Ultra-low temperature conservation of Brazilian Pine embryogenic cultures

This study aimed to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, enabling the ex situ conservation of the species. Embryogenic cultures were established from immature seeds and treated with variations of the cryoprotectant solutions SuDG, SoD and PVS2 prior to immersion in liquid nitrogen. Cell viability was evaluated after 30, 60 and 90 days of re-growth. The highest re-growth without morphological alterations and with normal biochemical composition was obtained with the PVS2 solution with 40 min immersion in ethanol (-20 °C). This procedure opens new horizons for the ex situ conservation of the species genetic.

cryopreservation; ex situ conservation; in vitro culture; plant germplasm conservation

Authorship

GRASIELA DEMARCHI

Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal, Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, Itacorubi, 88034-000 Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilLaboratório de Fisiologia do Desenvolvimento e Genética Vegetal, Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, Itacorubi, 88034-000 Florianópolis, SC, Brasil

VALDIR M. STEFENON

Centro Interdisciplinar de Pesquisas em Biotecnologia, Universidade Federal do Pampa, Campus São Gabriel, Av. Antonio Trilha, 1847, 97300-000 São Gabriel, RS, BrasilCampus São GabrielBrasilSão Gabriel, RS, BrasilCentro Interdisciplinar de Pesquisas em Biotecnologia, Universidade Federal do Pampa, Campus São Gabriel, Av. Antonio Trilha, 1847, 97300-000 São Gabriel, RS, Brasil

NEUSA STEINER

Laboratório de Fisiologia Vegetal, Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Rua Engenheiro Agrônomo Andrey Cristian Ferrei, s/n, Trindade, 88040-970 Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilLaboratório de Fisiologia Vegetal, Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Rua Engenheiro Agrônomo Andrey Cristian Ferrei, s/n, Trindade, 88040-970 Florianópolis, SC, Brasil

FELIPE N. VIEIRA

Laboratório de Camarões Marinhos, Departamento de Aquicultura, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, Itacorubi, 88034-000 Florianópolis, SC, BrasilUniversidade Federal de Santa CatarinaBrasilFlorianópolis, SC, BrasilLaboratório de Camarões Marinhos, Departamento de Aquicultura, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, Itacorubi, 88034-000 Florianópolis, SC, Brasil

LIRIO L. DAL VESCO

Universidade Federal de Santa Catarina, Campus Curitibanos, Rodovia Ulysses Gaboardi, Km 3, 89520-000 Curitibanos, SC, BrasilCampus CuritibanosBrasilCuritibanos, SC, BrasilUniversidade Federal de Santa Catarina, Campus Curitibanos, Rodovia Ulysses Gaboardi, Km 3, 89520-000 Curitibanos, SC, Brasil

MIGUEL P. GUERRA

Correspondence to: Miguel Pedro Guerra E-mail: mpguerra@cca.ufsc.br

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (1)

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Figure 1
Fresh mass of the A. angustifolia embryogenic cultures after 30, 60 and 90 days of re-growth in BM medium treated with SuDG, SoD and PVS2 cryoprotectant solutions. Columns with different letters in the same sub-culture (30, 60 and 90 days) are statistically different at 5% according to SNK test.

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Figure 2
Cytochemical analysis of the A. angustifoliaembryogenic cultures after 48 h of cryopreservation in liquid nitrogen. A, non-frozen embryogenic culture. Bar = 66.7 µm. B, embryogenic culture with responsive cells after cryopreservation. Bar = 1.257 mm. C, embryogenic culture without re-growth after 30 days of re-culturing. Bar = 0.587 mm. D, morphological analysis of embryogenic cultures stained with acetic carmim and Evans-blue, evidencing the intact cells of a bipolar somatic pro-embryo. Bar = 33.3 µm. E, morphological appearance of embryogenic cultures tolerant to cryopreservation, stained with acetic carmim and Evans-blue. Bar = 33.3 µm. F, morphological appearance of embryogenic cultures not tolerant to cryopreservation, stained with acetic carmim and Evans-blue. Arrows indicate suspensor cells with degraded walls and overflow of cellular content. Bar = 66.7 µm. G, embryogenic culture before freezing, stained with FDA. Bar = 18.84 µm. H-I, morphological appearance of embryogenic cultures tolerant to cryopreservation, stained with FDA. Arrows indicate viable cells. Bar = 18.84 µm.

