2007 International Nuclear Atlantic Conference - INAC 2007 Santos, SP, Brazil, September 30 to October 5, 2007 ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN ISBN: 978-85-99141-02-1

STRUCTURAL CHARACTERIZATION OF ANNATTO SEEDS (Bixa orellana) by transmission and scanning electron microscopy submitted to gamma radiation for dormancy break.

Marcia N. C. Harder, Neusa L. Nogueira, Valter Arthur, Mônica L. Rossi and Adriana P. M. Rodriguez

1 Centro de Energia Nuclear na Agricultura (CENA/USP-SP) Av. Centenário, 303 13.400-970 Piracicaba, SP [email protected] [email protected] [email protected] [email protected] [email protected]

ABSTRACT

The annatto (Bixa orellana) is the only species of the Bixaceae family. From the seeds an important food colorant is obtained, bixin, for the industry and domestic use. More recently studies have focused more extensively in medicinal purpose of the species. Due to structural and physiologic characteristics, the seeds have low germination rate, around 30 %. The irradiation of seeds with gamma radiation can promote the increase and/or acceleration of germination, better plant developmentand productivity, among other aspects. The radiation doses used for this purpose should not cause genetic modifications in the organism, hence experimentation is needed to define the appropriate doses. Absence of research done annatto related to the use of the irradiation aiming at the increase of germination rates lead to the structural characterization of the annatto seeds submitted to gamma radiation through transmission (TEM) and scanning electron microscopy (SEM). The objective of this study was to verify the effect of radiation on the seeds structures during the process of dormancy break. Dry seeds and seeds immersed in distilled water for 24 hours were submitted to gamma radiation from source of Co60 type Gammacell-220 at CENA/USP, at doses 100 Gy. After irradiation the seeds were processed for TEM and SEM. Preliminary results, showed structural modifications in the seeds.

1. INTRODUCTION

According to Joly [1] the annatto (Bixa orellana) is the only species of Bixaceae family, native from Tropical America and extremely cultivated. From these seeds an important food colorant is obtained for the industry and domestic use.

The annatto tree is an angiosperm, and during its development, the seed proceed by three different phases: histodifferention or morphogenesis, concomitant cellular expansion to the phase of accumulation reserve, and the final phase, that it corresponds to the dryness [2].

Dryness constitutes an integral event of the process of development of most seeds, especially the orthodox ones [3].

In this group, the development is completed when the seed has low humidity tenors. Possibly the dryness has a basic paper in the interruption of the essential development of the germination process, by the hydration of the quiescents seeds leads to germination [4].

In several seeds, during the dryness phase, the tegument become impermeable to the water. This dormancy type is quite common, mainly in arboreal legume [5]. Like this, the knowledge of the ontogenesis and suffered alterations by the tegument during the development of the seeds assumes great importance.

Corner [6] established a classification to seeds based on the origin of the tegument. The tissue that arise starting from the external tegument are called test and those that grow of the internal tegument constitute the tegmen. In several seeds, the test is the main responsible for the protection of the internal tissue and the tegmen doesn´t present any mechanical function frequently suffering compression by the endosperm and embryo. This type is quite common in the legumes. In other species the tegmen is the responsible by the protection of the embryo and of other internal tissue and if the test doesn´t disappear it specializes in other functions as the one of secretion of colorant as it happens in Bixa orellana [7]. Those colorants are identifies carotenoids as bixin and norbixin [8]. Like this in agreement with the exposed above the seeds of annatto can be classified as tegma plants.

The irradiation of plant seeds with certain radiation doses can promote the increase and/or acceleration of the germination, larger development of the plant, increase of the agricultural production, etc. The radiation doses used for obtaining those incentives doesn´t cause modifications in the genetic patrimony of the irradiated organism, therefore usually the dose level is low. Besides chronicle doses are many times more effective to cause stimulating effect than the sharp ones [9]. The stimulation of several biological functions of animals plants and microorganisms is known as radio-hormesis; the effect of the radiation is not so spread, but it was observed soon after the discovery of the radioactivity [10].

The inexistence of works related to the use of the irradiation with the purpose of the increase of the germination rate of annatto seeds and the lack of studies related to the own specie took to study of characterization of the annatto structures submitted to the gamma radiation, with the aim was verify the possible increase of the germination rate and some alteration in their structures promoted by the irradiation, with the target to dormancy break of the respective seeds.

2. MATERIALS AND METHODS

2.1. Material

Annatto seeds (Bixa orellana) was used given by CHR Hansen of Brazil, cv. Piave, from Valinhos, Sao Paulo.

2.2. Methods

2.2.1. Treatment The samples was submitted to the gamma radiation from a source of Co60 type Gammacell- 220, at CENA/USP, under a rate of dose of 10 Gy/hour, in this doses: 100 Gy and a portion

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also was submerged in distilled water by 24 hours, being compares among themselves and compared with the control no irradiated and no submerged in water.

2.2.2. Evaluation of the treatments

2.2.2.1. Transmission Electron Microscopy

Small portion of annatto seeds were fractured through bistoury and immerged in a modified solution of Karnosvsky composed for: glutaraldeyde 2%, parapholmaldeyde 2%, chloride of calcium 0.05 M in cacodilate buffer 0.1 M and post fixed by 1 hour in a osmium tetroxid in cacodilate buffer 0.1 M. After fast washes with saline solution 0,9% were colored “in block” with uranil acetate 2.5% in water overnight and dryness with growing series of acetone solution (25%; 50%; 75%) for 5 minutes, following for 2 treatments of 10 minutes each with acetone 90% and 3 treatments of 20 minutes with pure acetone [11].

