PLANT

CRYOPRESERVATION
AT THE HUNTINGTON
BOTANICAL GARDENS

Raquel Folgado Casado

Cryopreservation Research
Fellow

N EED FO R PLAN T CO N SERVATIO N

Rare and threatened species

Plant breeding

W H AT IS G ER M P LA S M ?

Germplasm broadly refer to the hereditary

material (total content of gene) transmitted
to the offspring through germ cell
It is also used to describe a collection of
genetic resources for an organism

G ER M P LA S M C O N S ER V ATIO N
Plant germplasm (genetic source material for
plant breeding)
They may include:
Seeds
Other plant propagules such as
Leaf
Stem
Pollen
Cultured cells
which can be grown into mature plant

C O N S ER V ATIO N O F P LA N T
M P LA
Menvironment
-G ER
In situ:
natural
(-)

High risks of losses, highly exposed

Ex situ:

botanical gardens, research centres, seed and

germplasm banks
Ex vitro:
Traditional method (field collections)
(-)

In vitro:

Infection risks, labour-intensive

Vegetatively propagated species

Normal growth
Slow growth (temp, O2 , H2O , medium ~)
(+) Sterile material, no risks by insects or weather

conditions
(-) Still labour-intensive, risks for contamination,
human error or somaclonal variation

Cryopreservation (-196C): Long-term germplasm conservation

(+) Unlimited storage time, reduced costs & space,
prevention of infection and genetic changes

W H AT IS C R YO P R ES ER V ATIO N ?

Cryopreservation (Greek, krayosfrost) literally mean in the frozen

state
Process of cooling, or freezing,
and storing cells, tissues or organs
to ultra-low temperatures and keep
them for a future use

S E O F C R YO P R ES ER V ATIO N
Conservatio
n of plant
germplasm

Conservatio
n of tissue
with specific
characterist
ics

Vegetative
ly
propagate
d species

Medicinal &
alcohol
producing
cell lines

Recalcitra
nt seed
species

Genetically
transformed
tissues
Transformati
on/Mutagen
esis
competent
tissues

Eradication
of viruses

Conservatio
n of plant
pathogens
and
symbionts
Pathogen
Fungi
Micorrhyz
al fungi
Nematod
es

IN V ITR O P LA N T M ATER IA L FO R
C R YO P R ES ER V ATIO N

M EC H A N IS M O F C R YO P R ES ER V ATIO N
Key: avoidance of the formation of
intracellular ice
Presence of salts and organic molecules in
the cells, the cell water requires much more
lower temperature to freeze (even up to
-68C)
At ultra-low temperature (such as -196C),
the metabolic processes and biological
deteriorations in the cells/tissues are
stopped.

TEC H N IQ U E O F C R YO P R ES ER V ATIO N

The cryopreservation of plant material

involves the following stages
1. Development of sterile tissue culture.
2. Addition of cryoprotectant and
pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival/viability
8. Plant regeneration

D EV ELO P M EN T O F S TER ILE

TISS U E C U LTU R E
Morphological and physiological characters
largely influence the ability of the explants to
survive in cryopreservation.

A D D ITIO N O F C R YO P R O TEC TA N T
Prevent the damage caused to cells by freezing
or thawing
Dimethyl Sulfoxide (DMSO)
Glycerol
Ethylene
Propylene
Sucrose
Mannose
Glucose
Proline
Acetamide

FR EEZ IN G
Slow freezing method :
at 0.5-5C/min from 0C to
-100C, and then transferred
to LN
Rapid freezing method :
-300 to -1000C/min
Stepwise freezing method:
Intermediate temperature for
30 min., and rapidly cool
Dry freezing method :
Dehydration of material before
freezing

S TO R A G E

Range of -72 to -196C

Over solid carbon dioxide (at -79C)
Low temperature deep freezers (at

-80C)
In liquid nitrogen (at -196C, gas phase
-150C)

The ultimate objective of storage

is to stop all the cellular metabolic
activities and maintain their
viability
For long term storage, temperature
at -196C in liquid nitrogen is ideal.

TH A W IN G
Warm water (temp 37 45C) bath with
vigorous swirling
Rapid thawing (at the rate of 500-750C min)
occurs, and this protects the cells from the
damaging effects ice crystal formation.

R EC U LTU R E A N D P LA N T R EG EN ER ATIO N

Cryoprotectant is removed and plant material

is cultured
Growth promoting and maintenance of
appropriate environmental conditions

Ultimate
purpose:
regenerate
desired plant

LIM ITATIO N S O F C R YO P R ES ER VATIO N

The expensive equipment needed to provide

controlled and variable rates of

cooling/warming temperatures
Formation of ice crystals inside the cell should

be prevented
Sometimes certain solutes from the cell leak

out during freezing

Cryoprotectant also affect the viability of cells

C R YO P R ES ER V ATIO N AT H B G
Succulents and
Cacti

Avocado
Meristems

Seeds: cacti
Meristems: Agave &
Aloe

Magnolia

Orchids

Meristems
Seeds

Seeds (Paphiopedilum)
Protocorms (clones of
interest)

Camellia

Amorphophallus
titanum

Clonal material

Pollen
Seeds
Embryos

SU CCU LEN TS AN D CACTI

Agave and Aloe: optimization of the plant
regeneration process

The tests with cacti seed are also promising

and therefore, we will be able to store some
species into liquid nitrogen

AVO CAD O (Persea am ericana)

7 Varieties in TC:
Mexicola (M)
Nabal (G)
Nimlioh (G)
Carlsbad (G)
Anaheim (G)
Zutano (Hybrid M x
G)
Mayo (Hybrid M x G)

Cryopreservation of
meristems
(collaboration with
group at University

M AG N O LIA spp
12 species introduced in TC:
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.
M.

grandiflora
macrophylla spp dealbata
macrophylla spp ashei
champaca
pacifica
doltsopa
aromatica
dawsoniana
denudata
acuminata var Subcordata
ernestii
x soulangeana
tripetala

O RCH ID S
Cryopreservation of seeds
Clones of interest from
Huntington living collection

CAM ELLIA
Introduction to TC:
C.
C.
C.
C.

hiemalis var.Kanjiro
japonica
hongconensis
grosii

TITAN ARU M (Am orphophallus titanum )

Pollen collected and freezed
Seeds collected and tested for

cryopreservation

O SM O TIC STRESS TO IM PRO VE CRYO PRESERVATIO N

TO LERAN CE

In sum m ary
The Huntington might be a Botanical Garden of

reference in America for the long-term

conservation
We intend to provide new tools for plant
conservation to other institutions and users
Cryopreservation technologies may be crucial
to the conservation of plant diversity worldwide

Thanks!!