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Development of V and D cryo-plate methods
Takao Niino (孝男 新野)Private adviser (Plant tissue culture & Cryopreservation)Former Senior Researcher, *Gene Research Center, University of Tsukuba JICA/JST SATREPS Project, Diversity Assessment and Development ofSustainable Use of Mexican Genetic Resources*Genebank, National Institute of Agrobiological Sciences (NIAS)
Vitrification is the only survival mechanism for maintaining the viability of hydrated cells and tissues in the temperature of LN by avoiding crystallization
Room temperature
Super low temperature
cooling warming Avoid the lethal intracellular freezing
(crystallization)
Requirements
Rapid cooling and Rapid warming
Sufficient dehydration
Every cryopreservation methods use this vitrification phenomenon
“The physical process by which the transition of water directly from the liquid phase into an amorphous phase or glass without the occurrence of crystallization (ice crystal).”
Conventional vitrification procedures including droplet vitrification (DV)
Subculture
Hardening
Preculture
Dehydration by PVS2 Require a precise control of treatment time
Immersion in LN
V
DV
Osmoprotection by LS solution
Repeated pipettingSmall size shoot tips are suspended in the various solutions employed Results in damage and loss of
shoot tips during the steps
Dehydrated shoot tips need to be transferred on aluminium strips
All these disadvantages may impede the implementationof large scale cryostorage.
Establishment an efficient and simple cryopreservation procedure.
We started development of a new, simple protocol from 2009.
Goal of development• The possibility of injuring and
losing shoot tips is reduced. • Shoot tips can be efficiently
treated with LS and PVS2.• Cooling and warming are
performed easily by direct immersion in LN and warming solution.
• High regrowth.
The main aim:The shoot tips to be cryopreserved should adhere on a carrier.Each steps of the cryopreservation protocol should be performed with this carrier including immersion into LNand warming solution.
Direct immersion
PUERTO LN
AIR DRY
PVS2LS
Rewarming
Device of cryo-plate
• The carrier (cryo-plate) should be hard, reusable with a high thermal conductivity.
• Adhere the shoot tips firmly with alginate gel to the cryo-plates throughout the whole procedure for efficient performance.
• Fit in 2 ml cryotube.• Direct immersion in LN or
warming solution easily.
Aluminium（A1050,99.5%)
Device of cryo-plate
No. 0 No. 1 No. 2 No. 3 No. 4
Well：No. 1: ø 1 mm, H 0.5 mm,No. 2: ø 1.5 mm, H 0.75 mm,No. 3: oval（2.5 mm x 1.5 mm), H 0.75 mm No. 4: ø 3.0 mm, H 0.75 mm
The cryo-plates are custom-made by Taiyo Nippon Sanso Corp Tokyo, Japan. Contact person: Mr. Kouji Sekita, e-mail: [email protected]
Vitrification method using cryo-plate(V cryo-plate method) of potato
In vitro shoot tips of potato
Excision shoot tips (1.5 mm long) and preculture on medium with 0.3 M Suc.
Adhere the shoot tips on cryo-plate
Polymerization of alginate gel
Osmoprotection by2 M Gly ＋ 0.8 M Suc. 30 minDehydration byPVS2 30 min
Setting of a cryo-plate incryotube
Immersion into LN Rewarming for
regeneration1 M Suc solution 15 min
Plating
Regrowth
Vitrification method using cryo-plate(V cryo-plate method)
Regrowth (%) of shoot tips of 16 potato varieties/lines cryopreserved using optimized the V cryo-plate procedure.(Yamamoto et al., 2015)
Variety/LineWhitefryer 100.0 ± 0North Chip 100.0 ± 0Kitahime 96.7 ± 5.8Tokachi Kogane 100.0 ± 0Ohoutsuku Chip 93.3 ± 5.8Snow March 100.0 ± 0May Queen 96.7 ± 5.8Natsufubuki 100.0 ± 0Danshakuimo 100.0 ± 0Snowden 100.0 ± 0Inca-no-Mezame 100.0 ± 0Waseshiro 100.0 ± 0S. chacoence 34 96.7 ± 5.8S. phureja 114 100.0 ± 0S. bulbocastanum 96.7 ± 5.8S. pinnatisectum 109 96.7 ± 5.8
Regrowth (%±SE.)
