POLLEN CRYOPRESERVATION

Transcription

1 COST ction F1104 Sustainable production of high--quality cherries for the European market Workshop: Long Term Preservation of Woody Species by Cryo-Techniques 26 & 27 March, 2015 rea di Ricerca CNR di Firenze, Italy POLLEN CRYOPRESERVTION Carla Benelli Istituto per la Valorizzazione del Legno e delle Specie rboree - CNR

2 Why to preserve pollen

3 Preserve nuclear genepool International exchange of germplasm Recalcitrant pollen Haploid production Pollen genebank llergy studies Biochemical and physiological research Flowering in different periods Impollination and breeding strategy Flowering in different and/or distant locations

4 dvantages lternative 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.

5 In many species pollen preservation at temperatures between 4 C and -20 C (short-period) Influence on longevity Temperature and moisture content To extend the period of storage the cryopreservation may be an aid tool

6 Cryopreservation pollen in Prunus spp.

7 IVLS s experience on pollen cryopreservation Persimmon Olive In collaboration with DISPS University of Florence

8 Diospyros spp. Most of persimmon production relies on parthenocarpy; nevertheless male fertility is a relevant issue for breeding and in specific environmental or productive conditions where fruit-set needs to be improved. The natural sex-expression of persimmon (polygamous dioic) is a bottlenecks in breeding linked to the effect of anthropic selection of progenies, which lead to a small amount of monoic cultivars. In non native areas of Persimmon, germplasm collections usually hold a very limited number of pollen donors

9 Olea europaea 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 pollen grains is common in olive trees.

10 Flowers from adult trees grown at the IVLS-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 C (LN) Petri dishes were used -80 C storage Cryovials were used to store pollen grains in LN For each treatment more than 500 (250 x 2 repetitions) pollen grains observed Pollen grains were thawed under a laminar flow for 2 hours at room temperature

11 Evaluation quality of pollen (at time 0 and after each conservation period) Viability and Germinability percentage were assessed

12 VIBILITY Fluorescein-diacetate/Propidium-iodide (FD-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 mercury lamp of 50 W at blue excitation ( nm) and cutting at 510 nm.

13 GERMINBILITY 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 the length of pollen tube was 2-fold the grain diameter (Pinney and Polito,1990)

14 Diospyros fresh pollen Control (T 0 ) B Viability Vitalità Germinability Germinabilità verage: Viability = 83.8% Germinability = 11.6% fter 5-6 days at room temperature pollen grains were not viable and showed no germination

15 Olive- fresh pollen Controll (T 0 ) B B C B B C Viability vitalità germinabilità Germinability verage: Viability = 41 % Germinability = 23 %

16 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

17 71 species/cultivars 60 had pollen viability after 1 year's cryopreservation Wisteria sinensis Wide-Scale Pollen Banking of Ornamental Plants Through Cryopreservation Xu J. et.al - Cryoletters, 2014

18 POLLEN CRYOBNK