Seed Science and Technology

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Volume 28 Number 1 2000

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Seed Science and Technology

Editorial Board Chief Editor

Dr A Bülow-Olsen

Associate Editors

DrMAmbrose Professor D Côme DrNWGalwey Professor J G Hampton Professor M B McDonald DrN Lisker Professor R E L Naylor Mr A A Pickett Professor N W Schaad Professor A M Steiner

11D JUL

2UOQ

Fernandez, E. M., Rosolem, C. A. and Oliveira, D. M. T. (2000), Seed Sei. & Technol., 27,185-192 28,

Peanut seed tegument is affected by liming and drying method E. M. FERNANDEZ, C. A. ROSOLEM and D. M. T. OLIVEIRA Crop Science Department, College of Agricultural Sciences, São Paulo State University, CP 237, 18603-970 Botucatu SP, Brazil (Accepted October 1998)

Summary Calcium plays a fundamental role in cell division and growth, and is an important constituent ofthe cell wall. An increase in Ca concentration in the tegument ofpeanut (Arachis hypogea L.) seeds in response to lime application can affect its stmcture. The tegument stmcture can also be affected by the drying method of the seeds. The effects of lime application and drying methods as affecting the peanut seed tegument stmcture were studied in seeds from a field experiment eondueted in São Manuel, São Paulo, Brazil. Peanut (ev. Botutatu, Valência Type) was grown in presence or absence of 2.1 Mg ha- I oflime and dried in an oven, in shade and in the field. The tegument anatomical features were deseribed and its stmcture was analysed. Pectic substances, lipidic reserves and starch accumulation were studied. The peanut tegument exhibited well differentiated exotesta, mesotesta and endotesta rich in pectates and covered by a cuticle. Tannin was not observed but there was lipid accumulation in mature teguments. Lignin was observed in the vascular bundles. Lime increased the tegument thickness and decreased the central cavity mainly in the exotesta cells when the period of seed drying was shortened. The effect of drying method upon the tegllment was more noticeable in seeds grown withollt lime. It can be inferred that liming increased the resistance ofthe tegument.

Introduction The peanut seed tegument differs from other leguminous plants because there is no palisade thick-walled celllayer and no hour-glass cells. The tissue is not differentiated, not far from an embryonary state (Corner, 1951). Some authors (Lush and Evans, 1980; Small, Kvein, Sumner and Csinos, 1989) described the peanut seed tegument as having two layers: the externaI or epidermic, and the internaI or parenchymatous, but Glueck, Clark and Smith (1977) found one additional internaI layer: the cells of the internai parenchyma. The free surface of the externallayer cells is formed by the excretion of waxes (Zambettakis and Bockelle-Morvan, 1976), and the externallayer or exotesta is formed by one layer of sclerenchymatous cells (Glueck et aI., 1977). In front view the cells are polygonal with three, four or five sides, and in cross-section they are conic (Zambettakis and Bockelle-Morvan, 1976; Glueck et aI., 1977). This particular cell arrangement and shape is used as a differential between cultivars (Zambettakis and Bockelle-Morvan, 1976). The median layer or mesotesta is formed by parenchymatous cells and is crossed by a vascular bundle (Zambettakis and Bockelle-Morvan, 1976, Glueck et ai. 1977). The mesotesta thickness varies depending on the cultivar (Glueck et ai., 1977). During the early development of the seed there is starch accumulation in the mesotestal 185