Regrowth of cryopreserved buds of 20 mat rush lines using V-Cryo-plate protocol
Variety Regrowth (% ± SEM)V-Cryo-plate D-Cryo-plate
Hiroshima 5 gou 86.7 ± 3.3 86.7 ± 3.3 Okayama Okei 83.3 ± 3.3 83.3 ± 3.3
Numakuwajisei B 83.3 ± 3.3 80.0 ± 5.8 Toshin 80.0 ± 5.8 86.7 ± 3.3
Okayama Ikei 80.0 ± 5.8 96.7 ± 3.3 Kijokazairai B 66.7 ± 3.3 96.7 ± 3.3 Kijokazairai A 66.7 ± 5.8 90.0 ± 5.8 Okayama Nkei 60.0 ± 5.8 93.3 ± 3.3
Onomichijisei C 56.7 ± 3.3 80.0 ± 5.8 Takasuzairai 1 50.0 ± 5.8 90.0 ± 5.8
Asanagihen 50.0 ± 5.8 73.3 ± 3.3 Sendaifutoi 50.0 ± 5.8 73.3 ± 3.3 Ohara 4 gou 46.7 ± 14.5 86.7 ± 3.3 Imoozairai 43.3 ± 13.3 86.7 ± 3.3 Kiyonami 40.0 ± 11.5 93.3 ± 5.8
Hiroshima 3 gou-2 26.7 ± 6.7 80.0 ± 0.0 Okayama 3 gou-1 26.7 ± 6.7 93.3 ± 3.3
Seto 7 gou 23.3 ± 8.8 76.7 ± 3.3 Hiroshima 4 gou-1 16.7 ± 3.3 86.7 ± 3.3 Okayama 2 gou-2 13.3 ± 3.3 93.3 ± 3.3
Average 52.5 86.3 Max-Min 86.7-13.3 96.7-73.3
Regrowth (%) of buds of mat rush lines “Hiroshima 5 gou” and “Okayama 2 gou(2)” after loading treatment (LS), dehydration with PVS2 (LS + PVS2) and cryopreservation (LS +PVS2 + LN) using the V cryo-plate protocol (Niino et al., 2013 rewrote).
Treatment by LS
Air dehydration using aluminum cryo-plates (1)
Cryo-plateAdhesion buds on cryo-plates
Place the preculture shoot tips in the well
Immersion in LN
Desiccation in laminar cabinet
1 2 3
Preculture
45
6
Rewarming
Air dehydration using aluminum cryo-plates (2)
Plating
Regeneration
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
0 1 2 3 4
Water contents
(%)
Time (hr)
Time course of water contents of buds with alginate gel on the plate
Water content： 0 hr 53%, 1 hr 33%, 2 hr 27%, 3 hr 25%, 4 hr 24%.
Excision shoot tips, Preculture
Adhere the shoot tips on cryo-plate
Osmoprotection by LS (2 M Gly＋0.8 M Suc)30 min
Immersion into LNRewarm and PlatingDehydration (150 min) by laminar air flow in cabinet or by silica gel in a Petri dish
Dehydration method using cryo-plate(D cryo-plate method) of mat rush
Regrowth of cryopreserved buds of 20 mat rush lines using V and D cryo-plate protocols (Niino et al., 2013)
Variety Regrowth (% ± SE)V Cryo-plate D Cryo-plate
Hiroshima 5 gou 86.7 ± 3.3 86.7 ± 3.3 Okayama Okei 83.3 ± 3.3 83.3 ± 3.3
Numakuwajisei B 83.3 ± 3.3 80.0 ± 5.8 Toshin 80.0 ± 5.8 86.7 ± 3.3
Okayama Ikei 80.0 ± 5.8 96.7 ± 3.3 Kijokazairai B 66.7 ± 3.3 96.7 ± 3.3 Kijokazairai A 66.7 ± 5.8 90.0 ± 5.8 Okayama Nkei 60.0 ± 5.8 93.3 ± 3.3
Onomichijisei C 56.7 ± 3.3 80.0 ± 5.8 Takasuzairai 1 50.0 ± 5.8 90.0 ± 5.8
Asanagihen 50.0 ± 5.8 73.3 ± 3.3 Sendaifutoi 50.0 ± 5.8 73.3 ± 3.3 Ohara 4 gou 46.7 ± 14.5 86.7 ± 3.3 Imoozairai 43.3 ± 13.3 86.7 ± 3.3 Kiyonami 40.0 ± 11.5 93.3 ± 5.8
Hiroshima 3 gou-2 26.7 ± 6.7 80.0 ± 0.0 Okayama 3 gou-1 26.7 ± 6.7 93.3 ± 3.3
Seto 7 gou 23.3 ± 8.8 76.7 ± 3.3 Hiroshima 4 gou-1 16.7 ± 3.3 86.7 ± 3.3 Okayama 2 gou-2 13.3 ± 3.3 93.3 ± 3.3
Average 52.5 86.3 Max-Min 86.7-13.3 96.7-73.3
