Cryopreservation of conifers: Challenges and bottlenecks

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1 Cryopreservation of conifers: Challenges and bottlenecks

2 Outline Forests and conifers Vegetative propagation and tree breeding Cuttings Organogenesis Somatic embryogenesis Success with cryopreservation of conifers Study cases: A. cephalonica and P. abies Challenges and bottlenecks

3 Forests and conifers Forests cover more than 4 billion ha of the Earth s surface In Europe, forests represent almost half of the land surface (102 million ha, ¼ of the world total), of which 65 % are conifers IUCN Red list includes 6,277 tree species that are threatened with extinction 1,002 tree species are recorded as Critically Endangered

4 Forests and conifers By 2040, the wood consumption of industrial wood will increase from 1.5 billion m 3 /year to 2.5 billion m 3 /year It has been suggested that between 50 and 70 % of industrial wood will have to come from planted forests wood will have to come from planted forests In Europe only 4 % are plantation forests (public acceptance) The use of vegetative propagation in forestry is the fastest, the most flexible and effective way to produce enough genetically improved material to meet future demand

5 Forests and conifers Domestication of food crops has an extensive history Trees were not domesticated except for the partial domestication of some fruit trees Tree improvement (domestication) began about more than a century ago with conventional breeding

6 Forests and conifers Characterized by long rotation times and long intervals in seed production They have numerous vegetative characteristics that change with maturation A higher average degree of heterozygosity than other plant species Large genome size, nearly 7x human Only 0.1 % consists of genes, little is known about gene and genome structure Arabidopsis (120 Mbp) Populus trichocarpa (450 Mbp) Humans (3 00 Mbp) Picea abies Mbp Pinus taeda Mbp

7 Vegetative propagation rooting of cuttings organogenesis somatic embryogenesis

8 Rooting of cuttings Success with Eucalyptus, Vegetative propagation plantations with superior selected trees were ready for harvesting after 7 years Conifers stem cuttings suitable for rooting are available only from young plants (difficult to predict their growth traits) from young plants (difficult to predict their growth traits) Proper tes ng requires al least ⅓ to ½ the rota on age

9 Organogenesis Vegetative propagation Has been very effective with some conifers, Pinus radiata However, for many conifer species have not reached a practical application, because of low plantlet formation rates, and excessive handling and costs

10 Somatic embryogenesis Vegetative propagation

11 Cryopreservation A key advantage of conifer SE over other micro and macropropagation methods is that, embryogenic cultures can be cryopreserved and stored while field tests are conducted to identify genotypes with significant genetic gain. The ability to maintain donor tissue juvenility through cryopreservation represents an immeasurable advantage over propagation programmes based on rooted cuttings and systems based on organogenesis.

12 Cryopreservation First reports were published in the late 1980 s; the target species being Picea glauca (Kartha et al. 1988) Picea abies and Pinus taeda (Gupta et al. 1987) How many coniferous species have been cryopreserved till now? Genus Araucaria (1) Araucaria angustifolia (Bert.) O. Kuntze Genus Abies (3) Abies alba Abies cephalonica Abies nordmanniana A. alba A. cephalonica A. alba A. numidica Genus Larix (6) Larix kaempferi Larix laricina Larix leptolepis Larix eurolepis Larix marschlinsii Larix occidentalis

13 Cryopreservation How many coniferous species have been cryopreserved till now? Genus Picea (8) Picea abies Picea engelmanni Picea glauca Picea glauca engelmanni complex Picea mariana Picea omorica Picea sitchensis Picea pungens Genus Pinus (10) Pinus armandii Pinus caribea Pinus kesiya Pinus nigra Pinus pinaster (also genetically modified) Pinus patula Pinus radiata Pinus roxburghii Pinus sylvestris Pinus taeda

14 Cryopreservation How many coniferous species have been cryopreserved till now? Pseudotsuga menziesii Sequoia sempervirens Genus Taxus (3) Taxus chinensis Taxus media Rehd. Taxus floridans Nutt. Tetraclinis articulata (Vahl.) Masters Torreya taxifolia Arn.

