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

Why topreserve pollen

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

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.

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

Cryopreservation pollen in Prunus spp.

IVALSA’s experience on pollen cryopreservation

Persimmon Olive

In collaboration with DISPSAA – University of Florence

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

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

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

Evaluation quality of pollen

(at time 0 and after each conservation period)

Viability and Germinability percentage were assessed

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.

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)

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

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

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

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

POLLEN

CRYOBANK