes 4 and 5, as well as Classes 2 and 3, were considered non-germinable and after the LERCAF test, did not produce normal seedlings. The percentages obtained by the germination test and the different seed classes according to the LERCAF test for all moisture contents and storage periods are shown in table 1. The results for the germination test showed that the treatments were separated into different quality categories, in which, for seeds with 33% moisture, the test classied the seeds with zero and two months of storage as higher quality and seeds with six months of storage as lower quality. Seeds with 28% moisture followed the previous standard; zero and two months of storage as higher quality and six months of storage as low quality. For seeds with 23, 18 and 13% moisture, the periods of zero, two and four months of storage stood out as higher quality, whereas only seeds with six months of storage showed lower quality
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Class 1

Class 2

Class 3

Class 4

Class 5

Figure 1. Coffee seeds classication using the LERCAF test. Class 1 - Seeds with clear endosperm and visible embryo (germinable); Class 2 - Seeds with light-brown endosperm (non-germinable); Class 3 - Seeds with dark brown endosperm (non-germinable); Class 4 - Seeds with black endosperm (non-germinable); Class 5 - Seeds with clear endosperm but without visible embryo (non-germinable).

in comparison with the others. Similar results were obtained by Vasconcelos et al. (1992), who found reduction in germination four months after storage. Results of the LERCAF test showed that seeds with 33, 28, 23, 18 and 13% moisture, which had still not been stored (zero months), showed 97, 98, 98, 99 and 99% of seeds in Class 1, respectively. The lack of seeds in Classes 2, 3 and 4 showed the initial high quality of the lots. In the second evaluation, after two months of storage, there was a decrease in germination for all studied moisture contents, which was determined by the decrease in the percentage of seeds in Class 1 and occurrence of Class 2. For seeds with 33 and 28% moisture, there was occurrence of Classes 4 and 3, respectively, but in a lower proportion when compared with Class 2. After four months of storage, for seeds with 28, 23 and 18% moisture, the percentage of seeds in Class 1 decreased in relation to the previous evaluation and the percentage of seeds in Classes 2 and 3 increased, but the proportion of seeds in Class 2 compared to Class 3 remained virtually the same, whereas for seeds with 33% moisture, besides the increase of seeds in Class 2, there was also increase of seeds in Class 4. After six months of storage, seeds with 28, 23, 18 and 13% moisture showed no seeds in Class 1, but there were large amounts of seeds in Class 2 and 3, with larger percentage of seeds in Class 3 than in Class 2. However, seeds with 33% moisture had a small percentage of seeds in Class 1 and a large percentage in Class 4. Results indicated that the newest seeds had larger percentage of seeds in Class 1 and the older ones had larger percentage of seeds in Classes 2, 3 and 4. It can be dened, therefore, that seeds with 33% moisture in advanced stage of deterioration caused by natural aging, move from Class 1 to Class 4, which means, from clear to black endosperm. Yet, seeds with 28, 23, 18 and 13% moisture move from Class 1 to Class 3 with an intermediate Class 2 between them. In this way, these seeds change from clear to a light brown and then to dark brown. There was no signicant variation between the studied moisture contents for Class-5 seeds, and this is because the occurrence of seeds without embryo is related to the production eld, the source plant or fertilization. Thus, since the seeds were obtained from a single crop, these similar values were already expected. The results from the different seed classes can determine germination estimates for the different studied moisture contents. Similarly to the germination test, the LERCAF test was able to separate the lots, in all studied moisture contents, into different categories. For 33% moisture, the seeds with zero months of storage showed larger number of
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J.B. ZONTA, E.F. ARAJO, R.F. ARAJO, M.S. REIS AND J. DA SILVA LIMA

Table 1. Results of LERCAF and germination tests during storage of coffee seed with different moisture contents. Treatments Moisture Content (%) Storage (months) 0 2 4 6 CV (%) 0 2 4 6 CV (%) 0 2 4 6 CV (%) 0 2 4 6 CV (%) 0 2 4 6 CV (%)
1

