Cryopreservation of Conifers: Challenges and Bottlenecks Outline

Cryopreservation of conifers: Challenges and bottlenecks 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 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 Forests and conifers

• By 2040, the wood consumption of industrial wood will increase from 1.5 billion m3/year to 2.5 billion m3/year

• It has been suggested that between 50 and 70 % of industrial 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 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 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

Humans Pinus taeda Arabidopsis Populus trichocarpa Picea abies (3 00 Mbp) 24 000 Mbp (120 Mbp) (450 Mbp) 20 000 Mbp Vegetative propagation

• rooting of cuttings • organogenesis • somatic embryogenesis Vegetative propagation

Rooting of cuttings • Success with Eucalyptus, • 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) • Proper tesng requires al least ⅓ to ½ the rotaon age Vegetative propagation

Organogenesis • 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 Vegetative propagation

Somatic embryogenesis 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. 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 Larix (6) Genus Araucaria (1) • Larix kaempferi • Araucaria angustifolia (Bert.) O. • Larix laricina Kuntze • Larix leptolepis • Larix × eurolepis Genus Abies (3) • Larix × marschlinsii • Abies alba • Larix occidentalis • Abies cephalonica • Abies nordmanniana • A. alba × A. cephalonica • A. alba × A. numidica Cryopreservation

• How many coniferous species have been cryopreserved till now?

Genus Picea (8) Genus Pinus (10) • Picea abies • Pinus armandii • Picea engelmanni • Pinus caribea • Picea glauca • Pinus kesiya • Picea glauca × engelmanni complex • Pinus nigra • Picea mariana • Pinus pinaster • Picea omorica (also genetically modified) • Picea sitchensis • Pinus patula • Picea pungens • Pinus radiata • Pinus roxburghii • Pinus sylvestris • Pinus taeda 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. 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 24 - 48 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 Cryopreservation

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)

• Shoot tips • Pinus kesiya (Kalita et al. 2012) • Sequoia sempervirens (Ozudogru et al. 2011, 2012), droplet vitrification • Tetraclinis articulata (Serrano-Martinez and Casas 2011) 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 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) 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) 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 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 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 Challenges and bottlenecks

• Cryopreservation as active part of multi-varietal forestry using SE 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  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) Acknowledgement

• University of Oulu, Finland

• University of Udine, Italy

• Finnish Forest Research Institute, Punkaharju Research Station, Finland

• National Forest Research Institute, Slovakia IUFRO Working party 2.09.02 Vegetative propagation of trees