Anatomical and histochemical characterization of seeds of <i>Cattleya intermedia</i> subjected to different storage conditions

Schvambach, Mariana Izabel; Santos, Cristina Magalhães Ribas dos; Souza, Priscila Fernandes de; Cabral, Nadhine Nostrani; Suzuki, Rogério Mamoru; Pescador, Rosete

doi:10.1590/2175-7860202273111

Mariana Izabel Schvambach ⁷7Author for correspondence: marianais1994@gmail.com

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Pós-graduação em Recursos Genéticos Vegetais, Florianópolis, SC, Brazil.

http://orcid.org/0000-0002-2989-7005

Cristina Magalhães Ribas dos Santos

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Depto. Fitotecnia, Florianópolis, SC, Brazil.

http://orcid.org/0000-0002-9118-6730

Priscila Fernandes de Souza

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Pós-graduação em Recursos Genéticos Vegetais, Florianópolis, SC, Brazil.

http://orcid.org/0000-0003-4096-4552

Nadhine Nostrani Cabral

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Pós-graduação em Recursos Genéticos Vegetais, Florianópolis, SC, Brazil.

http://orcid.org/0000-0003-0454-426X

Rogério Mamoru Suzuki

Instituto de Botânica, São Paulo, SP, Brazil.

http://orcid.org/0000-0003-1124-9875

Rosete Pescador

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Depto. Fitotecnia, Pós-graduação em Recursos Genéticos Vegetais, Florianópolis, SC, Brazil.

http://orcid.org/0000-0002-4667-9894

7Author for correspondence: marianais1994@gmail.com

Area Editor: Dra. Simone Teixeira

Figures (6)
Tables (5)

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Figure 1
a-j. Scanning electron microscopy and light microscopy of the flower, fruit and seed of Cattleya intermedia - a. Floral pieces; b. Fruits with floral remains. It is possible to identify the beginning of the opening to release the seeds; c. Seeds extracted from fruits; d. Seeds submitted to the tetrazolium test, showing viable embryos (stained by tetrazolium) and non-viable seeds (without the presence of the embryo); e. Thready seed, showing the testa, the chalazal and micropylar ends and the swollen region (key) where the embryo is located; f. Details of the testa; g. TBO: seed with cells from the testa, embryo and suspender, evidenced by the action of the reagent with cellulosic compounds and pectins; h. CBB: seed showing protein reserve in embryonic cells; i. Sudan IV: seed with embryonic cells evidenced by the action of the reagent with lipid reserves; j. PAS: seed with cells from the testa, embryo and suspender marked by the action of the reagent with starch and cellulose. Afru = opening of the fruits; TBO = toluidine blue; CBB = Coomassie brilliant blue; Cel = testa forming cell; Co = spine; Cr = reserve cell; Cst = superficial testa cell; Ec = chalazal end; Em = micropylar end; Emb = ellipsoid embryo; Fru = fruits; La = lip; Lof = frontal lobes; Lol = side lobes; PAS = periodic acid Schiff; Pe = petal; Pvc = cellulosic vegetable wall; Rac = starch reserve in embryonic cells; Re = floral remains; Ri = swollen region of the seed; Rlc = lipid reserve in embryonic cells; Rpc = protein reserve in embryonic cells; Se = seeds; Sed = dorsal sepal; Sef = seed with a threadlike shape; Sel = lateral sepal; Si = unviable seed; Sus = suspender; Sv = viable seed; Te = testa of the seed. Scale bar: a- c = 1 cm; d = 1 mm; e = 100 µm; f = 20 µm; g = 500 µm; h-j = 200 µm