15 Cryopreservation Most common method is controlled rate cooling incubation of embryogenic suspension in a proliferation medium with 0.2M and 0.4M sorbitol or sucrose for a period of h Cryoprotectans: 5-10 % (v/v) DMSO, 0.4 M sorbitol, PEG Vials are cooled in a programmable chamber or using alcohol-insulated containers (Mr. Frosty) plunged in liquid nitrogen and stored Afterwards are rapidly thawed for 1-2 min, at 37 C

16 Vitrification based protocols Embryogenic cell masses Araucaria angustifolia (Demarchi et al. 2014) Picea mariana (Touchell et al. 2002) Pinus armandii (Ishii et al. 2011) Somatic embryos Picea sitchensis (Gale et al. 2008) Cryopreservation Shoot tips Pinus kesiya (Kalita et al. 2012) Sequoia sempervirens (Ozudogru et al. 2011, 2012), droplet vitrification Tetraclinis articulata (Serrano-Martinez and Casas 2011)

17 Cryopreservation Cryopreservation of desiccated somatic embryos Picea glauca (Bomal and Tremblay 2000) (Kong and von Aderkas 2011) decrease of temperature during maturation, rapid cooling without cryoprotectant Picea mariana (Bomal and Tremblay 2000) Pseudotsuga menziesii (Kong and von Aderkas 2011) Picea glauca engelmanni (Percy et al. 2001) Encapsulation and dehydration Picea sitchensis (Gale et al. 2008), somatic embryos used Cryopreservation by desication of embryogenic cell masses Picea omorica (Hazubska-Przybyl el al. 2010) Picea abies (Hazubska-Przybyl el al. 2013) + ABA

18 Cryopreservation Cryopreservation of cotyledons from zygotic embryos Picea glauca (Toivonen and Kartha 1989) Pinus radiata (Hargreaves et al. 2004, 2005) Cryopreservation of dormant buds Pinus sylvestris (Kuoksa and Hohtola 1991)

19 Cryopreservation Cryopreservation of intact seeds Abies alba (Ahuja 1988, Chmielarz 2010) Larix decidua (Ahuja 1988) Picea abies (Ahuja, 1988, Suszka et al. 2005) Pinus canarensis, P. halepensis, P. nigra, P. pinaster, P. pinea, P. sylvestris, P. unicinata (Pita et al. 1997, Pita et al. 1998)

20 Study case Abies cephalonica The cryopreservation protocol based on slow cooling method for was developed for 8 embryogenic cell lines (Aronen et al. 1999) cold-hardening on MS-based proliferation medium with 0.2 M sucrose for 24 h and with 0.4 M for additional 24 h 10% PEG 6000, 10% glucose, 10% DMSO added drop wise for period of 30 min. reaching the final concentration of 5% Samples were frozen using a programmable controlled temperature cooling chamber All cell lines recovered after thawing The results emphased the necessity of monitoring genetic fidelity of cryopreserved material (a low level of background variation was observed in the DNA samples isolated from untreated and non-frozen material). 2 cell lines were stored for another 6-years

21 Study case 3 Abies cephalonica Long-term cryopreservation (Krajnakova et al. 2011) Cultures were able to proliferate and produce somatic embryos with morphological fidelity after 6 years in cryopreservation

22 Study case 3 Abies cephalonica Long-term cryopreservation (Krajnakova et al. 2011) The genetic fidelity analyses with RAPD markers indicated some changes when comparing the profiles derived prior and after long-term cryopreservation Necessity to carefully consider the marker selection as well as the importance to investigate the genetic stability during and after long-term cryopreservation

23 Challenges and bottlenecks Cryopreservation as active part of multi-varietal forestry using SE

24 Challenges and bottlenecks Reliable protocols for regeneration via SE A. cephalonica does not present advanced enough SE to enable plans for large scale clonal plantation forestry Reliable protocols for during the prolonged storage times genetic fidelity movement from individual marker analyses to high throughput genome analyses or specific genome area analyses or combination of specific genome markers and high throughput sequencing. will be available for a reasonable price, soon

25 Challenges and bottlenecks Development of new cryo-protocols without cryoptotectants More information is needed from field tests with cryo-material (only 10 publications) When a cryo-protocol is approved? Effects of environmental changes and timing (epigenetic changes) Advantage of being able to cryopreserve SE cultures may eventually lose some of its significance if cryopreservation is extended for use in the rooting of cuttings and organogesis process (Bonga 2015)

26 Acknowledgement University of Oulu, Finland University of Udine, Italy Finnish Forest Research Institute, Punkaharju Research Station, Finland National Forest Research Institute, Slovakia

27 IUFRO Working party Vegetative propagation of trees