A cryopreservation protocol for ex situ conservation of terrestrial orchids using asymbiotic primary and secondary (adventitious) protocorms

In Vitro Cellular & Developmental Biology - Plant - 2015
Betty Mauliya Bustam1,2,3, Kingsley Dixon1,4,2, Eric Bunn1,2
1School of Plant Biology, The University of Western Australia, Crawley, Australia
2Kings Park and Botanic Garden, West Perth, Australia
3Mathematics and Natural Sciences Faculty, Syiah Kuala University, Banda, Indonesia
4Department of Environment and Agriculture, Curtin University, Perth, Australia

Tóm tắt

In a bid to better conserve endangered terrestrial orchids, we detail cryogenic research using a widely distributed terrestrial orchid, Caladenia latifolia, as a model species for development of cryopreservation for primary (seed generated) and secondary (adventitious) protocorms. Primary protocorm cryopreservation (using droplet vitrification) involved a number of experimental lines of inquiry: investigation of a suitable plant vitrification solution (PVS) by comparing three variants of a standard PVS (2, 3 and 4), determining the most suitable primary protocorm developmental stage for successful cryopreservation, testing the effectiveness of a preculture medium treatment prior to cryopreservation, and investigating temperature preconditioning at the preculture stage as well as different components of the recovery medium. Primary protocorms were generated using asymbiotic in vitro germination media developed by the authors specifically for the test species (half-strength MS macroelements and microelements with 5% (v/v) fresh filter sterilized coconut water). Secondary protocorms were propagated using an in vitro proliferation medium (½ MS with 5 μM α-naphthaleneacetic acid + 2 μM 6-benzylaminopurine). A modified preconditioning step was developed, involving incubation on ½ MS with 0.2 M raffinose for 48 h at 15°C instead of 20°C. The standard recovery medium (½ MS 1 μM zeatin + 0.5 μM gibberellic acid) was replaced after the first week following warming from liquid nitrogen (LN), with asymbiotic germination medium (½ MS + 5% (v/v) coconut water) for the remainder of the recovery phase. This new step increased the survival of primary protocorms from 68 to 85%. The average post-cryostorage regeneration of plants from primary protocorms increased from 17 to 48% after a 6-wk incubation. A similar protocol increased the survival of secondary protocorms from 63 to 84%. Regeneration of plants from secondary cryostored protocorms increased from 11 to 26% after 14 wk. The protocols developed here provide a useful template for advancing cryoconservation of other orchid taxa, particularly rare and threatened species.

Từ khóa


Tài liệu tham khảo

Antony JJJ, Sinniah UR, Keng CL, Pobathy R, Khoddamzadeh AA, Subramaniam S (2011) Selected potential encapsulation-dehydration parameters on Dendrobium Bobby Messina protocorm-like bodies using TTC analysis. Aust J Crop Sci 5:1817–1822

Ashmore S, Azimi M, Drew RA (2001) Cryopreservation trials in Carica papaya. Acta Hort 560:117–120

Barraco G, Sylvestre I, Engelmann F (2011) Comparing encapsulation-dehydration and droplet-vitrification for cryopreservation of sugarcane (Saccharum spp.) soot tips. Sci Hort 130:320–324

Batty AL, Dixon KW, Brundrett M, Sivasithamparam K (2001) Constraints to symbiotic germination of terrestrial orchid seed in Mediterranean bushland. New Phytol 152:511–520

Batty AL, Brundrett MC, Dixon KW, Sivasithamparam K (2006) New methods to improve symbiotic propagation of terrestrial orchid seedlings from axenic culture to soil. Aust J Bot 54:367–374

Brundrett MC (2007) Scientific approaches to Australian temperate terrestrial orchid conservation. Aust J Bot 55:293–307

Bustam BM, Dixon KW, Bunn E (2014a) In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared to symbiotic germination. Bot J Linn Soc 176(4):556–566. doi:10.1111/boj.12216

Bustam BM, Dixon KW, Bunn E (2014b) Proliferation and harvesting of secondary protocorms as a novel means for improving propagation of terrestrial orchids. Aust J Bot 62(7):614–621. doi:10.1071/BT14291

Chang Y, Reed BM (2000) Extended alternating-temperature cold acclimation and culture duration improve pear shoot cryopreservation. Cryobiology 40:311–322

Clements MA, Muir H, Cribb PJ (1986) A preliminary report on the symbiotic germination of European terrestrial orchids. Kew Bull 41:437–445

Coates DJ, Dixon KW (2007) Current perspectives in plant conservation biology. Aust J Bot 55:187–193

Ellis D, Skogerboe D, Andre C, Helier B, Volk G (2006) Implementation of garlic cryopreservation techniques on the national plant germplasm system. Cryog Lett 27(2):99–106

Flachsland E, Terada G, Socchi A, Rey H, Mroginski L, Engelmann F (2006) Cryopreservation of seeds and in-vitro cultured protocorms of Oncidium bifolium Sims. (Orchidaceae) by encapsulation-dehydration. Cryog Lett 27:235–242

Gogoi K, Kumaria S, Tandon P (2013) Cryopreservation of Dendrobium eburneum Lindl. And C. hookerianum Rchb.f., two threatened and vulnerable orchids via encapsulation-dehydration. In Vitro Cell Dev Biol Plant 49:248–254

Hay FR, Merritt DJ, Soanes JA, Dixon KW (2010) Comparative longevity of Australian orchid (Orchidaceae) seeds under experimental and low temperature storage conditions. Bot J Linn Soc 164:26–41