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Figure 3
Content of protein and starch of the A. angustifoliaembryogenic cultures, cryopreserved using the PVS2 solution with 40 min in ethanol (-20°C). Columns with different letters in the same sub-culture (0, 16 and 32 days) are statistically different at 5% according to SNK test.

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TABLE I
Composition of the cryoprotective solutions evaluated

Figure 1 Fresh mass of the A. angustifolia embryogenic cultures after 30, 60 and 90 days of re-growth in BM medium treated with SuDG, SoD and PVS2 cryoprotectant solutions. Columns with different letters in the same sub-culture (30, 60 and 90 days) are statistically different at 5% according to SNK test.

Figure 2 Cytochemical analysis of the A. angustifoliaembryogenic cultures after 48 h of cryopreservation in liquid nitrogen. A, non-frozen embryogenic culture. Bar = 66.7 µm. B, embryogenic culture with responsive cells after cryopreservation. Bar = 1.257 mm. C, embryogenic culture without re-growth after 30 days of re-culturing. Bar = 0.587 mm. D, morphological analysis of embryogenic cultures stained with acetic carmim and Evans-blue, evidencing the intact cells of a bipolar somatic pro-embryo. Bar = 33.3 µm. E, morphological appearance of embryogenic cultures tolerant to cryopreservation, stained with acetic carmim and Evans-blue. Bar = 33.3 µm. F, morphological appearance of embryogenic cultures not tolerant to cryopreservation, stained with acetic carmim and Evans-blue. Arrows indicate suspensor cells with degraded walls and overflow of cellular content. Bar = 66.7 µm. G, embryogenic culture before freezing, stained with FDA. Bar = 18.84 µm. H-I, morphological appearance of embryogenic cultures tolerant to cryopreservation, stained with FDA. Arrows indicate viable cells. Bar = 18.84 µm.

Figure 3 Content of protein and starch of the A. angustifoliaembryogenic cultures, cryopreserved using the PVS2 solution with 40 min in ethanol (-20°C). Columns with different letters in the same sub-culture (0, 16 and 32 days) are statistically different at 5% according to SNK test.

TABLE I Composition of the cryoprotective solutions evaluated

	Control
T0	Non-cryopreserved cultures, free of cryoprotective treatments
	SuDG
T1	Sucrose 20% + DMSO 0% + Glycerol 20%
T2	Sucrose 20% + DMSO 10% + Glycerol 20%
T3	Sucrose 0% + DMSO 10% + Glycerol 20%
T4	Sucrose 20% + DMSO 10% + Glycerol 0%
T5	Sucrose 0% + DMSO 0% + Glycerol 20%
T6	Sucrose 0% + DMSO 10% + Glycerol 0%
T7	Sucrose 10% + DMSO 0% + Glycerol 0%
	SoD
T8	Sorbitol 0.3M + DMSO 10%
T9	Sorbitol 0.4M + DMSO 10%
T10	Sorbitol 0.4M + DMSO 0%
T11	Sorbitol 0.3M + DMSO 0%
T12	Sorbitol 0.3M + DMSO 5%
T13	Sorbitol 0.4M + DMSO 5%
	PVS2
T14	Ethylene glycol 15%+Sucrose 0.4M+Glycerol 30%+DMSO 15% (40 min in Ethanol -20°C)
T15	Ethylene glycol 15%+Sucrose 0.4M+Glycerol 30%+DMSO 15% (30 min in Ethanol -20°C)
T16	Ethylene glycol 15%+Sucrose 0.4M+Glycerol 30%+DMSO 15% (20 min in Ethanol -20°C)
T17	Ethylene glycol 15%+Sucrose 0.4M+Glycerol 30%+DMSO 15% (10 min in Ethanol -20°C)

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Ultra-low temperature conservation of Brazilian Pine embryogenic cultures

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[1] Correspondence to: Miguel Pedro Guerra E-mail: mpguerra@cca.ufsc.br