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Figure 2
a-l. Scanning electron microscopy of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Viable seeds, evidencing the testa, the chalazal and micropylar ends and the swollen region (key) where the embryo is located. Non-viable seeds are noted, with damage by dehydration; b. (four months) Viable seeds, evidencing the testa and swollen region (key). Predominance of dehydrated seeds; c. (six months) Viable seeds evidencing the swollen region, where the embryo is located. Predominance of dehydrated and non-viable seeds; d-f. Temperature of -20 °C - d. (two months) Viable seeds, evidencing the swollen region (key) and non-viable seeds, showing damage by dehydration (key); e. (four months) Viable seed, evidencing the swollen region (key) and non-viable seeds, dehydrated (key); f. (six months) Viable seed, evidencing the swollen region (key). Predominance of non-viable and dehydrated seeds (key); g-i. Temperature of -80 °C - g. (two months) Viable seed, evidencing the swollen region (key), where the embryo and un-viable seed are located, showing damage by dehydration (key); h. (four months) Viable seed, evidencing the swollen region (key), where the embryo and non-viable seeds are located, dehydrated (key); i. (six months) Viable seed, evidencing the swollen region (key), where the embryo is located. Predominance of non-viable seeds, showing damage by dehydration (key); j-l. Temperature of -196 °C - j. (two months) Viable seed, evidencing the swollen region (key), where the embryo and non-viable seed are located, showing damage by dehydration (key); k (four months) Viable seeds, evidencing the swollen region (key); l. (six months) Viable seeds, evidencing the swollen region (key), where the embryo and non-viable seed are located. Cel = testa forming cell; Dde = damage by dehydration; Ec = chalazal end; Em = micropylar end; Rte = rupture of the integument of testa; Si = non-viable seed; Sv = viable seed; Te = testa. Scale bar: a, e-l = 100 µm; b-d = 200 µm.

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Figure 3
a-l. Light microscopy and histochemical analysis with TBO of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seed highlighting reactive integument and embryo cells poorly reactive to TBO. The vacuolization of the suspender cells (arrowhead) can be noted by the lack of reaction with this dye; b. (four months) Seed showing embryo with cells poorly reactive to TBO, also evidencing some apparent vacuolization (arrowheads); c. (six months) Seed showing little reaction from the embryonic cells with TBO, and the lack of reaction with the suspender cells (arrowhead); d-f. Temperature of -20 °C - d. (two months) Seeds showing the chalazal end with superficial cells of testa highlighted by the reaction with TBO. The embryo reserve cells show evidence of vacuolization by the lack of reaction with the dye. The suspender and other cells with different intensities of reaction with TBO can be noted; e. (four months) Seed showing reactive embryonic cells and suspender with some apparent degeneration due to lesser reaction with TBO; f. (six months) Seed showing the dehydrated testa (key), embryonic cells reactive to TBO and the absence of suspender, by the lack of reaction with the dye (arrowhead); g-i. Temperature of -80 °C - g. (two months) Seeds showing different intensities of reaction from the embryonic cells with TBO, damage to the testa and apparent degeneration from the suspender cells due to the lack of reaction with TBO (arrowhead); h. (four months) Seed showing embryonic cells reactive to TBO and the lack of reaction with suspender cells (arrowhead); i. (six months) Seeds showing different reactions from the embryonic cells with TBO and apparent vacuolization. The absence of reaction with the suspender cells can be noted in one of the seeds, evidencing its degeneration (arrowheads); j-l. Temperature of -196 °C - j. (two months) Empty seed can be noted due to absence of reaction of internal structures with TBO. In the preserved seed the dye-reactive embryonic cells can be seen. The suspender cells, on the other hand, present different intensities of reaction, showing degeneration of cell walls; k. (four months) Seed showing embryonic cells with vacuolization evidenced by the lack of reaction with TBO. Degeneration process of the suspender, showing cells less reactive to the dye; l. (six months) Seeds showing different intensities of reaction of the embryonic cells with TBO. Degeneration of absence of the suspender can be observed, due to the less intensity of lack of reaction of the cells with TBO (arrowhead). Cr = reserve cell; Cst = superficial cell of the testa; Ec = chalazal end; Em = micropylar end; Emb = ellipsoid embryo; Emd = degenerating embryo; Rt = testa rupture; Se = seed; Sud = degenerating suspender; Sus = suspender; Sva = empty seed; Td = dehydrated testa; Te = testa. Scale bar: a-b, e-f, h, l = 500 µm; c-d, g, i-k = 200 µm.

