Agricultural biotechnology on plant tissue culture - orgonogenesis and cryopreservation of plant tissue culture and liquid nitrogen preservation

1. BABLU HRANGKHAWL
CAU/CPGS/B17/02
B.Sc.Agri 3rd year
College of Agriculture, Kyrdemkulai
CAU-IMPHAL
ORGANOGENESIS
AND CRYOPRESERVATION

2. ORGANOGENESIS
Cellular totipotency – The potential of a cell to
divide and develop into multicellular plant.
Regeneration: Refers to the development of
organised structures like roots, shoots, flower buds,
somatic embryos etc., from cultured cells/tissues.
 Regeneration may occur through either of two
processes:
1.Organogenesis
2.Embryogenesis

3. 1.Organogenesis:
It is a process involving re-differentiation of
meristematic cells present in callus into
shoot buds.
Root, shoot and leaves (but not embryo)
are the organs that are induced in plant
tissue culture. Since embryo is an
independent structure and does not have
vascular supply, it is not supposed to be
the plant organ.

4. These shoot buds are monopolar structures which in turn
give rise to leaf primordial and the apical meristem.
The buds have procambial strands connected with pre-
existing vascular tissue present in the explant or callus.
Shoot Regeneration:
First reported by White (1939) in tobacco. Arise from
meristemoids, which gives rise to leaf primordia and apical
meristem.

5. Meristemoids are considered to arise in areas that
accumulate starch.
As buds develop, procambial strands differentiate,
and become connected with the pre-existing vascular
tissue present in the explant/callus.

6. Events during shoot regeneration:
1. Shoot induction: Consists of molecular events that
irreversibly commit cells of the explant to proceed on a
particular developmental pathway.
Two phases:
a. Morphogenic competence acquisition: The explants
acquire the capability to perceive the inductive stimuli &
respond to them. Can be committed to form shoot/root.
b. Developmental determination phase: Cells become
irreversibly committed to a developmental path in
response to the inductive stimuli.

7. 2. Shoot differentiation & Development : Consists of
repeated cell divisions leading to specific pattern and
organ formation.

8. Factors affecting organogenesis:
1. Growth regulators: Promoted by cytokinin and inhibited
by auxins. GA-3 in general has inhibitory effect.
2. Explant taken from different plant varieties of same
species show different frequencies of shoot bud
differentiation.
3. Size and source of the explant: The larger the explant
(containing parenchyma, cambium and vascular tissue),
more is likelihood of shoot bud formation.
4. Physical factors: Light has been shown to have inhibitory
effect. The optimum temperature required may vary
with plant species.

9. Cryopreservation
There are various method of storage
1.Cryopreservation- generally storage in
liquid nitrogen.
2.Cold storage- storage at low
temperature and non-freezing
temperature
3.Low pressure- it involves partially
reducing the atmospheric pressure
4.Low oxygen storage- it involves
reducing the oxygen level but
maintaining the pressure.

10. Cryo is Greek word - (krayos – frost). It literally means
preservation in “frozen state.”
The principle - to bring plant cells or tissue to a zero
metabolism and non dividing state by reducing the temperature
in the presence of cryoprotectant.
It can be done :
1. Over solid carbon dioxide (at -79 degree)
2. Low temperature deep freezer (at -80 degree )
3. In vapor phase nitrogen (at -150 degree)
4. In liquid nitrogen (at -196 degree) Cryopreservation

11. Germplasm:Sum total of all genes and alleles of a crop and
its related species.
Different types:
1. Landraces
2. Obsolete varieties
3. Varieties in cultivation
4. Breeding lines (including mutant and transgenic lines)
5. Wild types/wild relatives of the crop.
Strategies of Germplasm conservation:
a) Freeze preservation (Cryopreservation)
b) Slow growth cultures
c) Desiccation
d) DNA clones

12. LONG TERM STORAGE
Cryopreservation: The preservation of cells, tissues or organs
at -196˚C using liquid nitrogen (LN) is called as
Cryopreservation.
Why Liquid nitrogen ?
Chemically inert
Relatively low cost
Non-toxic
Non-flammable
Readily available

13. Steps:
1. Selection of the explant
2. Pre Freezing treatment
3. Cryoprotection
4. Freezing
5. Storage
6. Thawing
7. Post treatment or re-culture.
8. Determination of survival/viability:

14. 1. Selection of the Explant:
The morphological and physiological conditions of the plant
material, prior to freezing, considerably influence its ability to
survive freezing at -196˚C
Small, richly cytoplasmic, meristematic cells survive better
than larger, highly vacuolated cells.
Young, globular embryos survive better than the older ones.

15. 2. Pre-freezing treatments:
a. Pre-culture : A brief culture of shoot apices before freezing
is beneficial. Freshly harvested shoot apices do not survive
super-cooling. For high frequency of survival of shoot apices
after freezing in LN, pre-culture for 48 hrs in the presence of
5% DMSO(Dimethyl Sulfoxide).
b. Desiccation : Exclusion of freezable water from the cells
before freezing. Amorphous freezing cryoprotectants are
used for this purpose. Dehydration optimum may vary with
the species and tissues.
c. Vitrification : A physical process by which a concentrated
aqueous solution cooled to low or ultra low temperature
directly solidifies into an amorphous ‘glassy’ state, without
crystallization. Cells applied with a highly concentrated
solution of osmotically active compounds, are protected from
internal damage from ice crystal formation during freezing.

16. 3. Cryoprotection:
They are chemical which prevent cryodestruction
These are sucrose, alcohols, glycols, some amino acid (proline), DMSO
(dimethyl sulfoxide) 5-8%, Glycerol.
Generally two cryoprotectant should be used together instead of
single one as they are more effective.
To protect cells against osmotic shocks, cryoprotectants should
be added gradually over a period of time (30-60 min). For glycerol,
prolonged treatment is necessary because of its low permeability.
4. Freezing:
I. Slow Cooling: @ 0.5 to - 4˚C/min from 0 to -100˚C
II. Rapid Cooling: @ >1000°C/min from -10 to -70°C
III. Pre freezing: Cooled gradually @ 1˚C/min or Stepwise @
5°C/min and held at that temperature for about 30 sec
and rapidly plunged into liquid nitrogen.

17. 5. Storage: The temperature should be ideally
always less than -130°C
6. Thawing: Rapid thawing is recommended.
Plunge into water (37° to 40°C for 90 sec and transferred
to ice bath)
7. Post treatment and re-culture:
Washed to remove the cryoprotectant.
The material is then re-culture into a fresh medium.

18. 8. Determination of survival/viability:
Regrowth of the plants from stored tissues or cells
is the only test of survival of plant materials.
Various viability tests include Fluorescien
diacetate (FDA) staining, growth measurement by
cell number, dry and fresh weight.
Important staining methods are: Triphenyl
Tetrazolium Chloride (TTC), Evan’s blue staining.

19. Application:
It is ideal method for long term conservation of material.
Disease free plants can be conserved and propagated.
Recalcitrant seeds can be maintained for long time.
Endangered species can be maintained.
Pollens can be maintained to increase longitivity.
Rare germplasm and other genetic manipulations can be
stored.