E. M. FERNANDEZ, C. A. ROSOLEM AND D. M. T. OLIVEIRA

cells (Glueck et aI., 1977), which is retranslocated during seed maturation (Patte, Johns, Singleton and Sanders, 1974; Glueck et aI. 1977). The internaI layer (endotesta) is fonned by a single layer of parenchymatous cells with thickened walls (Glueck et al., 1977). The water exchange between the seed and the environment is mediated by the tegument (Ketring, Benedict and Yeager, 1976), which stands also as a balTier to fungi infection (Zambettakis, 1975). The white tegument varieties are more susceptible to Aspergillus flavus (Singh, Mehan, Mengesha and Jambunathan, 1992) and Aspergillus parasiticus infection because they have a lower tannin content (Sanders and Mixon, 1978). Tannin is known as an aflatoxin production inhibitor (Pettit, Azeizen, Taber, Szerszen and Smith, 1989). Calcium plays a fundamental role in cell division and growth (Marschner, 1990) and is an impOltant constituent ofthe cell wall (Fry, 1986). Fernandez (1996) noticed an increase in Ca concentration in the tegument ofpeanut seeds, from 2.2 to 2.5 mg kg- I , in response to lime application. Therefore there may exist an effect of liming upon the tegument structure. The tegument structure can be affected by the drying method of the seeds, depending on the cultivar. The exotestal cells of a Spanish type cultivar shrank and showed a cracking appearance, and when the seeds were too dry the mesotestal cells practically disappeared. On the other hand, for a Valência type cultivar, the mesotestal cells had the spongeous feature accentuated next to the exotesta, the endotestal cells showed thinner walls, and an excess drying had no effect upon the tegument structure (Glueck et a!., 1977). High temperatures during drying cause detachment of the seed tegument (Wright and Steele, 1979). In this paper, possible interactions between lime application and drying methods as affecting the peanut seed tegument structure are discussed. Material and methods Seeds from a field experiment conducted on a Dark Red Latosol (Acrortox) at São Manuel, State of São Paulo, Brazil (22 o 46' S, 48 o 34' W, 740 m) were used in this study. Lime treatments with 0.0 and 2.1 Mg ha- 1 of dolomitic limestone (235 g kg- I CaO and 215 g kg-l MgO) were applied in plots 15 m long, with 5 rows ofpeanut, cv. Botutatu (Valência type). Drying methods were established in sub-plots 5 m long: 1) oven-dried, 2) drying in shade and 3) drying in the field. The experimental design was a randomised blocks with four rep!ications. Results of soil analyses as affected by liming, at planting time, are shown in Table 1. At planting, fertilizers we applied as follows: 20 kg ha- 1 ofN as ammonium sulphate, 35 kg ha- 1 ofP as simple superphosphate and 33 kg ha- 1 ofK as potassium chloride. The experiment was harvested 114 days after seedling emergence, by pulling the plants out ofthe soi! and shaking them out ofloose soil residues. According to the drying method, different procedures were followed until the seed water content was decreased to 100 g kg-l: 186

PEANUT SEED TEGUMENT IS AFFECTED BY L1MING AND DRYING METHOD

Table I. Results of soil analyses as affeeted by liming. Lime

pHCl)

Mgha- 1 0.0 2.1

4.2 4.9

K

O.M.

P(2)

H+AI

gkg- 1

mg kg-l

---------------------- emoIC') dm- 1 ----------------------

6.3 6.3

3.6 5.3

34.0 20.4

0.7 1.5

Ca

5.5 1,5

Mg

1.8 9.6

CEC

42.0 46.1

% 20 56

pH determined in CaCI 2 . P extraeted with anionie/eationie resin. (3) Soil Base Saturation.

(I)

(2)