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Figure 4
a-l. Light microscopy and histochemical analysis with CBB of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seeds showing embryos and embryonic cells poorly reactive to proteins. Arrowhead points to an absence of suspender; b. (four months) Seed evidencing testa, embryo and embryonic cells reactive to proteins. Degeneration of the embryo and absence of the suspender can be noticed (arrowhead); c. (six months) Seed with embryo and embryonic cells reactive to proteins. Degeneration of the embryo and absence of suspender can be noted (arrowhead); d-f. Temperature of -20 °C - d. (two months) Seeds showing embryo and embryonic cells with prominent nuclei evidenced by the reaction with CBB. Arrowhead indicates the absence of the suspender by the lack of reaction with this dye; e. (four months) Seed showing embryo and embryonic cells reactive to proteins. Degenerating embryo and absence of the suspender are observed (arrowhead); f. (six months) Seed with degenerating embryo, embryonic and suspender cells poorly reactive to proteins can be noted. Rupture of the testa is noticed (arrow); g-i. Temperature of -80 °C - g. (two months) Seed evidencing embryo, embryonic and suspender cells reactive to proteins. Embryonic cells with prominent nuclei and suspender cells reactive to proteins are observed. Embryonic cells with prominent nuclei are evidenced by the reaction with CBB; h. (four months) Seed showing embryo, embryonic and suspender cells reactive to proteins. Embryonic cells with prominent nuclei evidenced by the reaction with CBB can be noted; i. (six months) Seeds showing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, evidenced by the lack of reaction with the chemical is observed. j-l. Temperature of -196 °C - j. (two months) Seeds showing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, due to the lack of reaction with CBB are perceived; k. (four months) Seed evidencing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, due to the lack of reaction with the dye is observed; l. (six months) Seed showing embryo and embryonic cells reactive to proteins. Degeneration of the embryo and embryonic cells, evidenced by the lack of reaction with CBB are noted. Arrowhead indicates the absence of the suspender. Ced = embryonic cells in degeneration; Cen = embryonic cells with prominent nuclei; Cep = embryonic cells reactive to proteins; Csp = suspender cells reactive to proteins; Emb = embryo; Emd = degenerating embryo; Te = testa. Scale bar: 200 µm.

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Figure 5
a-l. Light microscopy and histochemical analysis with Sudan IV of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seeds showing testa, micropylar and chalazal ends. Arrow points to a lack of reaction to Sudan IV; b. (four months) Seed showing the testa, micropylar and chalazal end, lack of reaction to Sudan IV is noted (arrow); c. (six months) Seeds showing testa, micropylar and chalazal ends. Arrows indicate lack of reaction to Sudan IV; d-f. Temperature of -20 °C - d. (two months) Seed evidenced embryo reactive to Sudan IV is seen on the left. Absence of the suspender is perceived (arrowhead). On the right, a seed with an embryo poorly reactive to Sudan IV is noted, due the lack of reaction with the dye; e. (four months) Seed showing embryo and suspender cells reactive to Sudan IV. It can be seen embryonic cells in degeneration, due to the lack of reaction with the dye; f. (six months) Seed with reactive embryo for Sudan IV is seen on the left. Embryonic cells show degeneration caused by the lack of reaction with Sudan IV. On the right, a seed can be seen with no reaction to Sudan IV; g-i. Temperature of -80 °C - g. (two months) On the left, a seed with no reaction to Sudan IV is observed. On the right, it can be seen a seed showing embryo and suspender cells reactive to Sudan IV; h. (four months) Seed showing reaction to Sudan IV in the embryo and suspender cells; i. (six months) Seed showing embryo and suspender reactive to Sudan IV. Degeneration of the embryonic cells is perceived, due to the lack of reaction with the dye; j-l. Temperature of -196 °C - j. (two months) Seed showing embryo and suspender reactive to Sudan IV; k. (four months) On the left, seed showing embryo reactive to Sudan IV and suspender poorly reactive to Sudan IV. On the right, seed with no reaction to Sudan IV is noted; l. (six months) Seed with embryo reactive to Sudan IV and embryonic cells in degeneration can be observed, due to the lack of reaction with the dye. A damaged testa structure is noted (arrowhead). Ced = degenerating embryonic cells; Css = suspender cells reactive to Sudan IV; Ec = chalazal end; Em = micropylar end; Ems = embryo reactive to Sudan IV; Epr = embryo poorly reactive to Sudan IV; Sps = suspender poorly reactive to Sudan IV; Sus = suspender; Te = testa. Scale bar: a-b, g-j, k = 200 µm; c-f, l = 500 µm.

