pollen cryopreservation - inra · pollen cryopreservation carla benelli ... disadvantages only half...
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POLLEN CRYOPRESERVATION
Carla BenelliIstituto per la Valorizzazione del Legno e delle Specie Arboree - CNR
COST Action FA1104 ‘Sustainable production of high--quality cherries for the European market’
Workshop:Long Term Preservation of Woody Species
by Cryo-Techniques
26 & 27 March, 2015Area di Ricerca CNR di Firenze, Italy
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Why topreserve pollen
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Pollen genebank
Recalcitrant pollen
Preservenuclear genepool
International exchangeof germplasm
Allergy studies
Biochemical and physiological research
Haploid production
Impollinationand
breeding strategy
Floweringin different periods
Flowering in differentand/or distant locations
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Advantages Alternative for recalcitrant seed or
tissue culture impediments
Breeding programs and improve production
Relative easy procedure
Relative low cost
Facilitates global exchange
Least quarantine problems
Disadvantages Only half the genome will be preserved
Pollen of many species cannot be stored (recalcitrant pollen)
Limited pollen production
No standardized processing or viability-testing protocols.
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Influence on longevity
Temperature and moisture content
In many species pollen preservation at temperatures between
4°C and -20°C (short-period)
To extend the period of storagethe cryopreservation may be an aid tool
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Cryopreservation pollen in Prunus spp.
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IVALSA’s experience on pollen cryopreservation
Persimmon Olive
In collaboration with DISPSAA – University of Florence
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Diospyros spp.
Most of persimmon production relies on parthenocarpy; neverthelessmale fertility is a relevant issue for breeding and in specific environmentalor productive conditions where fruit-set needs to be improved.
The natural sex-expression of persimmon (polygamous dioic) is a bottle-necks in breeding linked to the effect of anthropic selection of progenies,which lead to a small amount of monoic cultivars. In non native areas ofPersimmon , germplasm collections usually hold a very limited number ofpollen donors
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Olive shows different factors of sterility which hinder productivity; among them,self- and inter-incompatibility and morphological aberrations are relevant.
Low fecundation capability due to a reduced viability and germinability of pollengrains is common in olive trees.
Olea europaea
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Petri dishes were used -80 °C storage
Cryovials were used to store pollen grains in LN
Pollen grains were thawed under a laminar flow for 2 hours at room
temperature
Flowers from adult trees grown at the IVALSA-CNR repository (Tuscany)Flowering shoots were collected in a sunny day (mid-May)
Samples of pollen grains were stored:
for 30, 90, 180 and 360 days
at – 80 °C and - 196 °C (LN)
For each treatment more than500 (250 x 2 repetitions) pollen grains
observed
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Evaluation quality of pollen
(at time 0 and after each conservation period)
Viability and Germinability percentage were assessed
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VIABILITY
• Fluorescein-diacetate/Propidium-iodide (FDA-PI) test (Greissl,1989);
• pollen grains incubated in dark for 24 hours at 24°C;
• observation at Leica DM 1000 microscope with a high-pressure mercurylamp of 50 W at blue excitation (450-490 nm) and cutting at 510 nm.
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GERMINABILITY
Medium for germination:
- sucrose (15%)- boric acid (50 mg/l H3BO3)-Phytoagar (0.6%)
-Incubation for 48 hours at 25°C
Pollen grains were considered germinated when thelength of pollen tube was 2-fold the graindiameter (Pinney and Polito,1990)
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0
10
20
30
40
50
60
70
80
90
100
Vitalità
Germinabilità
After 5-6 days at room temperature pollen grains were notviable and showed no germination
Average:
Viability = 83.8%
Germinability = 11.6%
Diospyros – fresh pollenControl (T 0 )
A A A AA
A A AA
B
Germinability
Viability
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0
5
10
15
20
25
30
35
40
45
50
vitalità
germinabilitàGerminability
AA
AB
A
AB
C
B
C
A
A
A
AAAA
B
Average:
Viability = 41 %
Germinability = 23 %
Olive- fresh pollenControll (T 0 )
Viability
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Ultra-low temperatures guarantee the survival of assessed accessions pollen grains after one year of storage.
Storage in LN resulted the best treatment in order to keep good levels of pollen viability and in vitro germinability
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Wide-Scale Pollen Banking of Ornamental Plants Through CryopreservationXu J. et.al - Cryoletters, 2014
71 species/cultivars
60 had pollen viabilityafter 1 year's cryopreservation
Wisteria sinensis 43.6 30.3 2.6
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POLLEN
CRYOBANK