1. Oven drying: fmits were manually separated from the plants and transferred to an air forced oven at 30+ 1°C, where they were spread in 5 cm thick layers. 2. Shade: the plants were immediately transferred to an open shelter and put upsidedown on wood benches. Care was taken to avoid contact of fmits with green plant material. 3. Field: The plants were gathered in bunches of 25-30 and put upside-down in the field. After drying, the fmits were manually dehulled and samples around 500 g of seeds were kept at -15°C until the analyses were performed. The tegument stmcture analysis was done only in the seeds dried in the field. This treatment was chosen because, according to the literature (Glueck et aI., 1977; Wright and Steele, 1979), it presented the highest potential for fungi infection. A sample of at least 10 seeds was taken from each treatment, and 25 cross-sections were made '"in the median portion of each seed. The sections were washed in distilled water, bleached in 20% sodium hypochloride for 20 minutes, washed with 1.0% acetic water and again in distilled water. The thickness of each of the three tegument layers were determined by using a micrometric ocular attached to an optical microscope. Seed teguments from all the subplots were dissociated according to a modification of the method proposed by Berlyn and Miksche (1976): small slices of tegument were macerated in a solution of equal parts of acetic acid and 30% hydrogen peroxide, transferred to an oven at 60°C for 5 hours and then washed with tap water and coloured using 50% alcoholic safranin to highlight the cell walls. The samples were then stored in refrigerator until they were observed through an optical microscope. Photomicrographs were taken. The terminology in describing anatomical features of the seed teguments was used as proposed by Comer (1976). The following microchemical tests were mn: 1) 12% floroglucinol plus chloridric acid, to localise lignified walls (Sass, 1951); 2) sudam IV, to identify lipidic reserves and the cutic1e; 3) lugol, to look for starch accumulation; 4) 10% ferric chloride plus calcium carbonate, to determine phenolic compounds; and 4) mthenium red, to highlight pectic substances (Johansen, 1940). 187


Results The peanut tegument exhibited well differentiated exotesta, mesotesta and endotesta (Figure la-d) covered by a cuticle, as indicated by sudam IV treatment (Figure la-b). The exotesta is formed by one single layer of polygonal cells, with four 01' five sides when in frontal view. In transversal view the cells are cube shaped. The primary wall is thickened, rich in Ca and Mg pectates, as shown by the ruthenium red treatment (Figure lc). There are invaginations towards the centre of the cells, forming a central cavity without wall thickening. This feature is variable, depending on Ca nutrition and dtying method. The degree of thickening in each cell decreases towards the mesotesta. There is no evidence of lignification in this tissue, as shown by the negative results of the acid floroglucinol test. The mesotesta is consisted of several layers of parenchymatous cells with a slight thickening with Ca and Mg pectates. Amphicribal vascular bundles run across the mesotesta (Figure lc). These bundles are surrounded by cells with thick walls containing high amounts of Ca and Mg pectates. In the vascular region, it was observed the only lignified tissue ofthe peanut tegument, the xylem. The endotesta consists of one layer of cells with thickened walls, rich in pectic compounds. The degree of thickening of these cells is higher than those of the mesotesta (Figure I c). There was no evidence of phenolic compounds, as shown by the ferric chloride test (Figure ld). The three regions of the tegument were thickened by liming (Table 2). Liming increased also the variance in thickness, mainly in the mesotesta. This region has a larger number of celllayers than the exotesta and the endotesta, which were unisseriated. The structure of the cells of the seed testa from each treatment was analysed after maceration. Only cells of the mesotesta and exotesta were examined because the endotesta cells were lost during the preparation processo It was tried to prepare the dissociations in a time as short as 2 h, without success in preserving the endotesta cells. When oven-dried, the exotesta cells of the seeds produced in the lime treatment (Figure 2a) had a more regular and tight distribution. They were bigger, with a smaller central cavity and thicker walls (transversal view) when compared with the exotestal cells Table 2. Exotesta, mesotesta, endotesta and total thickness of the tegument of peanut seeds dried in the field as affected by liming. Tegument tissue

Without Lime

WithLime

-------------------------- [lm --------------------------

Exotesta Mesotesta Endotesta Total

7.34±0.14 15.86±0.48 3.42±0.10 26.62±0.53

** Significant difference between lime treatments, t test (P < 0.01).

188

8.72±0.17** 24.76±1.l7** 4.26±0.15** 37.74±1.24**

PEANUT SEEO TEGUMENT IS AFFECTEO BY LIMING ANO DRYING METHOD

Figure 1. Cross-sections of the tegument of fie1d dried peanut seeds, showing the exotesta (ex), mesotesta (me) and endotesta (en). a-b: Sudan IV test, high1ighting the cutic1e (arrow) and severallipidic drop1ets (asterisk); c: region of the amphicribal bundles, after reaction to ruthenium red (note that exo, meso and endotesta present positive reaction); d: absence of reaction to tannins, after application of Ferric Chloride. Scale bars= 50~m.