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Figure 6
a-l. Light microscopy and histochemical analysis with PAS of the seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seed showing reaction to PAS in the testa structures, testa cells, embryo, embryonic and suspender cells; b. (four months) Seeds showing testa, embryo, embryonic and suspender cells evidenced by reaction to PAS are observed. Arrow indicates the lack of reaction with PAS in the suspender; c. (six months) Seed showing testa, embryo, embryonic and suspender cells reactive to PAS. Lack of reaction with the suspender cells can be perceived, pointing to its degeneration (arrow); d-f. Temperature of -20 °C - d. (two months) Seeds showing testa, embryo, embryonic and suspender cells reactive to PAS. Suspender cells show changes evidenced by the reaction to PAS (arrow); e. (four months) Seed showing reactive testa and embryo and embryonic cells poorly reactive to PAS. Alterations in the suspender cells can be noted evidenced by the reaction with the dye (arrow); f. (six months) Seed showing different intensities of reaction to PAS, reactive testa and embryo and embryonic and suspender cells poorly reactive to PAS can be observed. Changes in the suspender cells reactive to PAS are noted (arrow); g-i. Temperature of -80 °C - g. (two months) Seed showing different intensities of reaction in the embryo and embryonic cells with PAS; h. (four months) Seed evidencing embryo and embryonic cells poorly reactive to PAS. Alterations in the suspender cells evidenced by the reaction with the dye are noted (arrow); i. (six months) Seed showing different intensities of reaction in the embryo and embryonic cells with PAS. Changes can be seen in the cells of the embryo, due to less reaction with PAS (arrowheads); j-l. Temperature of -196 °C - j. (two months) Seed showing embryo, embryonic and suspender cells reactive to PAS. Alterations in the suspender cells reactive to the dye are noted (arrow); k. (four months) Seed showing testa, embryo and embryonic cells reactive to PAS. Changes in the embryonic cells (arrowhead), due to the lack of reaction with the dye and absence of reaction to PAS in the suspender (arrow) can be observed; l. (six months) Seeds showing different intensities of reaction to PAS in the embryo and embryonic cells, evidencing their degeneration. Changes in the embryonic cells (arrowhead), due to the poor reaction with the dye and seed with absence of reaction to PAS in the suspender (arrow) can be noted. Cepa = embryonic cells reactive to PAS; Cspa = suspender cells reactive to PAS; Ctpa = testa cell reactive to PAS; Emb = embryo; PAS = periodic acid-Schiff; Te = testa. Scale bar: a-d, l = 500 µm; e-k = 200 µm.

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Table 1
Description of viable and non-viable seeds of Cattleya intermedia submitted to different storage conditions observed through scanning electron microscopy.

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Table 2
Description of light microscopy and histochemical analysis with TBO of Cattleya intermedia seeds submitted to different temperatures and storage periods.

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Table 3
Description of light microscopy and histochemical analysis with CBB of Cattleya intermedia seeds submitted to different temperatures and storage periods.

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Table 4
Description of light microscopy and histochemical analysis with Sudan IV of Cattleya intermedia seeds submitted to different temperatures and storage periods.

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Table 5
Description of light microscopy and histochemical analysis with PAS of Cattleya intermedia seeds submitted to different temperatures and storage periods.

