Technique and Prospect of
Cryopreservation for
Germplasm Conservation
Retno Mastuti
What is plant germplasm?
is
the living tissue
from which new plants
can be grown.
Plant germplasm is usually
seed (orthodox
seeds)
,
or it can be
another plant part
--- a stem, a
leaf, or pollen, for example, or even just a
few cells that can be cultured into a whole
plant.
Germplasm conservation – is it
urgent ?
increasing population poses a constant
threat to this biodiversity.
Many species are critically endangered, are
endangered, vulnerable, extinct etc.
an immediate attention from mankind and
especially biologists as genetic diversity
Germplasm Conservation :
The availability
of useful germplasm at
any time
genetic improvements
→→
increases in
productivity and resistance to pests,
diseases and adverse growing conditions.
conserving biodiversity of
indigenous
plant
species. In addition to commonly occurring
species,
threatened, rare
or
endangered
Germplasm Conservation
In vivo
Live plants
In vitro
Cryo-conserved material stem, a leaf, or pollen, or
even just a few cells
in situ
In natural habitat and production environment: National parks, agriculture
Etc.
ex situ
Seed banks, botanical gardens, Arboreta,
The plant materials can be conserved in two
main ways
• It is particularly useful where it is not possible to maintain a seed bank : recalcitrant
seeds, vegetative propagation (bulbs, tubers, corms etc.), non-viable seeds due to
damage caused by grazing or diseases, and large and fleshy seeds.
Short or medium conservation using in vitro methods using slow growth storage.
Long term conservation (stop growth storage) for
cryopreservation.
This method is useful for many species like those of temperate woody plants, fruit trees,
horticultural and numerous tropical species
Slow Growth Method
•
Stores at
non-freezing
condition
•
Water in the tissues : in liquid condition BUT, all biochemical
processes are delayed
•
Growing process are reduced to a minimum by limitation of
a combination of factors like
temperature
(5-10
°
C),
nutrient
medium
(1/10 strength MS plus sucrose 1 %),
hormone
(ABA
5-10 mg/l),
osmotic inhibitor
(3-6 % manitol) etc.
•
Reduction of
inoculum size
: extension of lag phase and
delay the onset of rapid cell growth
•
The
major problem
: loss of water from liquid and solid
media
→
good seal, replacement of seal
•
The tissues still have to be sub cultured regularly
→
contamination
Cryopreservation = Stop the growth
preservation in the frozen state = to be storage at very
low temperature; liquid nitrogen -196
°
C.
Cells in a completely inactive state
→
low
temperature : slows down metabolic processes and
biological deterioration
Freeze preservation = transfer of water present in the
cells to solid state
Pure water – water cells
Two considerations
: Degree of freeze tolerance &
formation of ice crystals within the cells
Cryopreservation:
storage of biological material at ultra-low temperature,
liquid nitrogen (-196°C)
At this temperature, all cellular divisions and metabolic processes are stopped
The plant material can be stored without alteration or modification for unlimited period of time
cultures are stored in a small volume, protected from
contamination, and require a very limited maintenance
safe and cost efficient long-term conservation of different types of germplasm
Why liquid nitrogen? - Chemically inert - Relatively low cost - Non toxic
- Non flamable - Readily available
Cryopreservation Techniques
•
Some materials have
natural
dehydration processes
; can
be cryopreserved without any
pretreatment
•
Other materials (cell
suspensions, calluses, shoot
tips, embryos, etc.)