Achievement of cryopreservation methods using cryo-plates
References
Av. Range Av. Range
Mat rush 20 53 13-87 20 86 73-97 Shoot tip Niino et al (2013, 2014)
Dalmatia chrysanthemumn 7 77 65-90 - Shoot tip Yamamoto et al (2011)
Mint 17 89 73-100 - Shoot tip Yamamoto et al (2012)
Carnation 4 95 93-97 - Shoot tip Sekizaw a et al (2011)
Strawberry 15 81 70-97 - Shoot tip Yamamoto et al (2012)
Mulberry 13 87 73-100 10 79 73-90 Shoot tip, WB Yamamoto et al (2012)
Potato 17 99 93-100 17 92 80-100 Shoot tip Yamamoto et al (2015)
Sugarcane 12 70 57-100 12 52 20-100 Shoot tip Tariq et al. (2015, 2016)
Perilla 1 80 80 - Shoot tip Matsumoto et al (2014)
Date palm - 2 86 75-96 PEMs Salma et al (2014)
Clinopodium odorum 1 71 71 1 29 29 Shoot tip Engelmann-Sylvestre & Engelmann (2015)
Persimon - 10 87 67-97 WB Matsumoto et al (2015)
Bluberry - 10 80 43-100 WB,shoot tip Dhungana et al (2015)
Cleome rosea 1 100 100 - Shoot tip de Silva Cordeiro et al (2015)
Cherry plum 1 65 65 1 70 70 Shoot tip Vujovic et al (2015)
Liverwort 1 100 100 1 100 100 Gemmae Tanaka et al (2015)
Chrysanthemumn - 7 88 67-100 Shoot tip Tanaka et al (2016)
Arundina graminifolia - 1 77 77 Protocom Cordova & Thammasiri (2016)
Petiveria alliacea - 1 85 85 SEs de Almeida Pettinelli et al (2017)
Ulluco 1 43 43 11 90 73-97 Shoot tips Valle Arizaga et al (2017)
Rakkyo 1 100 100 Tanaka et al (2018)
Dendrobium signatum Rchb.f 1 56 56 1 50 50 Pollinia Jitsopakul et al (2018)
Arundina graminifolia 1 71 71 1 82 82 Seeds Thammasiri et al (2018)
Cassava 1 77 77 Shooy yips Yamamoto et al (2018)
Chayote 11 57 43-77 Shoot tips Valle Arizaga et al (2018)
PlantD cryo-plate
No. oflines
Regrowth (%) SpecimensV cryo-plate
No. oflines
Regrowth (%)
Main advantages of V and D cryo-plate
• Handling of shoot tips is very easy. The possibility of injuring and losing shoot tips is considerably reduced.
• Shoot tips on cryo-plate can be efficiently treated by LS and PVS2
• Cooling and warming are performed easily by immersing the cryo-plate.
• High regrowth• The window of optimal dehydration duration is broad.• All staff can implement this procedure after a little
practice• Addition of D cryo-plate: Larger specimens can be
used, avoiding use of PVS2
MethodRegrowth (% ± SEM)
Direct immersion Immersion in cryotube
V-Cryo-plate 80.0 ± 5.8 40.0 ± 5.8
D-Cryo-plate 93.3 ± 3.3 53.3 ± 6.7
Effect of cooling rate on regrowth of cryopreserved mat rush buds by V-Cryo-plate method and D-Cryo-plate
Note: Buds (Ohara 4 No.2) were excised, precultured for overnight at 25°C on MS with 0.3 M sucrose, loaded in 2.0 M glycerol and 1.0 M sucrose solution for 30 min at 25°C, dehydrated by PVS2 for 40min at 25°C (V-Cryo-plate method) and desiccated for 3 hrs at 25°C in clean bench (D-Cryo-plate method). Ten shoot tips were tested for each of the three replicates. Different letters indicate significant differences (P<0.05) using the Tukey Test.