Figure 3 a-l. Light microscopy and histochemical analysis with TBO of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seed highlighting reactive integument and embryo cells poorly reactive to TBO. The vacuolization of the suspender cells (arrowhead) can be noted by the lack of reaction with this dye; b. (four months) Seed showing embryo with cells poorly reactive to TBO, also evidencing some apparent vacuolization (arrowheads); c. (six months) Seed showing little reaction from the embryonic cells with TBO, and the lack of reaction with the suspender cells (arrowhead); d-f. Temperature of -20 °C - d. (two months) Seeds showing the chalazal end with superficial cells of testa highlighted by the reaction with TBO. The embryo reserve cells show evidence of vacuolization by the lack of reaction with the dye. The suspender and other cells with different intensities of reaction with TBO can be noted; e. (four months) Seed showing reactive embryonic cells and suspender with some apparent degeneration due to lesser reaction with TBO; f. (six months) Seed showing the dehydrated testa (key), embryonic cells reactive to TBO and the absence of suspender, by the lack of reaction with the dye (arrowhead); g-i. Temperature of -80 °C - g. (two months) Seeds showing different intensities of reaction from the embryonic cells with TBO, damage to the testa and apparent degeneration from the suspender cells due to the lack of reaction with TBO (arrowhead); h. (four months) Seed showing embryonic cells reactive to TBO and the lack of reaction with suspender cells (arrowhead); i. (six months) Seeds showing different reactions from the embryonic cells with TBO and apparent vacuolization. The absence of reaction with the suspender cells can be noted in one of the seeds, evidencing its degeneration (arrowheads); j-l. Temperature of -196 °C - j. (two months) Empty seed can be noted due to absence of reaction of internal structures with TBO. In the preserved seed the dye-reactive embryonic cells can be seen. The suspender cells, on the other hand, present different intensities of reaction, showing degeneration of cell walls; k. (four months) Seed showing embryonic cells with vacuolization evidenced by the lack of reaction with TBO. Degeneration process of the suspender, showing cells less reactive to the dye; l. (six months) Seeds showing different intensities of reaction of the embryonic cells with TBO. Degeneration of absence of the suspender can be observed, due to the less intensity of lack of reaction of the cells with TBO (arrowhead). Cr = reserve cell; Cst = superficial cell of the testa; Ec = chalazal end; Em = micropylar end; Emb = ellipsoid embryo; Emd = degenerating embryo; Rt = testa rupture; Se = seed; Sud = degenerating suspender; Sus = suspender; Sva = empty seed; Td = dehydrated testa; Te = testa. Scale bar: a-b, e-f, h, l = 500 µm; c-d, g, i-k = 200 µm.

Figure 4 a-l. Light microscopy and histochemical analysis with CBB of seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seeds showing embryos and embryonic cells poorly reactive to proteins. Arrowhead points to an absence of suspender; b. (four months) Seed evidencing testa, embryo and embryonic cells reactive to proteins. Degeneration of the embryo and absence of the suspender can be noticed (arrowhead); c. (six months) Seed with embryo and embryonic cells reactive to proteins. Degeneration of the embryo and absence of suspender can be noted (arrowhead); d-f. Temperature of -20 °C - d. (two months) Seeds showing embryo and embryonic cells with prominent nuclei evidenced by the reaction with CBB. Arrowhead indicates the absence of the suspender by the lack of reaction with this dye; e. (four months) Seed showing embryo and embryonic cells reactive to proteins. Degenerating embryo and absence of the suspender are observed (arrowhead); f. (six months) Seed with degenerating embryo, embryonic and suspender cells poorly reactive to proteins can be noted. Rupture of the testa is noticed (arrow); g-i. Temperature of -80 °C - g. (two months) Seed evidencing embryo, embryonic and suspender cells reactive to proteins. Embryonic cells with prominent nuclei and suspender cells reactive to proteins are observed. Embryonic cells with prominent nuclei are evidenced by the reaction with CBB; h. (four months) Seed showing embryo, embryonic and suspender cells reactive to proteins. Embryonic cells with prominent nuclei evidenced by the reaction with CBB can be noted; i. (six months) Seeds showing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, evidenced by the lack of reaction with the chemical is observed. j-l. Temperature of -196 °C - j. (two months) Seeds showing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, due to the lack of reaction with CBB are perceived; k. (four months) Seed evidencing embryo, embryonic and suspender cells reactive to proteins. Degeneration of the embryo and embryonic cells, due to the lack of reaction with the dye is observed; l. (six months) Seed showing embryo and embryonic cells reactive to proteins. Degeneration of the embryo and embryonic cells, evidenced by the lack of reaction with CBB are noted. Arrowhead indicates the absence of the suspender. Ced = embryonic cells in degeneration; Cen = embryonic cells with prominent nuclei; Cep = embryonic cells reactive to proteins; Csp = suspender cells reactive to proteins; Emb = embryo; Emd = degenerating embryo; Te = testa. Scale bar: 200 µm.