from the treatment without lime (Figure 2b). When in frontal view the wallthickening was inegular, showing bigger projections into the cells. The mesotestal cells of the limed treatment (Figure 2c) were smaller, shorter and showed a more evident thickening in some parts of the walls when compared with cells from the treatment without lime (Figure 2d). In the shade dried seeds there were also differences within lime treatments. The differences observed in the exotestal cells (Figure 2e-f) were not so clear, once the edges ofthe cell walls ofthe no lime treatment (Figure 2f) were more inegular, with a smaller central cavity when compared with those from the oven-dried seeds. The seeds from the limed treatment showed no difference when dried in the oven 01' in the shade. The mesotestal cells of the seeds from the limed treatment (Figure 2g) were shorter and wider than those from the no lime treatment (Figure 2h). Comparing these seeds with those dried in the oven, there was no difference in the limed treatment, but in the no lime treatment the seeds dried in the oven presented shorter cells with rougher edges. In field, differences within lime treatments were also observed (Figure 2i-l). The exotestal cells from the no lime treatment (Figure 2j) were similar to those dried in the oven (Figure 2b). The cells ofthe mesotesta ofthe lime treatment (Figure 2k) are bigger and have thicker walls when compared with those from the no lime treatment (Figure 21). The exotestal cells from the lime treatment were very similar in the three drying 189


Figure 2. Tegument dissociations slices, showing the effect oflime upon the exotesta (plus lime: a, e, i, minus lime: b, f, j) and the mesotesta (plus lime: c, g, k, minus lime: d, h, I) within drying treatments: oven: a-d, shade: e-h and field: i-I. The arrows show the central cavity ofthe exotestal cells. Scale bar= 50 ~m.

methods (Figure 2a, e, i), but those from the no lime treatment were different (Figure 2b, f, j), The seeds dried in the oven and in the field showed no clear difference, The seeds dried in the shade had a smaller central cavity than the others, The mesotestaI cells of the lime treatment in the three drying methods are alike (Figure 2c, g, k), with walls thicker than those from the no lime treatment (Figure 2d, h, 1), 190

PEANUT SEED TEGUMENT IS AFFECTED BY LIMING AND DRYING METHOD

On the other hand the cells from the no lime treatment showed differences due to the drying method. The mesotestal cells from oven-dried seeds were the biggest and those from shade-dried seeds were the smallest. The mesotestal cells from the shade-dried seeds were narrower than in the other two treatments.

Discussion As reported by Comer (1976) the peanut tegument is not characteristic of the Leguminosae family because its tissues are not differentiated. In our experiment the palisade layer and the hour-glass cells were not observed. The observation of three cell regions in the tegument agrees with the Glueck et ai. (1977) report, but is different from observations by Zambettakis and Bockelle-Morvan (1976), Lush and Evans (1980) and Small et aI. (1989), that reported the existence of only two regions. The apparent controversy can be explained by the fact that the endotesta is very thin and sometimes is detached when the cutting is performed. Exotesta and mesotesta characteristics similar to our observations were described by Zambettakis and Bockelle-Morvan (1976) and Glueck et aI. (1977). The Botutatu cultivar that was analysed is very similar to cv. 6 described by Zambettakis and BockelleMorvan (1976). Glueck et ai. (1977) reported that the exotesta is consisted of sc1enchymatic cells, but tis sue lignification was not observed in this experiment. According to Patte et aI. (1974) the tegument is a reserve accumulation region to provide for the seed initial development, and Glueck et ai. (1977) reported that the reserves are stored in the mesotesta. In our description the three regions of the testa presented large amounts of Ca and Mg pectates. In mature seeds, starch accumulationwas not observed because they were probably consumed during the seed development (Patte et ai., 1974; Glueck et ai., 1977), but lipidic droplets were found throughout the peanut seed testa. So, besides storing starch in the early development stage of the seeds, the testa serves also as an oil storage organ when the seeds mature. The red colour of the tegument is usually associated with the presence of tannins (Sanders and Mixon, 1978; Pettit et aI., 1989), which was not observed in this experiment. In summary, lime increased the testa thickness and decreased the central cavity mainly in the exotesta cells when the period of seed drying was shortened. It can be inferred that liming increased the resistance of the tegument, which may increase the resistance to fungi infection.

