Cryopreservation of M
agnolia m
acrophyllavar. asheishoot tips
by droplet vitrification
Raquel Folgado*, Tim Thibault* and Bart Panis~
*The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA
(USA)
~BioversityInternational, Leuven (Belgium
)
Photo by Rick Cantrellhttp://w
ww
.fnps.org/plants/plant/magnolia-m
acrophylla-var-ashei
Shoot tips
•A
loe•
Agave
•M
agnolia•
Avocado•
Oak
Seeds
•C
acti•
Orchids
•M
agnolia
Pollen
•Titan arum
•C
ycads
Em
bryos•
Oaks
•C
ycads
Cryopreservation at The H
untington
Provide protocols for long-term conservation of plants
Conservation
of PlantGerm
plasm-
In situ:natural environm
ent(-)
High risks of losses, highly exposed
-Ex situ:
botanical gardens, research centres, seed and germ
plasm banks
Ex vitro:Traditional m
ethod (field collections)(-)
Infection risks, labour-intensiveIn vitro:
Vegetatively propagated species�
Norm
al growth
�S
low grow
th (tempÌ
, O2 Ì
,H2 O
Ì, m
edium ~)
(+)S
terile material, no risks by insects or w
eather conditions(-)
Still labour-intensive, risks for contam
ination,hum
an error or somaclonalvariation
Cryopreservation
(-196ºC): Long-term
germplasm
conservation(+)
Unlim
ited storage time, reduced costs &
space,prevention of infection and genetic changes
At ultra-low tem
perature (such as -196°C), the m
etabolic processes and biological deteriorations in the cells/tissues are stopped.
Cryopreservation
(Greek,
krayos-frost)literally
mean
“inthe
frozenstate”
Process
ofcooling,orfreezing,and
storingcells,tissues
ororgans
toultra-low
temperatures
andkeep
themfor
afuture
use
•Over solid carbon dioxide (at -79⁰C
)•Low
temperature deep freezers (at -80⁰C
)•In liquid nitrogen (at -196⁰C
, gas phase -150⁰C)
Cryopreservation
Cryopreservation
Uses of cryopreservation
Conservation of plant
germplasm
Vegetativelypropagated
species
Recalcitrant seed species
Conservation of tissue w
ith specific
characteristics
Medicinal &
alcohol
producing cell lines
Genetically transform
ed tissues
Transformation/
Mutagenesis
competent
tissues
Eradication of viruses
Conservation of plant
pathogens and sym
bionts
Pathogen Fungi
Micorrhyzal
fungi
Nem
atodes
Ex situ conservation of m
agnolias using cryopreservation
•~1/2 of the M
agnoliaceae taxa are threatened worldw
ide•
Developm
ent of efficient methods of ex situ conservation plays a
crucial role•
Conservation of w
oody plants in field clonal banks is costly and risky•
Tissue culture helps to ensure the ex situ preservation of threatened M
agnoliaceae•
Prior to the im
plementation of protocols for the cryopreservation of
Magnolia spp., establishm
ent of micropropagation
from adult trees is
needed•
The proliferation of Magnolia
shoots is difficult due to the oxidation of phenolic com
pounds during micropropagation, recovery from
cryopreservation and rooting before ex vitro acclim
ation
-Key: avoidance of
the formation of
intracellular ice
Vitrification: involve dehydration w
ith highly concentrated cryoprotectant m
ixtures
Droplet-vitrification:
reduced damage
during cooling and re-w
arming
Photoby
Kate Lain
PhotosbyKate Lain
Photoby
Kate Lain
http://ww
w.huntington.org/W
ebAssets/Templates/content.aspx?id=21900
Cryopreservation by droplet vitrification
Ashe´s M
agnolia
•S
mall tree
orlargeshrub, 15 -30 feettall
•E
ndemic
toeight
countiesof the
Florida P
anhandle.
http://floridata.com/Plants/M
agnoliaceae/Magnolia%
20macrophylla%
20subsp.%20ashei/1071
http://florida.plantatlas.usf.edu/Plant.aspx?id=1241http://w
ww
.arboretum.harvard.edu/plants
MI
4 Wks
Shoot proliferation
1-2 W
ks
Steriliza
tionInitiation
Tissue culture of Magnolia m
acrophyllavar. ashei4 W
ks4 W
ks
NAA
higherlow
er
BA
High
Med
Low
Optim
ization of medium
for shoot tips recovery
Shoot tip grow
th, 4 w
eeks after dissection
Cryopreservation using droplet-vitrification
30, 45 & 60 m
in
Shoot-tip response to droplet-
vitrification and LN exposure
Lowest hyperhydricity
was
found in shoot tips exposed to P
VS2 for 45 m
in
Norm
al
CallusHyperhydric
Post-rew
arming regeneration
rates were sim
ilar in explants treated 45 and 60 m
in
Conclusions
•D
roplet-vitrification is a suitable method for the long-term
conservation of M
agnoliam
acrophyllavar. ashei
•R
ecovery of plants from cryopreservation still needs to
be optimized
•The recovery from
cryopreservation depends on:–
State of plantlets (preculture)
–U
se of antioxidants (before, during and/or after protocol)–
Recovery m
edium (nutrients and horm
ones)
Outcom
e•
At The H
untington, we intend to provide new
tools for plant conservation to other institutions and users, for the long-term
conservation of magnolias
•In vitro repository at The H
untington:M
. acuminata
var. subcordata, M. cam
pbelliisppm
ollicomata, M
. figo, M.
garrettii, M. grandiflora, M
. liliflora, M. m
acrophylla, M. m
acrophyllavar. ashei,
M. m
acrophyllavar. dealbata, M
. officinalis, M. pacifica
spppacifica, M
. pacificaspp
tarahumara, M
. sphaerantha, M. tam
aulipana, M. x soulangeana, M
. delavayii,
•C
ryopreservation technologies may be crucial to the
conservation of Magnoliaceae
diversity worldw
ide
Acknow
ledgments
•D
r. Michael S
. Dosm
ann•
Kathryn
Richardson
•D
r. Bart P
anis
•D
r. Jim Folsom
•S
ean Lahmeyer
•Tim
Thibault•
Taylor La Val•
JosetteTin
TH
E H
UN
TIN
GT
ON