Later the speciments was included in Spurr resin, like the instructions of the manufacturer.

The ultrafine sections (60-90 nm) were deposited on copper grids coated with colodium, contrasted with uranil acetate 2.5% and lead citrate [12]. These sections were examined under a Zeiss EM-900 transmission electron microscope electronic and digital photographed.

2.2.2.2. Scanning Electron Microscopy

Samples of annatto were fastened in solution of glutaraldeyde 2% in sodium cacodilate buffer 0,2 M for 24 hours. After they were washed 3 times, being 10 minutes each in sodium cacodilate buffer 0,1 M. The dryness was made in growing series of ethanol in water in following concentrations: 35%; 50%; 60%; 70%; 80%; 90% one time for 15 minutes each and 100% 3 times, 20 minutes each. Afterwards, the samples were taken to the drying apparet to the critical point (Balzers CPD 050) of NAP/MEPA/ESALQ/USP, mounted in “stubs”, and metallic with gold during 260 seconds to the evaporator (MED 010 Balzers). The sample were observed under a Zeiss LEO 435 VP scanning electron microscope and digital images were obtained [13; 14].

Another part of the samples followed the same steps above and later broken with criofracture, according to the method of Tanaka & Nagaya [13], where the samples of annatto seeds were fastened in a solution of modified Karnovsky for 16 hours to 4º C and substituted by glycerol 30% for 30 minutes.

After cold the accessories, in liquid nitrogen, the samples were induced to the cleavage. The small criofractures obtained were mounted in “stubs” with silver glue and post fix with steam of osmium tetroxid 1% by 1 hour. Afterwards the samples were metallic with gold for 260 seconds in the evaporator (MED 010 Blazers) of NAP/MEPA/ESALQ/USP. The samples were observed under a Zeiss LEO 435 VP scanning electron microscope and images were obtained.

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3. RESULTS

Pieces of the annatto seeds were observed and compare their structures using the transmission and scanning electron microscopy.

Figure 1. Comparation of all treatments by Scanning electron microscopy: the left to the right: 0 to 10 Gy

It was observed the external part (resins) and the internal structures showing, coming in a porous way, with some oval structures (probably grains of starch).

In the seeds that received treatment the external part was observed (resins) and the structures of the part intern, coming in a more compact way, with to presences of the oval structures visualization of the place where is the embryo.

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Figure 2. Comparation of all treatments by Transmission electron microscopy: the left to the right: 0 to 10 Gy

In all pictures of the witness were difficult the identification of the nucleus of the cells, perfect visualization of the cellular membranes besides short space among them (intercell space), eventual presence of plasmodesms.

The seeds that received treatment were easy identification of the nucleus of the cells, perfect visualization of the cellular membranes, the space among them (intercell space) it is larger than the witness and easy visualization, perfect plasmodesms visualization.

4. CONCLUSIONS

Preliminary results, showed structural modifications in the seeds.

The use of auxiliary methods, as the light microscopy is necessary for more conclusive comparative determinations.

At first the one that can be said is that both the gamma radiation, and water provokes modifications in the structures of the seeds and because this there is an increase on the germination rate.

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ACKNOWLEDGMENTS

To CHR Hansen of Brazil by the samples.

Specially to Elliot Watanake Kitajima – NAP/MEPA – ESALQ/USP, for maintaining the electron microscope facilities.

REFERENCES

1. A. B. Joly, Botânica: introdução à taxonomia vegetal, Ed. Nacional, Sao Paulo, Brazil (1993). 2. V. Thaker, “Changes in water content, sugars and invertase activity in developing seeds of Hibiscus esculentum”, Acta Physiologiae Plantarum, 21, pp. 155-159 (1999). 3. A. R. Kermode, J. D. Bewley, J. Dasgupta, S. Misra, “ The transition from seed development to germination: a key role for desiccation?”, HortScience, 21, pp. 1113-1118 (1986). 4. J. D. Bewley, M. Black, Seeds: Physiology of development and germination, Plenum Press, New York, USA (1994). 5. M. P. Rolston, “Water impermeable seed dormancy”, Botanical Review, 44, pp. 365-396 (1978). 6. E. J. H. Corner, The seeds of dicotyledons, Cambridge University Press, Cambridge, England, UK (1976). 7. R. N. Chopra, H. Kaur, “Embryology of Bixa orellana Linn”, Phytomorphology, 15, pp. 211-215 (1965). 8. J. F. Reith, “Properties of bixin and norbixin and the composition of annatto extracts”, Journal of Food Science, 36, pp. 861-864 (1971). 9. T. D. Lukey, Hormesis with Ionizing Radiation, CRC Press, 10. J. E. Bovi, V. Arthur, J. Tessarioli Neto, “Uso de baixas doses da radiação gama de cobalto-60 em sementes de rabanete e seus efeitos no desenvolvimento da planta, produção e armazenamento de raízes”, Revista de Agricultura, 75, pp. 373-386 (2000). 11. J. J. Bozzola, L. D. Russel, Electron Microscopy, Jones & Bartett Publ., Boston, USA (1992). 12. E. S. Reynolds, “The use of the lead citrate at high pH as an electron-opaque stain in electron microscopy”, Journal of Cell Biology, 17, pp. 208 (1963). 13. K. Tanaka, T. Nagaya, Scanning electron microscopy illustrated, Ed. Asakura, Toquio, Japan (1980). 14. W. de Souza, Manual sobre técnicas básica em microscopia eletrônica, Sociedade Brasileira de Microscopia Eletrônica, Rio de Janeiro, Brazil (1989).

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