Cryopreservation of lIex Immature Zygotic Embryos

LUIS MROGINSKI; NATALIA DOLCE; PEDRO SANSBERRO; CLAUDIA LUNA; ANA GONZALEZ; HEBE REY

Plant Embryo Culture Methods and Protocols

Trevor A. Thorpe and Edward C. Yeung

Lugar: Colgary, Alberto; Año: 2011; p. 215 - 225

Tropical I!exspecies have recalcitrant sceds. This chap tcr describes pro tocola tal' long- rerrn conservaría n oftal' long- rerrn conservaría n of Ilex brnsiliensis; 1. brcvicuspis; l. rilt1JIOSa, l. mícrodonia; l . i nteq erri ma, 1. parnJ]uarienJis, 1. pseudoboxus; 1. taubcvtintuv; and l . theezans through crvoprcscrvation of zygotic rudimcntarv emb ryos ar rhe hearr dcvclopmcntal stagc. Thc cmbryos are asepticallv removed from thc sccds and prcculturcd (7 days) in thc dar k at 27 ± 2°e on solidificd quartcr-strcngth Murashigc and Skoog médium wirh 3%sucrose and 0. 1 mg/L zeatin. Thc embryos are then cncapsulared in 3'){! calcium alginate beads and pretreatcd at 24-h in rcrvals in liqnid medium supplcrucnrcd wirh progrcssivclv increasing sucrose concenrratio ns (0,5, 0.75, and 1 .\1). The beads are deh ydrare d for 5 h wirh silica gel ro 25%water contcnt (fresh weight basis) and rhen placed in stc rilc 5-mL crvovials. Th en thc bcads are cirber plungcd rapidlv in liq uid nitrogen whcrc thcy are kepr for 1 h (rapid cooling), or cooled at I°C/min ro -30°C and then immcrs cd in liquid nitrogcn for 1 h (slow cooling). ?Afie- cryoprcscrvation , rhe beads Me rewarmed by immersion ofthe crvovials for 1 min in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moVtaubcvtintuv; 1. brcvicuspis; l. rilt1JIOSa, l. mícrodonia; l . i nteq erri ma, 1. parnJ]uarienJis, 1. pseudoboxus; 1. taubcvtintuv; and l . theezans through crvoprcscrvation of zygotic rudimcntarv emb ryos ar rhe hearr dcvclopmcntal stagc. Thc cmbryos are asepticallv removed from thc sccds and prcculturcd (7 days) in thc dar k at 27 ± 2°e on solidificd quartcr-strcngth Murashigc and Skoog médium wirh 3%sucrose and 0. 1 mg/L zeatin. Thc embryos are then cncapsulared in 3'){! calcium alginate beads and pretreatcd at 24-h in rcrvals in liqnid medium supplcrucnrcd wirh progrcssivclv increasing sucrose concenrratio ns (0,5, 0.75, and 1 .\1). The beads are deh ydrare d for 5 h wirh silica gel ro 25%water contcnt (fresh weight basis) and rhen placed in stc rilc 5-mL crvovials. Th en thc bcads are cirber plungcd rapidlv in liq uid nitrogen whcrc thcy are kepr for 1 h (rapid cooling), or cooled at I°C/min ro -30°C and then immcrs cd in liquid nitrogcn for 1 h (slow cooling). ?Afie- cryoprcscrvation , rhe beads Me rewarmed by immersion ofthe crvovials for 1 min in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moVtaubcvtintuv; and l . theezans through crvoprcscrvation of zygotic rudimcntarv emb ryos ar rhe hearr dcvclopmcntal stagc. Thc cmbryos are asepticallv removed from thc sccds and prcculturcd (7 days) in thc dar k at 27 ± 2°e on solidificd quartcr-strcngth Murashigc and Skoog médium wirh 3%sucrose and 0. 1 mg/L zeatin. Thc embryos are then cncapsulared in 3'){! calcium alginate beads and pretreatcd at 24-h in rcrvals in liqnid medium supplcrucnrcd wirh progrcssivclv increasing sucrose concenrratio ns (0,5, 0.75, and 1 .\1). The beads are deh ydrare d for 5 h wirh silica gel ro 25%water contcnt (fresh weight basis) and rhen placed in stc rilc 5-mL crvovials. Th en thc bcads are cirber plungcd rapidlv in liq uid nitrogen whcrc thcy are kepr for 1 h (rapid cooling), or cooled at I°C/min ro -30°C and then immcrs cd in liquid nitrogcn for 1 h (slow cooling). ?Afie- cryoprcscrvation , rhe beads Me rewarmed by immersion ofthe crvovials for 1 min in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moV± 2°e on solidificd quartcr-strcngth Murashigc and Skoog médium wirh 3%sucrose and 0. 1 mg/L zeatin. Thc embryos are then cncapsulared in 3'){! calcium alginate beads and pretreatcd at 24-h in rcrvals in liqnid medium supplcrucnrcd wirh progrcssivclv increasing sucrose concenrratio ns (0,5, 0.75, and 1 .\1). The beads are deh ydrare d for 5 h wirh silica gel ro 25%water contcnt (fresh weight basis) and rhen placed in stc rilc 5-mL crvovials. Th en thc bcads are cirber plungcd rapidlv in liq uid nitrogen whcrc thcy are kepr for 1 h (rapid cooling), or cooled at I°C/min ro -30°C and then immcrs cd in liquid nitrogcn for 1 h (slow cooling). ?Afie- cryoprcscrvation , rhe beads Me rewarmed by immersion ofthe crvovials for 1 min in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moV-30°C and then immcrs cd in liquid nitrogcn for 1 h (slow cooling). ?Afie- cryoprcscrvation , rhe beads Me rewarmed by immersion ofthe crvovials for 1 min in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moVmin in a wate r bath at 30°C. Finally, rhc beads are transferred onto culture mcdium (1/ 4.\15, 3(%sucrosc, and 0. 1 mg/L zcatin, solidificd with 0.8% agar) and incubared in a grow rh room ar 27 ±2cC undc r a 14-h ligh t (116 ~moVcC undc r a 14-h ligh t (116 ~moV m2/s) and 10-h dark pho topcriod . Máximum recovery pcrccnrages bctween 1S and 83% (depending 011 rhe spccics and thc trcatmcnt) were obrained wirh thc cryopreserved cmbrvos.2/s) and 10-h dark pho topcriod . Máximum recovery pcrccnrages bctween 1S and 83% (depending 011 rhe spccics and thc trcatmcnt) were obrained wirh thc cryopreserved cmbrvos.