1. Rapid cooling and warming Direct immersion in LN and I M sucrose solution
Comparison of regrowth after cryopreservation using different protocols
Crop V cryo-plate D cryo-plate Droplet vitrification Vitrification Reference52.5% 86.3% 63.3%
13.3-86.7% 73.3-96.7% Non-data 30.0-90.0%20 lines 20 lines 40 lines96.7% 93.3% 82.5% 57.0%
93.0-100.0% 83.0-100.0% 64.0-94.4% 47.0-71.0%16 lines 16 lines 12 lines 14 lines80.0% 57.7% 68.0%
70.0-97.0% Non-data 50.0-66.6% 50.0-93.3%15 lines 3 lines 8 lines
Niino et al (2013) Niinoet al (2007)
Strawberry
Mat rush
PotatoYamamoto et al (2015), Hirai& Sakai (1999), Kim et al(2006)
Pinker et al (2009),Yamamoto et al (2012), Niinoet al (2003)
Optimization of procedures
• When testing the V and D cryo-plate method with a new plant species, we need a slight change of condition of each treatment.
Preparation of material to be cryopreserved Preconditioning Excision Preculture Mounting the shoot tips on cryo-plate Osmoprotection Dehydration with PVS or air flow Storage Regeneration
PreconditioningPreconditioning is necessary to obtain uniform and
actively growing shoot tips for cryopreservation experiments.
In sugarcane, in vitro grown shoots containing shoot tips were cut to a size of 5–7 mm and plated on the medium in Petri dishes for 1 week to obtain uniform material before excision.
In mat rush, small explants were cut from multiple shoots and cultured for 1 week in the standard conditions. The shoots grown in the Petri dishes were then cold-hardened at 5℃ for 1 to 2 months. Buds with a basal stem were dissected from the cold-hardened shoots.
The necessity of cold-hardening is species-specific.
Need No need
V cryo-plate
Dalmatian chrysanthemum,Mat rush, Strawberry,
Potato,Sugarcane,Clinopodium odorum ,Liverwort, Mint, Carnation,Perilla, Cleomaceae , Cherryplum, Plum, Mulberry
D cryo-plate
Mat rush, Chrysanthemum,Persimmon, Ulluco
Sugarcane, Clinopodiumodorum , Liverwort, Cherryplum, Plum, Date palm,Blueberry, Petiveria alliaceaL.
Cold-hardeningProtocol
PrecultureWith the V and D cryo-plate methods, preculture is a crucial step to obtain high regrowth after cryopreservation for all plant species tested.
Effect of cold-hardening and precultureon regrowth of cryopreserved shoot tipsof potato using D cryo-plate protocol(Valle Arizaga et al., 2017, modified).
Plant namePreculture
D cryo-plate V cryo-plate
Mat rush 0.3 M Suc, 16 h at 25℃ 0.3 M Suc, 16 h at 25℃
Potato 0.3 M Suc, 16 h at 24-25℃ 0.3 M Suc, 16 h at 25℃
Sugarcane 0.5 M Suc, 1 day at 25℃ 0.5 M Suc, 1 day at 25℃
Clinopodium odorum 0.3 M Suc, 1 day at 27℃ 0.3 M Suc, 1 day at 27℃
Liverwort 0.3 M Suc, 1 day at 23℃ 0.3 M Suc, 1 day at 25℃
Chrysanthemum 0.3 M Suc, 3 day at 4℃
Cherry plum & Plum 0.3 M Suc, 1 day at 23℃
Persimmon 0.3 M Suc, 2 days at 25℃
Date palm 0.3 M Suc, 3 days at 27℃
Blueberry 0.3 M Suc, 1 day at 25℃
Ulluco 0.3 M Suc, 16 h at 25℃
Petiveria alliacea L. 0.5 M Suc, 1 day at 20-22℃