LERCAF Test (%) Germination Test (%) 92 a1 83 a 54 b 10 c 9243 94 a 88 ab 78 b 00 c 7325 93 a 90 a 86 a 0b 6488 94 a 91 a 91 a 0b 6101 94 a 92 a 91 a 0b 5932 Germinable 1 97 a 85 b 57 c 14 d 4265 98 a 93 b 80 c 0d 3084 98 a 93 ab 88 b 0c 5773 99 a 96 ab 93 b 0c 2260 99 a 95 b 93 b 0c 2376 2 0 10 15 2 0 5 13 20 0 5 6 29 0 2 6 37 0 3 5 45 Non-germinable Classes 3 0 0 0 0 0 1 4 78 0 0 4 68 0 0 1 60 0 0 0 54 4 0 3 26 84 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 3 2 2 0 2 1 3 2 2 2 2 1 1 2 0 3 1 2 2 1 -

33

28

23

18

13

Means followed by the same letter are not significantly different by the Tukey test at 5% probability.

germinable seeds, with germination estimated at 97%, whereas seeds with six months of storage showed 14% of germination. The same pattern was observed for seeds with the other moisture contents; seeds with 28 and 23% moisture with zero months of storage had 98% of germination, whereas seeds with six months of storage had 0% of germination. Seeds with 18 and 13% moisture had initial germination of 99% and after six months of storage the germination was estimated at 0%. Seeds with zero months of storage had better quality than the lots with six months of storage. These gures show that the LERCAF test was efcient to estimate germination of coffee seeds, since the results were very similar to those obtained by the germination test. To conrm the test efciency, the Pearsons correlation coefcient (r) was calculated between the germination obtained by the germination test and viability obtained by
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100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100

Germination test (%)

LERCAF test (%)


Figure 2. Spatial distribution correlating all data from LERCAF and germination tests without separation by moisture contents or storage periods.

LERCAF test. A dispersion graphic involving all values found in both tests was constructed to illustrate the high correlation found between the two tests. The evaluation of test feasibility showed that there was positive and highly signicant correlation (r> 0.99) between the test results. The high correlations found between the two tests is another contribution to indicate the high potential of the method for fast determination of coffee seed quality, bringing to the coffee industry benets such as early commercialization of seed lots, greater convenience and lower cost compared with the tetrazolium (Dias and Silva, 1998) and electrical conductivity (Costa and Carvalho, 2006) tests. During the development of the LERCAF test, Reis (2004) evaluated relatively new seed lots and found that damaged / deteriorated bean parts were greenish. In this work, the effect of NaClO on seed colour was studied during storage, i.e., naturally aged seeds. Sera et al. (2006) found that old seeds released brown exudates after six hours of water soaking and this result was negatively correlated with seed germination capacity. But, no brown exudates were found when evaluating new seeds with high germination capacity. The brown or black seed endosperm observed after the LERCAF test may have occurred because of the leaching of substances during the process of seed deterioration, i.e., loss of ions such as potassium. This fact was reported by Amorim (1978), Prete (1992), Prete et al. (2000a, b) during electrical conductivity tests of defective seeds. Here, this deterioration may have been accelerated by the presence of NaClO in the soaking solution. According to Prete et al. (2000), this brown exudate is an indication of cell deterioration, which was conrmed and probably accelerated, with the brown or black endosperm colour after the use of NaClO.

Conclusions 1. The LERCAF test is effective to estimate coffee seed germination during storage; 2. Seed lots with high germination capacity show larger percentage of seeds with clear endosperm; 3. Seed lots with low germination capacity show larger percentage of seeds with brown or black endosperm.
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J.B. ZONTA, E.F. ARAJO, R.F. ARAJO, M.S. REIS AND J. DA SILVA LIMA

Acknowledgements To CNPq for nancial support. References


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