Figure 6 a-l. Light microscopy and histochemical analysis with PAS of the seeds of Cattleya intermedia subjected to different temperatures and storage periods - a-c. Temperature of 25 (± 2 °C) - a. (two months) Seed showing reaction to PAS in the testa structures, testa cells, embryo, embryonic and suspender cells; b. (four months) Seeds showing testa, embryo, embryonic and suspender cells evidenced by reaction to PAS are observed. Arrow indicates the lack of reaction with PAS in the suspender; c. (six months) Seed showing testa, embryo, embryonic and suspender cells reactive to PAS. Lack of reaction with the suspender cells can be perceived, pointing to its degeneration (arrow); d-f. Temperature of -20 °C - d. (two months) Seeds showing testa, embryo, embryonic and suspender cells reactive to PAS. Suspender cells show changes evidenced by the reaction to PAS (arrow); e. (four months) Seed showing reactive testa and embryo and embryonic cells poorly reactive to PAS. Alterations in the suspender cells can be noted evidenced by the reaction with the dye (arrow); f. (six months) Seed showing different intensities of reaction to PAS, reactive testa and embryo and embryonic and suspender cells poorly reactive to PAS can be observed. Changes in the suspender cells reactive to PAS are noted (arrow); g-i. Temperature of -80 °C - g. (two months) Seed showing different intensities of reaction in the embryo and embryonic cells with PAS; h. (four months) Seed evidencing embryo and embryonic cells poorly reactive to PAS. Alterations in the suspender cells evidenced by the reaction with the dye are noted (arrow); i. (six months) Seed showing different intensities of reaction in the embryo and embryonic cells with PAS. Changes can be seen in the cells of the embryo, due to less reaction with PAS (arrowheads); j-l. Temperature of -196 °C - j. (two months) Seed showing embryo, embryonic and suspender cells reactive to PAS. Alterations in the suspender cells reactive to the dye are noted (arrow); k. (four months) Seed showing testa, embryo and embryonic cells reactive to PAS. Changes in the embryonic cells (arrowhead), due to the lack of reaction with the dye and absence of reaction to PAS in the suspender (arrow) can be observed; l. (six months) Seeds showing different intensities of reaction to PAS in the embryo and embryonic cells, evidencing their degeneration. Changes in the embryonic cells (arrowhead), due to the poor reaction with the dye and seed with absence of reaction to PAS in the suspender (arrow) can be noted. Cepa = embryonic cells reactive to PAS; Cspa = suspender cells reactive to PAS; Ctpa = testa cell reactive to PAS; Emb = embryo; PAS = periodic acid-Schiff; Te = testa. Scale bar: a-d, l = 500 µm; e-k = 200 µm.

Temperature	Months	Observed description
25 (± 2 °C) (room)	two	Seed dehydration (Fig. 2a)
25 (± 2 °C) (room)	four	Seed dehydration (Fig. 2b)
25 (± 2 °C) (room)	six	Dehydration was more intense (Fig. 2c)
-20 °C (freezer)	two	The viable seeds were characterized by having the region close to the swollen embryo, while the non-viable seeds showed damage by dehydration (Fig. 2d-f)
-20 °C (freezer)	four
-20 °C (freezer)	six
-80 °C (ultra freezer)	two	The viable seeds were characterized by having a swollen area, close to the embryo, and the non-viable seeds showed damage by dehydration (Fig. 2g-i)
-80 °C (ultra freezer)	four
-80 °C (ultra freezer)	six
-196 °C (cryopreservation)	two	Viable seeds, with the region of the embryo swollen and non-viable showing damage by dehydration (Fig. 2j-k)
-196 °C (cryopreservation)	four
-196 °C (cryopreservation)	six	In the viable seed, a rupture in the integument was observed (Fig. 2l)