contain
high amounts of cellular free
water
•
are thus extremely
sensitive
to freezing injury
since most
of them are not inherently
freezing-tolerant
•
Cells have thus to be
dehydrated artificially
to
protect them from damage
caused by crystallization of
intra cellular water into ice
Cryopreservation
Classical techniques
• Freeze-induced dehydration:
slow cooling down to a defined prefreezing temperature,
followed by rapid immersion in liquid nitrogen
• pembekuan pd suhu di bawah titik beku air hingga -40°C
Teknik Pembekuan lambat / 2 tahap :
- inkubasi pd krioprotektan (total konsentrasi 1-2 M) : dehidrasi moderat
- pembekuan lambat : mis. 1°C/menit sampai – 35°C →
pembekuan dalam nitrogen cair
- pelelehan (thawing)
• Two considerations : Degree of freeze tolerance & formation of ice crystals within the cells
New techniques
• Vitrification: the transition of water directly from the liquid phase into an amorphous phase or glass,while avoiding the
formation of crystalline ice
• more appropriate for complex organs (shoot tips, embryos) which contain a variety of cell types, each with unique
requirements under conditions of freeze-induced dehydration
- fase transisi air dari bentuk cair menjadi bentuk non kristalin / amorf, tembus pandang (glassy) pd suhu di atas titik beku air (tidak beku = non freezing) → inkubasi pd krioprotektan (total konsentrasi 5-8 M pd 0-25°C - pembekuan
Freezing
•
Slow freezing
: decrease of 0.1 – 10 C/min from 0
C to -100 C then transfer to liquid nitrogen. Slow
cooling permit flow of water from the cells to the
outside. Extracellular ice formation instead of
lethal intracellular freezing
•
Rapid freezing
: - 300 C to – 1000 C/min or more.
The quicker the freezer is done, the smaller the
Addition of cryoprotectant
•
The effect of temperature on plants depend on
genotype, environment and physiology
•
Hardening process : growing the plants for
shorter duration of 1 week at a lower
temperature
•
Glycerol, dimethyl sulfoxide (DMSO), glycols
(ethylene, diethylene, propylene), acetamides,
sucrose, mannose, ribose, glucose,
Cryopreservation Requirements
1)
Preculturing
a rapid growth rate to create cells with small vacuoles and low water content
2)
Cryoprotection
Glycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form
of ice crystals
3)
Freezing
The most critical phase :
- Slow freezing allows for cytoplasmic dehydration
- Quick freezing results in fast intercellular freezing with little dehydration
4)
Storage
Usually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur
above -100
°
C
5)
Thawing
Usually rapid thawing to avoid damage from ice crystal growth
6)
Recovery
(don’t forget you have to get a plant)
- Thawed cells must be washed of cryoprotectants and nursed back to
normal growth
- Avoid callus production to maintain genetic stability
Two types of cryopreservation techniques: Classical
Classical technique : Freeze-induced dehydration
slow cooling down to a defined
prefreezing temperature
cells and the external medium initially supercool, followed by ice formation in the medium
cell membrane acts as a physical barrier and prevents the cell interior
remain unfrozen but supercooled
the extracellular solution is converted into ice, resulting
in the concentration of intracellular solutes aqueous vapour pressure exceeds
the frozen external compartment, cells equilibrate by loss of water to
external ice most or all intracellular
freezable water is removed
reducing or avoiding detrimental intracellular ice formation upon
subsequent immersion of the specimen in liquid nitrogen
Classical technique
Pregrowth of sample Cryoprotection slow cooling (0.5 –2.0°C min 21) to a determined prefreezing temperature (usually around -40°C) rapid immersion of samples in liquid nitrogen storage Rapid thawing recoveryOptimized methodology for the cryopreservation of sugarcane calli with embryogenic structures.
New Cryopreservation Technique
Pregrowth of sample
Cryoprotection
rapid cooling - all factors that affect
intracellular ice formation are avoided
-dehydration rapid immersion of samples in liquid nitrogen storage Rapid thawing recovery
Seven different vitrification-based procedures
•
(1) encapsulation –dehydration;
•
(2) vitrification; vitrification;
•
(3) encapsulation - vitrification
•
(4) dehydration;
•
(5) dehydration;
•
(6) pregrowth – dehydration ; and
Source tissue
Pre growth
Cryopreservation : cryoprotectant, freezing
Storage
Regrowth
Regeneration
Plants Dehydration (high osmotic pressure)
Liquid nitrogen
Liquid nitrogen
They are kept for about two years in an aseptic environment in test tubes containing semi-solid culture medium at a temperature of 6-8 °C, low light, and in presence of an osmotic regulator slow their growth. The plantlets grow normally whenever they are planted again in normal environment.