References Berlyn, G. P. and Miksche, J. O. (1976). Botanical Microtechnique and Cytochemist/y. Iowa State University, Ames. Comer, E. J. H. (1951). The leguminous seed. Phytomorphology, 1,117-150. Comer, E. J. H. (1976). The seed of dicotyledons. University Press, Cambridge. Fernandez, E. M. (1996). Produtividade e qualidade de amendoim (Arachis hypogaea L.) em fimção da calagem e do método de secagem.[Yield and peanut quality as affected by liming and drying method]. Doc-

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--E. M. FERNANDEZ. C. A. ROSOLEM AND D. M. T. OLIVEIRA

tor Thesis, College of Agricultural Sciences, São Paulo State University, Botucatu, Brazil. Fry, S. C. (1986). Cross-linking ofmatrix polymers in the growing cell walls ofangiosperms. Annual Review ofPlant Physiology, 37,165-186. Glueck, J. A., Clark, L. E., Smith, O. D. (1977). Testa comparisons offour peanut cultivars. Crop Science, 17, 777-782. Johansen, D. A. (1940). Plant microtechnique. McGraw Hill Book Company, New York. Ketring, D. L.; Benedict, C. R., Yeger, M. (1976). Growing season and location effects on water uptake and drying rates of peanut seeds from genotypes resistant and susceptible to invasion by Aspergillus jlavus. AgronomyJournal, 68, 661-665. Lush, W. M. and Evans, L. T. (1980). The seed coats of cowpea and other grain legumes: stmcture in relation to function. Field Crops Research, 3, 267-286. Marschner, H. (1990). Mineral Nutrition ofHigher Plants. Academic Press, San Diego. Parte, H. E., Johns, E. B., Singleton, J. A.and Sanders, T. H. (1974). Composition changes of peanut fmit parts during maturation. Peanut Science, 1, 57-62. Pertit, R. E., Azeizen, H. A., Taber, R. A., Szerszen, J. B. and Smith, O. D. (1989). Screening groundnut cultivars for resistance to Aspergillus jlavus, Aspergillus parasiticus and aflatoxin contamination. In Proceedings for the International Workshop Ajlatoxin Contamination of Groundnut, pp209-303. International Crops Research Institute for the Semi-Arid Tropics, Patanchem. Sass, J. E. (1951). Botanical microtechnique. Iowa State Press, Ames. Sanders, T. H. and Mixon, A. C. (1978). Effect of peanut tannins on percent seed colonisation and in vitro growth by Aspergillus parasiticus. Mycopathologia, 66, 169-173. Singh, A. K., Mehan, V. K., Mengesha, M. H. and Jambunathan, R. (1992). Imbibition rates, leachates and fungai colonisation of seeds of selected groundnut germplasm !ines with different seed testa colours. Oleagineux, 47, 579-582. Small, H., Kvien, C. S., Sumner, M. E. and Csinos, A. S. (1989). Solution calcium concentration and application date effect on pod calcium uptake and distribution in Flomnner and Tifton-8 peanut. Journal of Plant Nutrition, 12,37-52. Wright, F. S. and Steele, J. L. (1979). Potential for direct harvesting ofpeanuts. Peanut Science, 6, 37-42. Zambettakis, C. H. (1975). Etude de la contamination de quelques variétés d'arachide par Aspergillusjlavus. Oleagineux, 30,161-167. Zambettakis, C. H. and Bockelle-Morvan (1976). Recherches sur la stmture du tégument séminal de la graine d'arachide et son inflnence sur la penetration de l'Aspergillus jlavus. Oleagineux, 31, 219-228.

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