Cryopreservation of woody species and cryotherapy

Transcription

1 Cryopreservation of woody species and cryotherapy Maurizio Lambardi Istituto per la Valorizzazione del Legno e delle Specie Arboree

2 Plant cryo-meetings 20 years ago.and today!!!

3 CIMMYT Svalbard Global Seed Vault About 1750 seed banks in the world ICARDA Ex situ conservation of genetic resources Form of conservation Accessions Distribution Seed banks Cereals Others % 45% In field collections % In vitro + cryopres.???? --- Source SoWPGR-2, FAO 2010 Total %

4 Florence, July 2001 CRYOPRESERVATION OF WOODY PLANT GERMPLASM Milestone n 1: 1960, Akira Sakai Akira Sakai Populus sieboldii Salix koriyanagi

5 Milestone n 2: 1990, Akira Sakai Jean Dereuddre

6 Milestone n 3: 1982, Luis Mroginski & K.K. Kartha Droplet-freezing techniques The strips are inserted in cryovials filled with liquid nitrogen Explants are treated in drops of DMSO ( droplet-method ) or PVS2 ( droplet-vitrification ) over strips of aluminium foils Thawing at room temperature and plating The racks are immersed in liquid nitrogen

7 AXILLARY BUDS Treatment with PVS2 solution Encapsulation- -dehydration Droplet methods Encapsulation- -vitrification Dehydration Dehydration + slow cooling (-1 C/h) SEEDS AND EM- BRYONIC AXES, POLLEN Two-step freezing (-1 C/min) DORMANT BUDS CELL AND CALLUS CULTURES

8 Broad-leaf trees Conservation of clonal germplasm high importance! Fruit species, timber species (e.g., poplar), outdoor ornamentals Shoot tips (axillary/apical microbuds) Conservation of seedpropagated species Forest species Seeds, embryonic axes Embryogenic callus Dormant (winter) buds

9 Conifers Conservation of seedpropagated species high importance! Forest species Seeds, embryonic axes Embryogenic callus Conservation of clonal germplasm Outdoor ornamentals, conifers with a well-developed micropropagation technique Shoot tips (axillary/apical microbuds)

10 Shoot tips (=meristematic apexes from axillary and apical microbuds) Largely utilized for the conservation in LN of fruit species Can be used naked or incapsulated in Ca-alginate Suitable for using with several cryo-techniques (PVS2-based vitrification, encapsulation-dehydration, encapsulation-vitrification, droplet-methods, cryo-v-plate) Useful in cryotherapy Advantages Tens of explants from one single jar of shoots Thousands of explants can be preserved even in small dewars

11 High post-thaw recovery with optimized procedures Genus Actinidia Diospyros Malus Olea Prunus Plant species (n Cultivar/rootstock) a Cryopreservatio n technique b Maximum regrowth (%) A. chinensis (1) Vitrif 52 Xu et al. (2006) A. deliciosa (1) En-dehy 45 Zhai et al. (2003) Actinidia spp. (9) En-dehy 95 Bachiri et al. (2001) Actinidia spp. (3) En-dehy 56 Wu et al. (2001a) Diospyros spp. (2) Vitrif 30 Ai and Luo (2003) Diospyros spp. (3) Droplet-vitrif 80 Niu et al. (2009) M. domestica (5) En-dehy 88 Paul et al. (2000) M. domestica (4) Droplet 80 Wu et al. (2000) M. domestica (2) En-dehy 78 Kushnarenko et al. (2006) M. domestica (1) Vitrif 80 Liu et al. (2008) M. domestica (4) Vitrif / En-dehy 80 Kushnarenko et al. (2009) M. domestica (4) Droplet-vitrif 70 Halmagyi et al. (2010a) M. domestica (2) Droplet-vitrif 68 Halmagyi et al. (2010b) M. domestica (1) En-dehy 72 Forni et al. (2010) M. domestica (4) Droplet 70 Condello et al. (2011) M. pumila (3) En-dehy 87 Hao et al. (2001) M. pumila (1) Vitrif (PVSL) c 81 Liu et al. (2004) O. europea Vitrif 15 Benelli et al. (2001) O. europea Vitrif 30 Nisi et al. (2006) O. europea Vitrif 38 Lynch et al. (2007) P. cerasifera Droplet-vitrif 20 Vujovic et al.(2011) P. domestica Vitrif 57 De Carlo et al. (2000) P. dulcis Vitrif 88 Channuntapipat et al. (2000) P. dulcis En-dehy 62 Shatnawi et al. (2000) P. dulcis Droplet 58 De Boucaud et al. (2002) P. dulcis Vitrif 67 Al-Ababneh et al. (2003) P. dulcis En-dehy 60 Al-Ababneh et al. (2003) P. dulcis Vitrif 80 Wirthensohn et al. (2006) Prunus hybrids TSF 74 De Boucaud et al. (2002) P. persica Droplet 32 De Boucaud et al. (2002) Prunus rootstock Droplet-vitrif 52 De Boucaud et al. (2002) P. salicina Vitrif 60 Zhao et al. (2008b) Ref.