Source tissue : raising sterile tissue
cultures
Various type of tissues : apical & lateral
meristem, plant organ, seeds, cultured plant
cells, somatic embryos, protoplasts,
calluses, etc.
Small, richly cytoplasmic, meristematic cells
survive better than the larger, highly
vacuolated cells.
Cell suspension : late lag phase or
exponential
Callus : old cells and blackened area should
be avoided
Na-alginate is used for the
encapsulation of the embryo
Teknik-teknik Kriopreservasi Baru :
Jenis Penjelasan
Vitrifikasi bahan tanaman diperlakukan dengan senyawa krioprotektif dan dehidrasi dengan larutan vitrifikasi, lalu diikuti dengan pembekuan cepat, pelelehan, dan pembuangan krioprotektan serta pemulihan kultur.
Enkapsulasi-dehidrasi
(dikembangkan pd produksi benih sintetik)
bahan tanaman dienkapsulasi pada kapsul alginat, lalu ditumbuhkan pada medium yang diperkaya dengan sukrosa dan dikeringkan secara parsial dalam
laminar air flow cabinet atau gel silika hingga kandungan air sekitar 20% dan diikuti oleh pembekuan cepat.
Enkapsulasi-vitrifikasi
kombinasi antara teknik vitrifikasi dan enkapsulasidehidrasi, yaitu bahan tanaman dienkapsulasi dengan kapsul alginat, lalu dibekukan dengan teknik vitrifikasi.
Desikasi teknik yang paling sederhana, yaitu mengeringkan bahan tanaman dalam
laminar air flow cabinet, gel silika atau flash drying hingga kandungan air 10-20%, kemudian diikuti oleh pembekuan cepat.
Pratumbuh penanaman bahan tanaman ke dalam media yang mengandung krioprotektan, lalu diikuti oleh pembekuan cepat.
Pratumbuh-desikasi Menanam bahan tanaman ke dalam media yang mengandung krioprotektan, lalu mengeringkannya dalam laminar air flow cabinet atau gel silika dan diikuti oleh pembekuan cepat.
Dropplet-freezing Diawali dengan praperlakuan bahan tanaman ke dalam media cair yang mengandung krioprotektan, lalu meletakkan pada Al-foil yang disertai dengan droplet krioprotektan dan diikuti oleh pembekuan cepat.
Kelebihan dan kekurangan
Lama
Peralatan terprogram, cukup mahal
Teknik pembekuan pd kultur sel, sulit diaplikasikan pd
unit sel yg lebih besar (tunas, embrio, dll.)
Berhasil pd sistem kultur yg tidak terdiferensiasi &
species toleran suhu dingin
Tidak / kurang berhasil diterapkan pada spesies tropis.
Baru
Tidak perlu alat canggih, prosedur lebih sederhana
Memungkinkan untuk unit sel yang besar
Berhasil diterapkan pada species dgn skala yg lebih luas
(tropis dan subtropis) dan sistem kultur yg lebih
kompleks (embrio somatik, suspensi sel dan meristem
apikal)
Peluang kerusakan sel tanaman
selama pembekuan dan pelelehan
karena (1) :
Eksposur
bahan tanaman
pada suhu rendah
: inaktivasi
protein yg sensitif thd suhu dingin
Formasi kristal es
dpt merusak sel krn. (Grout, 1995) :
- daya mekanis kristal es yang tumbuh,
- gaya adhesi kristal es thd membran,
- interaksi elektris yg disebabkan oleh perbedaan
solubilitas ion pada fase es dan cair
- Formasi gelembung udara intraseluler
- Luka khemis yg berhubungan dgn peroksidase lipid
- Perubahan pH pd lokasi tertentu
Peluang kerusakan sel tanaman
selama pembekuan dan pelelehan
karena (2) :
Sel terdehidrasi
:
Terdehidrasi terlalu kuat →
plasmolisis kuat
berakibat perubahan pH, interaksi mikromolekuler
& peningkatan konsentrasi zat elektrolit
Saat pelelehan
:
Kontraksi osmotik dpt menyebabkan endositotik
vesikulasu irreversibel yg mengakibatkan sel lisis
