1. In vitro conservation of groundnut
germplasm
by
M.M. Abdulmalik & I.S. Usman
Department of Plant Science, Institute for Agricultural Research
Ahmadu Bello University Zaria

2. Introduction
• Groundnut (Arachis hypogaea L.) is an important
source of protein and edible oil in the world.
• Nigeria ranks third after India and China in terms of
production (FAOSTAT 2010).
• Production is constraint by low quality of seeds as
these deteriorate rapidly in storage particularly in the
tropics (Delouche et al., 1973).
• Conventional methods used by our subsistence
farmers to store seeds (RMRDC, 2004) are
inadequate for long term storage.
• Newly improved varieties are fast replacing our
traditional varieties.

3. • Groundnut germplasm are conventionally stored in
gene banks and seeds are the most preferred
propagule used.
• Due to their high lipid content and thin seed coat
they cannot tolerate the gene bank conditions for
longer periods like true orthodox seeds.
• This has led to the suggestion that the groundnut
should be considered as suborthodox (Vasquez-
Yanes and Arechiga 1996, Gagliardi, et al., 2000).
• Studies on groundnut seeds viability in storage in
IAR revealed that all the entries evaluated had
viability of 50% or less by 1 year and 25% by 2 years
of storage.

4. • The IAR maintains its varieties and cultivars
by planting every season.
• Not only is this laborious, time consuming
and expensive but also plants are exposed to
the possible risk of pest, disease and
environmental stresses.
• Thus, cryopreservation is considered as
important complimentary strategies for ex
situ conservation.

5. • Cryopreservation or storage in liquid nitrogen at temperature
of –1960C, at which all the cells are in a state of suspended
animation, is the most promising method of invitro germplasm
storage
• advantages
– enables long term storage of the plant material
– require less space
– less labor
– it’s cheap at the long run
– genetic stability
– elimination of viral disease.
• Cryopreservation techniques
– Desiccation
– Vitrification
– Encapsulation vitrification
– Encapsulation dehydration
– Droplet freezing

6. Material and Methods
• Plant material- Seeds of four
groundnut (Arachis hypogaea
L.) varieties.
• Surface sterilized of seeds
-5min in70% alcohol,
-20min in 10% NaOCl+ 2-3
drops of tween 20
-rinsed thrice with sterile
distilled water
- 10min in 5% NaOCl + 2-3
drops of tween 20
-washed three times with
sterile distilled water.
• Seeds were soaked in sterile
distilled water for 3hr.
• Embryonic axes were excised

7. Vitrification technique
• Preculturing of embryonic axes
on solidified MS medium
supplemented with 0.3M
sucrose for 24hr.
• treatment with a loading
solution (2M glycerol plus 0.4M
sucrose dissolved in MS
medium) for 15min at 25°C.
• Treated embryonic axes were
transferred to 2ml cryovials
and 1ml PVS2 was added
dehydrated for 2hr.
– PVS2
(30% (w/v) glycerol, 15%
(w/v) ethylene glycol and
15% (w/v) dimethylsulfoxide
(DMSO) in MS medium with
0.4M sucrose)

8. Desiccation
• Embryonic axes were excised
and subjected to desiccation
under the air current of a
laminar flow cabinet for 4hr.
• Moisture content was
determined on fresh weight
basis after drying in a 100°C
oven for 24hr (3 replicates of
10 embryonic axes per
duration).
• Desiccated and non
desiccated (control) embryonic
axes were placed in 2ml sterile
cryovials.

9. • Cryovials were
directly immersed into
liquid nitrogen (-
196°C) and held for
1hr.

10. • Thawing took place in
a water bath at 40°C
for 2min.

11. • Embryonic axes were
cultured individually in
test tubes containing
10ml of MS medium
(Murashige and
Skoog, 1962)
supplemented with
15mg/L 6-
benzylaminopurine
(BAP) and solidified
with 8g/L agar.

12. • Cultures were
maintained in a
growth chamber at
26±2°C under 16hr
light/8hr dark photo
period provided by
white inflorescence.

13. • Regenerated
microshoot of
groundnut after
4weeks

14. • Microshoots were
subcultured in MS
media supplemented
with 1mg/L NAA for
rooting

15. • Hardening-off &
acclimatization
– Conditioning plantlets
to the external env’t•
i.e. before transfer to
field conditions

16. • Groundnut plantlet in
the screen house

17. Expected Impact
• Genebank managers have
a complimentary
biotechnology conservation
technique
• Breeders have wide array
of groundnut germplasm
• Improved groundnut seeds
that are high yielding and
resistant to pest and
diseases and better
adapted to changing
climates made available to
farmers
• Groundnut pyramids of
Kano flourishing

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