12 Shoot tips (=meristematic apexes from axillary and apical microbuds) Problematics Cryo-techniques are poorly efficient in species with low proliferation rates (e.g., walnut, chestnut, olive, ) Results are often cultivar-dependent Slow recovery of shoot culture lines in post-cryopreservation The from field-to field is highly time-consuming

13 Cryopreservation of dormant buds Towill L.E. e Ellis D.D., Cryopreservation of dormant buds. In: Reed B.M. (ed) Plant Cryopreservation. A Practical Guide. Springer, New York, pp Over 2,200 accessions of APPLE cryopreserved at the USDA/NCGPR of Fort Collins, U.S.A. Uni-nodal segments, cold hardened at 4 C and dehydrated up to a MC of 35-26% (6-8 wks) Slow cooling up to -30 C (-1 C/hour) -5 C Winter collection of scions and cold harde ning at -5 C (8 wks) Chip budding After 1-2 wks Thawing and rehydration in damp peat at 2 C (2 wks) After 6 wks Rootstock: Malus floribunda, cv Radian -196 C

14 Advantage: tissue culture not required!! Tissue culture-based technique: vitrification of shoot tips -196 C Dormant bud-based cryopreservation technique

15 Dormant bud cryotechnique vs. Tissue culture-based cryotechnique Lambardi M., Previati A., Benelli C., Da Re F., De Carlo A., Ellis D., Acta Hort., in press (1st Int. Symp. on Cryopreservation in Horticultural Species. Leuven, 5-8 April 2009). from the field to LN in almost 1/2 time from LN to the field in 1/4 of the time about 50% of the handlabour required

16 Problematics - 1 The technique requires to be optimized for each cv, especially as for moisture content of uninodal sections before LN but.. only one trial per year can be carried out, due to grafting time a large number of goodquality rootstocks is necessary every year Good healing Failure

17 Pre-grafting tests

18 Problematics - 2 the species-specificity of the technique Pear apple Cherry Olive Not idoneous for species that cannot be reproduced by budding Olive grafting

19 Problematics - 3 Dormant buds vs. PVS2 vitrification Dewar of 120 lt of liquid nitrogen 7 : 1 8 racks x 10 cryoboxes x 25 cryovials x 10 shoot tips = 20,000 units of conservation 8 racks x 10 cryoboxes x 7 bags x 5 segments = 2,800 units of conservation

20 Cryopreservation of seeds and embryonic axes

21 When seed cryopreservation is very easy Abies alba Pinus cembra Fagus sylvatica % Abies viable non viable empty total +LN viable ctrl ctrl (-LN) 15 A40CV ARTCV A40AL ARTAL Pinus viable non viable empty total +LN viable ctrl ctrl (-LN) 1 CN40CV CNRTCV CV40CV CVRTCV CN40AL CNRTAL CV40AL CVRTAL Fagus viable non viable empty total +LN viable ctrl ctrl (-LN) 9 FN40CV FNRTCV FN40AL FNRTAL FV40AL FVRTAL TTC test

22 Germination (%) Pistacia vera L. Desiccation: silica gel, 8 h Thawing: 37 C, 5 min Cryopreservation: direct immersion in LN Germination: hormone-free MS Natural germinability: 100% 90% germinability after seed cryopreservation MC (%) 20 16,9 14,5 12,5 11,7 11,4 9,8 Time (h) CONTROL LN+ Seedling from a cryopreserved seed Ozden-Tokatli Y, Ozudogru EA, Gumusel F, Lambardi M (2007) CryoLetters, 28:

23 Germinability (%) Cryopreservation of polyembrionic seeds 100 Foetifera 80 75% C. volkameriana C. aurantium x C. paradisi C. sinensis C. sinensis 'Oblongus' C. aurantium 'Virgatum' C. limon 'Foliis variegatis' C. aurantium 'Foetifera' C. lumia 'Pyriformis' C. medica C. limon C. limonmedica 'Perettone' C. limonmedica 'Fiorentina' C. limetta di Rangpur C. meyer -LN +LN C. aurantium Virgatum De Carlo A, Lambardi M, Ozudogru EA (2011). Meth. in Molec. Biol., 710. Springer, pp Lambardi M, Halmagyi A, Benelli C, De Carlo A, Vettori C (2007). Adv. Hort. Sci. 21:

