Technique and Prospect of Cryopreservation for ...retnomastutibiologi.lecture.ub.ac.id/files/2016/03/...pd suhu di atas titik beku air (tidak beku = non freezing) → inkubasi pd krioprotektan

Technique and Prospect of Cryopreservation for

Germplasm Conservation

Retno Mastuti

Biology Department

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 once lost can never be regained.

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 endangeredspecies are made available for study.

Germplasm Conservation

In vivoLive plants

In vitroCryo-conserved materialstem, a leaf, or pollen, or

even just a few cells

in situIn natural habitat and

production environment:National parks, agriculture

Etc.

ex situSeed 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

- pelelehan

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

intracellular ice crystals are.

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,

polyvinylyrolidone, proline, etc.

Cryopreservation Requirements1) Preculturing

a rapid growth rate to create cells with small vacuoles and low water content2) Cryoprotection

Glycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form of ice crystals

3) FreezingThe most critical phase :- Slow freezing allows for cytoplasmic dehydration- Quick freezing results in fast intercellular freezing with little dehydration

4) StorageUsually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur above -100°C

5) ThawingUsually 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 New

Classical technique : Freeze-induced dehydration

slow cooling downto 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 ofintracellular solutes

aqueous vapour pressure exceeds the frozen external compartment, cells equilibrate by loss of water to

external ice

most or all intracellularfreezable water is

removed

reducing or avoiding detrimental intracellular ice formation upon subsequent immersion of the specimen in liquid nitrogen

Classical techniquePregrowth of

sample

Cryoprotection

slow cooling (0.5 –2.0°C min 21) to a determined

prefreezingtemperature (usually

around -40°C)

rapid immersion of samples in liquid

nitrogen

storage

Rapid thawing

recovery

Optimized methodology for the cryopreservation of sugarcane calli with embryogenic structures.

New Cryopreservation Technique

Pregrowth of sample

Cryoprotection

rapid cooling - all factors that affect

intracellular iceformation 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

• (7) droplet freezing.

Chawla, 2004

Source tissue

Pre growth

Cryopreservation : cryoprotectant, freezing

Storage

Regrowth

Regeneration

Plants

Dehydration (high osmotic pressure)

Liquid nitrogen

Liquid nitrogen

Viability stain

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.

In vitro conservation of potato plantlets

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

encapsulation-dehydration

Some examples of artificial seeds and the emergence 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 dengandroplet 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 bebasRadikal 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 plant.

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 the number of subcultures

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’

(Tambunan dan Mariska, 2003)

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

� Tipe dan keadaan fisiologis bahan yang disimpan

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 metode kriopreservasi (Mariska dkk. 1996).

� 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)

= Koleksi Kerja

Problematics of seed conservation

1. Plants don’t produce seeds → vegetatively propagated (banana)

2. Plants which have sterile genotypes and genotypes produced orthodox seeds, highly heterozygous (potato or sugarcane)

3. Plants which produce recalcitrant seeds

� Orthodox seeds : seeds that can be dehydrated to low moisture contents and and can thus be stored at low temperature for extended periods

� Recalcitrant seeds : seeds that cannot be dried to sufficiently low moisture level to permit their storage at low temperature

The disadvatages of traditionally ex situ / field conservation:

→ Distribution and exchange from field is high risk and difficult