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Slow Growth Storage

Slow growth storage is a method for medium-term conservation of in vitro plant cultures at above freezing temperatures, which reduces plant metabolism by modifying culture conditions. This technique is crucial for commercial micropropagation as it minimizes contamination risks, enhances production organization, and facilitates the transfer of materials between laboratories. Key factors affecting storage include temperature, light conditions, medium composition, and the health of stock cultures.

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0% found this document useful (0 votes)
31 views19 pages

Slow Growth Storage

Slow growth storage is a method for medium-term conservation of in vitro plant cultures at above freezing temperatures, which reduces plant metabolism by modifying culture conditions. This technique is crucial for commercial micropropagation as it minimizes contamination risks, enhances production organization, and facilitates the transfer of materials between laboratories. Key factors affecting storage include temperature, light conditions, medium composition, and the health of stock cultures.

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defnesucoskun572
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Slow Growth Storage

Prof. Dr. Elif Aylin Özüdoğru


Fall 2024-2025, İstanbul
[email protected]
Conservation of Plant Biodiversity

In situ Ex situ

Rain forests, national Field collections


parks, farms… (botanic gardens)

Plant tissue Seed banks


culture

In vitro collections

Slow growth
storage
(°C , O2 , H2O Cryopreservation
, medium ~) (-196°C)
✓ Slow growth storage (=conservation at above freezing temperatures)

✓ Cryopreservation (=conservation at cryogenic temperatures,-196°C)


✓ Slow growth storage (=conservation at above freezing temperatures)

It is a technology that provides us medium-term


conservation of in vitro shoot cultures (usually up to
several year).

Plant metabolism is reduced by modifying the culture


conditions and/or medium compositions.
(°C , O2 , H2O , medium ~)
✓ Slow growth storage (=conservation at above freezing temperatures)

Important for commercial micropropagation laboratories,


because:
it allows a better organization of production
number of subcultures is markedly reduced
risks of contamination are markedly reduced
the laboratory offer (species, cultivars) is greatly widened
it makes easier the transfer of material between long-
distance laboratories
Subculture cycle

3-4 3-4 3-4


Weeks Weeks Weeks

1st...................4th..................8th......................12th

Several months
?
Conservation at low temperature
Conservation of shoot culture at low temperature (=slow growth storage)

Factors affecting the storage in vitro


of shoot cultures at low temperature

• Quality of stock cultures


• Storage temperature (2-10°C)
• Darkness/Photoperiod conditions
• Medium composition
• Containers for conservation

determining the

MAXIMUM CONSERVATION TIME


Shoot lines introduced to slow growth
storage MUST be:
• in healthy conditions (= no symthoms of
vitro-pathologies, e.g., hyperhydricity)
• tested for being virus-free and latent
contamination-free (bacteria)
• coming from a not-too-many cycles of
subculturing
• introduced to conservation 7-10 days
after last subculturing
Lambardi and Ozudogru, 2013
Conservation:
✓ 4°C - darkness
✓ DKW, 0.5 M BA

Initial material
32.4

62.4

After 12-month storage

After 1st subculture of post-storage


Lambardi et al., 2006.
Gisela 5®
RGR = (Ln FWfinal -Ln FWinitial) x 100 / n° days of storage

Sucrose 30 g/lS30 Sucrose 45 g/lS45


5 5
4,33
4 3,64 4

3 3
2,13
2.01 2
2
1
1 0,27
0,32
0
0
standard culture after 16 mon post-storage standard culture after 16 mon post-storage
Inizio
conditions 16
thsmesi
at 4°C Rec.
regrowth Inizio
conditions 16 mesi
ths at 4°C Rec.
regrowth

Sucrose 60 g/l
S60
Sucrose 30 g/l + Mannitol 15S30+M15
g/l
5
5 4,83 4,31 4,43
3,81 4
4
3
3

2 2

1 1
0,29 0,19
0
0
standard culture after 16 mon post-storage
Inizio
conditions
16 mesi
ths at 4°C
Rec.
regrowth
standard culture
Inizio
after 16 mon
16 mesi
post-storage
Rec.
conditions ths at 4°C regrowth
Gisela 5® 12 months: morphological evaluation of survival and
quality (% of jars with material still conservable)

100
+/- ++ +++ +
90 100 100 100
80
70
60 66,7
50
%

40
30
20
10
0

S30 S45 S60 S30+M15

+++, excellent; ++, good; +, sufficient; -, poor

16 months: “workable” material after slow growth storage (WM = gr/jar)

15
12,65
12
8,85
9

6 5,52
3,81
3

0
S30 S45 S60 S30+M15

WM (Workable Material) = (no jars still in storage/total n° of initial jars) x (average gr of WM per jar)
Gisela 5® After 20 months

…after 3-4 subcultures


Slow growth storage:

- Gas permeability +
Slow growth storage
Storage conditions:

✓ 4°C
✓ darkness
✓ different containers
✓ 6 months
Glass jars,
500 cc

Plastic tubes,
80 cc

‘StarPac’ plastic bags


‘Rosa Sant’Antonio di Padova’ Conservation at 4°C in the dark: 4 months

Transfer to standard culture conditions


PR
2.1
4 weeks in: MS, 30 g/l
sucrose, 2 µM BA;
20°C, 16h, 45 µM m-2 s-1
2.1

Standard
1.7 Proliferation Rate:
2.0
‘Domenica’ Conservation at 4°C in the dark: 4 months

Transfer to standard culture conditions


PR
2.5
4 weeks in: MS, 30 g/l
sucrose, 2 µM BA;
20°C, 16h, 45 µM m-2 s-1
1.9

Standard
2.0 Proliferation Rate:
3.7
Conservation at 4°C in the dark: 6 months

‘Rosa Sant’Antonio
di Padova’

‘Domenica’

Recovering of etiolated material

2-3 subcultures
Stop complaining!
I told you! We don’t have
money to buy a cold cham-
ber for the laboratory!

Thanks for your


SLOW GROWTH STORAGE
patience and
attention

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