24 Contenuto in acqua(%) Contenuto in acqua (%) Contenuto in acqua (%) Contenuto in acqua (%) Contenuto in acqua (%) Dehydration trend MC 60 % C. aurantium C. Foetifera aurantium 50 MC % C. volkameriana C. limon (Volkameriana) MC % C. lumia Pyriformis C. lumia Disidratazione Dehydration (ore) (h) Disidratazione Dehydration (ore) (h) Disidratazione Dehydration (ore) (h) MC % C. aurantium x C. paradisi C. aurantium x C. paradisi MC % C. sinensis C. sinenis Disidratazione Dehydration (ore) (h) Disidratazione Dehydration (ore)(h)

25 Problematic Seeds with large cotyledons cannot be cryopreserved entire Cryopreservation of embryonic axes

26 Cryopreservation of embryogenic callus lines Ash Cypress Horsechestnut Olive Norwey spruce

27 Slow cooling Conifers Effective protocols developed for 14 species from 3 genera Explant Embryogenic callus or suspension cells Preculture M sorbitol Pre-freezing 5% DMSO (30 min) sorbitol/sucrose/peg Direct immersion in LN Cooling rate C/min or -70/-80 C Thawing 37 C-45 C Ozudogru E.A., Lambardi M., In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)

28 Broadleaf trees: slow cooling Explant Embryogenic callus or somatic embryos Pre-freezing 10% DMSO (30 min) (+ 1M sucrose) Cooling rate C/min Thawing RT-40 C Ozudogru E.A., Lambardi M., In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)

29 Broadleaf trees: one-step freezing Effective protocols developed for 11 species from 10 genera Explant Embryogenic callus or somatic embryos (G/H/T/Cot) Preculture M sucrose (4 C) Pre-freezing PVS2 ( min) ENC+DEHY/DEHY Thawing RT-45 C Ozudogru E.A., Lambardi M., In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)

30 Cryopreservation by SLOW COOLING CRYOPROTECTION: WPM gr/l sucrose + 7.5% DMSO, min, at 0ºC SLOW COOLING to -40 C (-1 C/min)

31 60 MINUTE-TREATMENT (Sucrose + DMSO) WPM g/l sucrose day 0 WPM g/l sucrose day 42 WPM g/l sucrose WPM g/l sucrose

32 TTC (=TEZ) TEST (2,3,5- triphenyltetrazolium chloride) 1.0% TTC, overnight at 30 C and darkness day 42 day 0 day 14 2 months

33 Horsechestnut (Aesculus hippocastanum) CRYOPRESERVATION Explants: Portion of embryogenic callus Crio-technique: PVS2-based (90 at 0 C) Recovery from LN: >90%

34

35 Introducing cryotherapy.

36 Traditional approach to recover virus-free plants 37 C Thermotherapy Shoo-tip excision ( mm) Shoot-tip colture Virus-free plant Shoot development

37 Relations among virus eradication, meristem size and shoot development

38 Virus eradication by cryotherapy Courtesy of Qiaochun Wang REVIEW PAPER Wang Q., Panis B., Engelmann F., Lambardi M. & Valkonen J.P.T., Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation. Annals of Applied Biology, Vol. 154:

39 Virus Species Eradication in postcryopreservation (%) Reference Plum Pox Virus (PPV, Sharka) Prunus 75 Brison et al., 1997 Banana Streak Virus (BSV) Tomato Mosaic Virus (TMV) Grapevine Virus A (GVA) Banana 90 Helliot et al., 2002 Banana 30 Helliot et al., 2002 Grape 97 Wang et al., 2003 Potato Virus Y (PVY) Patato 95 Wang et al., 2006 Potato LeafRoll Virus (PLRV) Sweet Potato Feathery Mottle Virus (SPFMV) Sweet potato chlorotic stunt virus (SPCSV, Crinivirus) Raspberry Bushy Dwarf Virus (RBDV) Sweet Potato Little Leaf Phytoplasma Patato 86 Wang et al., 2006 Sweet potato 100 Wang et al., unpublished Sweet potato 100 Wang et al., unpublished Raspberry 35 Wang et al., unpublished Sweet potato 100 Wang et al., 2007