krn. Bahanmembran yg baru tdk mampu
memfasilitasi deplasmolisis
Formasi radikal bebas
Radikal bebas : merusak fraksi lipid pada membran
dan menghasilkan lipid peroksida yg akan terurai
menjadi senyawa produk oksidasi sekunder yang
toksik
In vitro conservation
The tissue culture techniques can be
used to collect, maintain and store
different plant tissues like shoot
apices, stem cuttings, buds, embryos
etc., depending upon the type of
Advantages of in vitro conservation :
Plant species that are in danger of
being extinct enable to be conserved
Vegetatively propagated plants :
saving in storage space and time
Plants that can not reproduced
generatively
Possible to
reduce growth
: decreases
Two main approaches for in vitro storage
germplasm :
Slow growth
method : reduction on growth
rates of cells and tissues
Stop
the growth : inhibition of growth of
cells and tissues by ‘
cryopreservation’
Faktor-faktor yg Mempengaruhi
Keberhasilan Kriopreservasi :
Kecepatan pembekuan
- Terlalu lama : sel terlalu terdehidrasi shg konsentrasi zat
elektrolit dalam sel menjadi tinggi
- Terlalu cepat : sel kurang mengalami dehidrasi shg terjadi
formasi es intraselular yg bersifat letal
Jenis dan konsentrasi krioprotektan
- Krioprotektan : memelihara keutuhan membran dan
meningkatkan potensial osmotik media shg cairan di dalam sel
mengalir keluar dan terjadi dehidrasi
-
Permeating agent
(masuk ke dalam sel) : DMSO, gliserol (pd
suhu tertentu)
-
Non permeating agent
(tdk masuk ke dalam sel) : sukrosa dan
gula alkohol (manitol, sorbitol, dll.)
Suhu akhir pembekuan
Teknik Pelestarian / Penyimpanan
secara in vitro meliputi :
1.
penyimpanan jangka pendek
(penyimpanan
dalam keadaan tumbuh),
2.
penyimpanan jangka menengah
(penyimpanan dengan metode pertumbuhan
lambat atau pertumbuhan minimal), dan
3.
penyimpanan jangka panjang
dengan
Medium-term storage
: seed in paper envelopes at
4
°
C and 20% relative humidity.
Medium-term storage
(1 to 4 years)
- increasing intervals between subcultures
- reduce growth, temperature and light reduction
- modifying the culture medium, mainly by reducing
the sugar and/or mineral elements concentration
- limit the evaporation of the culture medium
- reduction of the oxygen level
Long-term storage
: seed is preserved in laminated
envelopes at -20
°
C.
Cryopreservation
(a type of freezing) in or
over liquid nitrogen at -196
°
C
- Ultra low temperatures
- Stops cell division & metabolic processes
- Very long-term
Konservasi benih ortodoks plasma nutfah tanaman
pangan (padi, jagung, kedelai, sorgum dan
kacang-kacangan) dilakukan secara:
– metode penyimpanan di ruang dingin dengan kondisi suhu
14 sd. 18 derajat Celcius untuk penyimpanan jangka
pendek (
short term
).
– penyimpanan di dalam
cold storage
dan
chiller
bersuhu 0
sd. -5 derajat Celcius untuk penyimpanan jangka
menengah
(medium term
).
– penyimpanan di dalam
freezer
bersuhu -18 sd. -20 derajat
Celcius untuk penyimpanan jangka panjang (
long term
).
Untuk plasma nutfah ubi-ubian (ubikayu, ubijalar
dan ubi-ubian minor), konservasi dilakukan dalam
bentuk tanaman di lapang (
field gene bank
) .
Storage of cultures has involved
explants of various types
Shoots with roots have demonstrated
better survival than those without
microtubers are suited to storage for
potato
encapsulation of explants in
alginate beads.
(Tambunan dan Mariska, 2003)