Dalmatian chrysanthemum 0.5 M Suc, 2 days at 5℃
Strawberry 2.0 M Gly, 0.3 M Suc, 2 days at 5°C
Mint 0.088 M Suc, 1 day at 25℃
Carnation 0.3 M Suc, 2 day at 25℃
Perilla 0.3 M Suc, 1 day at 25℃
Cleomaceae 0.3 M Suc, 1 day at 27℃
Cherry plum & Plum 0.3 M Suc, 1 day at 23℃
Mulberry 0.3 M Suc, 0.2 mg/L BA, 1 day at 25℃
0
20
40
60
80
100
Reg
row
th (%
)
With Without
Cold-hardening and preculture
Cold-hardening
Preculture
Size of shoot tips Plant name MaterialShoot tip length (mm)
D cryo-plate V cryo-plate
Mat rush In vitro buds 1.5-2.0 1.5-2.0
Potato In vitro shoot 2.0-2.5 1.5
Sugarcane In vitro shoot 1.5-2.0 1.5-2.0 Clinopodium
odorum In vitro shoot ～1.0 ～1.0
Liverwort In vitro gemmae 0.3 0.3
Chrysanthemum In vitro shoot 1.5-2.0 Cherry plum &
Plum In vitro shoot 1.5
Persimmon Dormant buds 1
Date palmProembryo-genic masses
3.0-5.0
Blueberry In vitro shoot 1
Ulluco In vitro shoot 1.0-1.5 Petiveria alliacea
L.Somatic embryos SEs
Dalmatian chrysanthemum In vitro shoot 1.0-1.5
Strawberry In vitro shoot 1.0-2.0
Mint In vitro shoot 1.0-1.5
Carnation In vitro shoot 1.0-1.5
Perilla In vitro shoot 1.0
Cleomaceae In vitro shoot ～1.5 Cherry plum &
Plum In vitro shoot 1.5
Mulberry In vitro shoot 1.0-1.5
Effect of size of shoot tips of potato variety ‘Sayaka’ to be cryopreserved on regrowth (%) by V cryo-plate and the D cryo-plate (Yamamoto et al., 2015, modified).
Effect of shoot tipssize of mat rushon regrowth (%)after LN using 3different protocols(Niino et al., 2013,modified).
Osmoprotection(V cryo-plate)
Effect of concentration of sucrose in LS solution on regrowth of cryopreserved shoot tips of potato variety ‘Sayaka’ using the V cryo-plate method (Yamamoto et al., 2014; modified).
Optimal osmoprotection conditions of severalplants using the V cryo-plate method.
Plant name OsmoprotectionClinopodium odorum 2.0 M Gly, 0.4 M Suc, 30 min, 27°CCleomaceae 2.0 M Gly, 0.4 M Suc, 30 min, 27°CCherry plum & Plum 2.0 M Gly, 0.4 M Suc, 30 min, 27°CPerilla 2.0 M Gly, 0.6 M Suc, 20 min, 25°CMulberry 2.0 M Gly, 0.6 M Suc, 30 min, 25°CPotato 2.0 M Gly, 0.8 M Suc, 30 min, 25°CStrawberry 2.0 M Gly, 0.8 M Suc, 30 min, 25°CMint 2.0 M Gly, 0.8 M Suc, 30 min, 25°CMat rush 2.0 M Gly, 1.0 M Suc, 30 min, 25°CLiverwort 2.0 M Gly, 1.0 M Suc, 30 min, 25°CDalmatian chrysanthemum 2.0 M Gly, 1.4 M Suc, 30-60 min, 25°CCarnation 2.0 M Gly, 1.4 M Suc, 90 min, 25°CSugarcane 2.0 M Gly, 1.6 M Suc, 30 min, 25°C
Effect of sucrose concentration in LS on regrowth (%) of mat rush line ‘Kitakei 2’buds cryopreserved (+LN) using the V cryo-plate procedure (Niino et al., 2013, modified).
Osmoprotection(D cryo-plate)
Effect of different LS solutions on regrowth of cryopreserved shoot tips of ulluco by D cryo-plate protocol (Valle Arizaga et al., 2017, modified).
Effect of different duration of LS (1.0M Suc + 2M Gly) treatment on regrowth of cryopreserved shoot tips of ulluco using the D cryo-plate method (Valle Arizaga et al., 2017, modified).