Temperature	Months	Observed description
25 (± 2 °C) (room)	two	Seed showed the integument and embryo cells, but little reactive to TBO vacuolization of the suspender cells is also observed, due to the lack of reaction with the dye (Fig. 3a)
25 (± 2 °C) (room)	four	Seeds showed little reactive embryo, with apparent vacuolization in the embryo cells, due to the degeneration process (Fig. 3b)
25 (± 2 °C) (room)	six	It can be noted little reaction from the embryo cells with the TBO, in addition to an absence of reaction in the suspender cells, due to the ir degeneration (Fig. 3c)
-20 °C (freezer)	two	Seeds with the chalazal end and the superficial cells of the testa evidenced by reaction with TBO. There is evidence of vacuolization in the embryo cells, due to the lack of reaction with the dye, in addition to the suspender and the other cells with different intensities of reaction with the TBO (Fig. 3d)
-20 °C (freezer)	four	Seeds show reactive embryo cells and the suspender with apparent degeneration, due to less reaction with the reagent (Fig. 3e)
-20 °C (freezer)	six	Seed evidenced the dehydrated testa, and the embryo cells reactive to TBO. The absence of the suspender is observed, due to the lack of reaction with the TBO (Fig. 3f)
-80 °C (ultra freezer)	two	Embryo cells with different intensities of reaction with TBO. Damage to the testa structure and apparent degeneration of the suspender cells were also observed, due to the lack of reaction with TBO (Fig. 3g)
-80 °C (ultra freezer)	four	Embryo cells reactive to TBO and the absence of reaction in the suspender cells (Fig. 3h)
-80 °C (ultra freezer)	six	Seeds presented different reactions with TBO in the embryonic cells and their apparent vacuolization. Absence and degeneration of the suspender structure is observed, due to the lack of reaction with the suspender cells in one of the seeds (Fig. 3i)
-196 °C (cryopreservation)	two	It is possible to notice empty seeds, due to the lack of reaction with the TBO in the internal structures of the seeds, while in the the preserved seed, it is noted reaction of the embryo cells with the TBO and the suspender cells show different intensities of reaction, evidencing degeneration of the cell walls (Fig. 3j)
-196 °C (cryopreservation)	four	Vacuolization in the embryo cells is observed, they are evidenced by the lack of reaction with the TBO and the suspender in degeneration, showing cells less reactive to the dye used (Fig. 3k)
-196 °C (cryopreservation)	six	Embryo cells with different intensities of reaction with the TBO, in addition to seeds in degeneration or lack of suspender, due the less intensity or lack of reaction of the cells with the dye used (Fig. 3l)

Temperature	Months	Observed description
25 (± 2 °C) (room)	two	Embryos poorly reactive to proteins, due to the degeneration of these structures. Absence of suspender can be observed, due to the lack of reaction with the dye (Fig. 4a)
25 (± 2 °C) (room)	four	Seeds show the testa, and embryo cells reactive to proteins. It also can be noted the degeneration of the embryo and suspender structure, due to the lack of reaction with CBB (Fig. 4b)
25 (± 2 °C) (room)	six	Seed with embryo cells reactive to proteins. Again, embryo degeneration and absence of suspender can be noted, due to the less intensity or lack of reaction of the cells with the dye (Fig. 4c)
-20 °C (freezer)	two	Embryo cells with prominent nuclei were evidenced by the reaction with CBB, as well as the absence of the suspender structure due to the lack of reaction with CBB (Fig.4d)
-20 °C (freezer)	four	Embryo cells reactive to proteins. An embryo in degeneration and absence of suspender can be noted, due to the lower intensity or lack of reaction of the cells with the dye (Fig. 4e)
-20 °C (freezer)	six	Seed with embryo in degeneration can be observed, while the embryon and cells appear to be less reactive to CBB. A rupture of the testa is also noticed (Fig. 4f)
-80 °C (ultra freezer)	two	Structures of the embryo, are observed by the reaction with CBB. The embryo cells show ed prominent nuclei due to the intense reaction with this dye (Fig. 4g-h)
-80 °C (ultra freezer)	four
-80 °C (ultra freezer)	six	Degeneration of the embryo evidenced by the lack of reaction with CBB (Fig. 4i)
-196 °C (cryopreservation)	two	Embryo reactive to proteins. Degeneration of the embryo cells can be noted, due to the lack of reaction with CBB (Fig. 4j-k)
-196 °C (cryopreservation)	four
-196 °C (cryopreservation)	six	Degeneration of the embryo is noticed, as well as the absence of the suspender, due to the lower intensity or lack of reaction of the cells with this dye (Fig. 4l)