Plant name OsmoprotectionClinopodium odorum 2.0 M Gly, 0.4 M Suc, 20 min, 27°CDate palm 2.0 M Gly, 0.4 M Suc, 20 min, 27°CPetiveria alliacea L. 2.0 M Gly, 0.4 M Suc, 20 min, 20-22°CCherry plum & Plum 1.9 M Gly, 0.5 M Suc, 30 min, 27°CBlueberry 2.0 M Gly, 0.6 M Suc, 30 min, 25°CMat rush 2.0 M Gly, 1.0 M Suc, 30 min, 25°CLiverwort 2.0 M Gly, 1.0 M Suc, 30 min, 23°CChrysanthemum 2.0 M Gly, 1.0 M Suc, 30 min, 23°CPersimmon 2.0 M Gly, 1.0 M Suc, 30 min, 25°CPotato 2.0 M Gly, 1.0 M Suc, 45 min, 24°CUlluco 2.0 M Gly, 1.0 M Suc, 90 min, 25°CSugarcane 2.0 M Gly, 1.2 M Suc, 30 min, 25°C
Dehydration(V cryo-plate)
Effect of exposure time to PVS2 solution onregrowth of cryopreserved shoot tips ofpotato variety ‘Sayaka’ by V cryo-platemethod (Yamamoto et al., 2014; modified)
Effect of exposure time to PVS2 solutionon regrowth of cryopreserved mintshoot tips by V cryo-plate method(Yamamoto et al., 2012; modified)
Plant name DehydrationPerilla PVS 2, 15 min, 25°CCarnation PVS 2, 25 min, 25°CPotato PVS 2, 30 min, 25°CSugarcane PVS 2, 30 min, 25°CCherry plum & Plum PVS A3, 30 min, 27°CMulberry PVS 2, 30 min, 25°CDalmatian chrysanthemum PVS 7M, 40 min, 25°CMat rush PVS 2, 40 min, 25°CStrawberry PVS 2, 50 min, 25°CMint PVS 2, 50 min, 25°CClinopodium odorum PVS 2, 60 min, 0°CCleomaceae PVS 2, 90 min, 0°CLiverwort PVS2 is not necessary
Dehydration(D cryo-plate)
Effect of dehydration method and ofdehydration duration on moisturecontent (MC, % FW) and onregrowth (%) of cryopreserved budsof mat rush using the D cryo-platemethod (Niino et al., 2014 modified)
Effect of desiccation time on regrowth ofcryopreserved ulluco shoot tips by D cryo-plate method (Valle Arizaga et al., 2017modified)
Plant name DehydrationPersimmon Laminar flow 30 min, 25°CSugarcane Laminar flow 45 min, 25°CUlluco Laminar flow 60 min, 25°CBlueberry Laminar flow 60 min, 25°CClinopodium odorum Laminar flow 60 min, 27°CPotato Silica gel 90 min, 25°CDate palm Laminar flow 90 min, 23°CChrysanthemum Laminar flow 90 min, 23°CPetiveria alliacea L. Laminar flow 140 min, 20-22°CMat rush Laminar flow, silica gel, 150 min, 25°CCherry plum & Plum Laminar flow, silica gel, 180 and150 min, 25℃Liverwort Laminar flow, 80-240 min, 23°C
Other key factors to obtain higher regrowth after rewarming
Setting shoot tips on the cryo-plates When using the V and D cryo-plate protocols, it is necessary to ensure that shoot tips adhere firmly to the cryo-plates throughout the whole procedure for efficient performance.
@The shape and size of shoot tips.@The size of the wells. @The addition of sucrose and/or glycerol to the alginate gel.
@Avoiding shoot tips dropping from the cryo-plate.
Mounting the BEMCOT paper on the cryoplatesUse second-used cryo-plate
Other key factors to obtain higher regrowth after rewarming
@ Dark or low light intensity condition@ Media with progressively decreased sucrose levels@ Add antioxidants in recovery media@ Add adequate plant hormones in recovery media@ Combination with micrografting
Post-LN growth condition
During cryopreservation steps
@ Add antioxidants (ascorbic acid) in solutions or media
Long-term storage in LN tank
Capped cryo tube
Set the cryotubes on the cryo-cane and the
cryo-cane in rack
Liquid phase tank
Special cryo-cane and canister for liquid phase LN tank
1,600 accessions in a LN tank
Genetic Resources Center, NARO, Japan
Long term storage room of orthodox seedsCapacity: 400,000 cansTemperature: -18℃Humidity: 30%
Long term storage room of vegetative propagated crops Capacity: 22 LN tanks Vapor phase (-170℃): 6 tanksLiquid phase (-196℃): 7 tanks
Cryopreservation should beconsidered as a backup to fieldcollections to insure againstloss of plant germplasm. The Vand D cryo-plate methods havethe possibility to becomesuitable protocols forrecalcitrant plants. We expectthat the V and D cryo-plateprocedures contribute tofacilitate the implementation ofcryobanking.
Conclusion
Thank you for your attention!E-mail: [email protected]
The book “Manual of Cryopreservation Methods using Cryo-plate” was published on April 2017.
E-mail: [email protected]
Practical session (8-9 Nov. 2019)
Tools: cryo-plates, Petri dish, filter paper, micropipette (1 mL, 20–100 µL),tip, cryo-cane, cryotubes (2 mL), cryotube stand, pipetting reservoir or alternatives, timers, LN vessel, LN
Chemical solutions: plant vitrification solutions (PVSs), loading solutions, 1.0–1.2 M sucrose solution, 2% (w/v) Na-alginate solution with or without sucrose, 0.1% calcium chloride solution with or without sucrose、sucrose solution
V cryo-plate method of in vitro grown carnation
In vitro shoot tips MS medium (3% sucrose, 0.4% gellan gum)Preconditioning5 mm shoot with a lateral bud, 2 weeks at 25℃
Excision shoot tips 1.0-1.5 mm long Preculture MS with 0.3 M Suc for 2 days at 25℃. Adhere the shoot
tips on cryo-plate2.0–2.5 μL 2% (w/v)Na-alginate solution with 0.4 M sucrose
Polymerization of alginate gel0.1 M calcium solution with 0.4 M sucrosefor 15 min at 25℃
V cryo-plate method of in vitro grown carnation
DehydrationPVS2 for 25 min at 25℃orPVS3 for 50 min at 25℃
Osmoprotection 2 M Gly ＋ 1.4 M Suc. for 90 min at 25℃
Setting of a cryo-plate incryotube
Immersion into LN
Vitrification method using cryo-plate of carnation(V cryo-plate method)
Regeneration
Rewarming 1 M Suc solution for 15 min at 25℃
PlatingRegrowth
Vitrification method using cryo-plate of carnation(V cryo-plate method)
V cryo-plate method of in vitro grown strawberry
In vitro shoot tips MS medium (3% sucrose, 0.4% gellan gum)Preconditioning5 mm shoot with a lateral bud, 2 weeks at 25℃Cold-hardening3-4 weeks at 5℃
Excision shoot tips 1.5-2.0 mm long Preculture MS with 2M glycelol + 0.3 M Suc for 2 days at 5℃. Adhere the shoot
tips on cryo-plate2.5 μL 2% (w/v)Na-alginate solution
Polymerization of alginate gel0.1 M calcium solution for 15 min at 25℃
Case study of chayote
JICA/JST SATREPS Project (Aug 2013-Aug 2018, 5 years) ‘Diversity Assessment and Development of Sustainable Use of Mexican Genetic Resources’(SATREPS: Science and Technology Research Partnership for Sustainable Development Project)
Cryopreservation of in vitro shoot tips of chayoteProcedure by D cryo-plate (Mounting to LS treatment)
Pouring Na-Alginate
Transfer to dehydrationOsmoprotectionPolymerization
Placing shoot tips Pouring Ca solution
Cryopreservation of in vitro shoot tips of chayoteProcedure by D cryo-plate (Dehydration to LN)
Dehydration
Long term storageStorage Immersion to LN
Dried shoot tips Setting the cryo-plate
Cryopreservation of in vitro shoot tips of chayoteProcedure by D cryo-plate (Dehydration to LN)
Taking out cryo-plate
Incubation in darkPlatingRemoving from cryo-plate
Rewarming Diluting in 1 M suc. sol.
dark
Regrowth of cryopreserved in vitro shoots tips of 11 chayote accessions using the optimized D cryo-plate procedure
Cryopreservation of in vitro shoot tips of chayoteRegenerated shoot tips after LN exposure by D cryo-plate
A (H635-12), B(551-10) and C(346-06); 5 days shoot tips after plating. Scale bars indicate 1 mm. D and E; shoots 4 months after plating. F; plantlet 8 months after plating.