Temperature	Months	Observed description
25 (± 2 °C) (room)	two	Seeds show the testa, micropylar and chalazal end, however, there is no reaction with Sudan IV in the embryo (Fig. 5a-c)
25 (± 2 °C) (room)	four
25 (± 2 °C) (room)	six
-20 °C (freezer)	two	Seeds with the embryo reactive to Sudan IV and the absence of the suspender can be observed, due to the lack of reaction with this dye. A seed with the embryo poorly reactive to Sudan IV can be noted (Fig. 5d)
-20 °C (freezer)	four	Seed shows the embryo cells reactive to Sudan IV, however, degeneration of these structures is seen, due to the lack of reaction with the chemical (Fig. 5e)
-20 °C (freezer)	six	Seed with the embryo reactive to the product can be noted, however, the embryo cells show degeneration, due to the lack of reaction with Sudan IV. Also, a seed with total lack of reaction with Sudan IV in the embryonic area is observed (Fig. 5f)
-80 °C (ultra freezer)	two	Seed with lack of reaction to Sudan IV and a seed with the embryo and the suspender cells reactive to the dye (Fig. 5g)
-80 °C (ultra freezer)	four	The reaction to Sudan IV in the embryo cells is evidenced (Fig. 5h)
-80 °C (ultra freezer)	six	Degeneration of the embryo cells is noted, due to the lack of reaction with the dye (Fig. 5i)
-196 °C (cryopreservation)	two	Embryo structure and the suspender reactive to Sudan IV (Fig. 5j)
-196 °C (cryopreservation)	four	Seeds with the embryo reactive to Sudan IV and the suspender poorly reactive to the dye can be observed, evidencing the degeneration. Yet, it is noticed a seed with a complete lack of reaction to Sudan IV in the embryo (Fig. 5k)
-196 °C (cryopreservation)	six	Seed with the embryo reactive to Sudan IV is perceived, however, the embryo cells are in degeneration, due to the lack of reaction to the dye (Fig. 5l)

Temperature	Months	Observed description
25 (± 2 °C) (room)	two	Reaction to PAS in the structures of the testa cells, embryo, embryo cells and suspender cells (Fig. 6a)
25 (± 2 °C) (room)	four	Absence of the suspender, due to the lack of reaction with PAS (Fig. 6b)
25 (± 2 °C) (room)	six	Degeneration in the suspender cells can be seen, due to the lack of reaction with the dye (Fig. 6c)
-20 °C (freezer)	two	Seeds show testa, embryo, and suspender cells, due to the reaction with the dye. Changes in embryo cells can be seen, evidenced by the reaction with PAS (Fig. 6d)
-20 °C (freezer)	four	Seed with a reactive testa can be observed, while the embryo cells seem to be poorly reactive to PAS, alterations in the suspender cells evidenced by the reaction with the dye can also be noted (Fig. 6e)
-20 °C (freezer)	six	Seeds show different intensities of reaction with PAS, reactive testa and embryonic and suspender cells poorly reactive to the chemical are noted, evidencing these structures degeneration. The suspender cells show changes evidenced by the reaction to PAS (Fig. 6f)
-80 °C (ultra freezer)	two	Different intensities of reaction with PAS in the embryo cells (Fig. 6g)
-80 °C (ultra freezer)	four	Seeds showed to be poorly reactive to PAS in the embryo structure and embryonic cells. It was possible to note changes in the suspender cells, evidenced by the reaction to PAS (Fig. 6h)
-80 °C (ultra freezer)	six	Seeds showed different intensities of reaction to PAS in the embryo cells, as well as alterations in the embryonic cells, caused by the less reaction with the dye, that way pointing to degeneration of these cells (Fig, 6i)
-196 °C (cryopreservation)	two	Embryo and suspender cells reactive to PAS, some changes in the suspender cells are observed (Fig. 6j)
-196 °C (cryopreservation)	four	Seeds show reaction to PAS in the embryo and suspender cells, also showing alterations in the embryonic cells and absence of the suspender, due to the less or lack of reaction with PAS (Fig. 6k)
-196 °C (cryopreservation)	six	Seeds show different intensities of reaction to PAS for the embryo cells, which indicates their degeneration. Changes in the embryo cells and absence of the suspender are noted, due to the lack or less reaction with the dye (Fig. 6l)

[1] 7Author for correspondence: marianais1994@gmail.com

Brasil

Brasil

Anatomical and histochemical characterization of seeds of Cattleya intermedia subjected to different storage conditions

Abstract