Heringer AS, Steinmacher DA, Fraga HPF, Veira LN, Ree JF, Guerra MP (2013) Global DNA methylation profiles of somatic embryos of peach palm (Bactris gasipaes Kunth) are influenced by cryoprotectants and droplet-vitrification cryopreservation. Plant Cell Tiss Organ Cult 114:365–372

Hirano T, Godo T, Mii M, Ishikawa K (2005) Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep 23:534–539

Hooi TH, James J, Julkiflee A, Poobaty R, Gnasekaram P, Subramaniam S (2010) A novel approach for preliminary PVS2 vitrification optimization parameters of Dendrobium sonia-28 orchid with evan blue staining. Adv Environ Biol 4(2):284–290

Institute for statistics and Mathematics of WirtshaftuniversitatWein (2014) www.r-project.org

Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757

Jitsopakul N, Thammasiri K, Ishikawa K (2008) Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration. Cryog Lett 29:253–260

Jusatitis M, Sorensen B (1993) Germination of Pterostylis arenicola—an endangered greenhood from South Australia. Orchadian 11:18–22

Kaczmarczyk A, Funnekotter B, Menon A, Phang PY, Al-Hanbali A, Bunn E, Mancera RL (2012) Current issues in plant cryopreservation. In: Katkov II (ed) Current frontiers in cryobiology. InTechOpen, p 417–437

Kaczmarczyk A, Turner SH, Bunn E, Manceera RL, Dixon KW (2013) Development of cryopreservation for Loxocaryacinerea—an endemic Australian plant species important forpost-mining restoration. Cryog Lett 34:508–519

Khoddamzadeh AA, Sinniah UR, Lynch P, Kadir AA, Kadzimin SB, Mahmood M (2011) Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f) Christenson by encapsulation-dehydration. Plant Cell Tiss Organ Cult 107(3):471–481

Kim HH, Lee SC (2012) ‘Personalisation’ of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects. Cryog Lett 33(4):271–279

Kim HH, Lee YG, Shin DJ, KO HC, Gwag JG, Cho EG, Engelmann F (2009) Development of alternative plant vitrification solutions in droplet-vitrification procedures. Cryog Lett 30(5):320–334

Lee YI, Hsu ST, Yeung C (2013) Orchid protocorm-like bodies are somatic embryos. Am J Bot 100(11):2121–2131

Liu H, Feng CL, Luo YB, Chen BS, Wang ZS, Gu HY (2010) Potential challenges of climate change to orchid conservation in a wild orchid hotspot in southwestern China. Bot Rev 76:174–192

Maneerattanarungjoy P, Bunnang S, Monthatong M (2007) In vitro conservation of Cleistoma areitinum (Rchb.f.) Garay, rare Thai orchid species by an encapsulation-dehydration method. Asian J Plant Sci 6:1235–1240

McCarty JP (2001) Ecological consequences of recent climate change. Conserv Biol 15:320–331

Merritt DJ, Hay FR, Swarts ND, Sommerville KD, Dixon KW (2014) Ex situ conservation of orchid germplasm. Int J Plant Sci 175:46–58

Mohanty P, Das M, Kumaria S, Tandon P (2012) High-efficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl. Plant Cell Tiss Organ Cult 109:297–305

Nicholls N (2004) The changing nature of Australian droughts. Climate Change 63:1473–1480

Nikishina TV, Popova EV, Vakhrameeva MG, Varlygina TI, Kolomeitseva GL, Burov AV, Popovich EA, Shirokov AI, Shumilov VY, Popov AS (2007) Cryopreservation of seeds and protocorm of rare temperate orchids. Fiziolog Rastenii 54:137–143

Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus-officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci 91(1):67–73

Nurfadillah S (2010) Conservation of critically endangered orchid Drakaea elastica Lindl. in the context of nutritional requirements of saprophytic competency of the mycorrhizal fungus and its propagation. MSc Thesis. The University of Western Australia, Perth

Phillips RD (2010) Landscape, pollinator and mycorrhizal specificity and their contribution to rarity in Drakaea (hammer orchid). PhD Thesis. The University of Western Australia, Perth

Popova EV, Nikishina TV, Kolomeitseva GL, Popov AS (2003) The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia. Russ J Plant Physiol 50:750–755

Pornchuti W, Thammasiri K (2008) Cryopreservation of protocorms of Dendrobium cariniferum RCHB.F. Acta Hort 788:63–68

Ramsey RR, Dixon KW, Sivasithamparam K (1986) Patterns of infection and endophytes associated with Western Australian orchids. Lindleyana 1:203–214

Reed BM, Okut N, D’Achino J, Narver L, DeNoma J (2003) Cold storage and cryopreservation of hops (Humulus L.) shoot cultures through application standard protocol. Cryog Lett 24:389–396

Rong HS, Hua YM (2012) High-efficiency encapsulation-vitrification protocols for cryopreservation of embryonic calli of the oriental medicinal plant Anemarrhena asphodeloides BUNGE. Cryog Lett 33:190–200

Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30–33

Senula A, Keller ERJ, Sanduijav T, Yohannes T (2007) Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol. Cryog Lett 28:1–12

Sinniah UM, Gantait S (2013) Cryopreservation of immature Parkiosa speciosa Hassk. Zygotic embryogenic axes following desiccation or exposure to vitrification solution. Acta Physiol Plant 35:629–2634

Sommerville KD, Siemon JP, Wood CB, Offord CA (2008) Simultaneous encapsulation of seed and mycorrhizal fungi for long term storage and propagation of terrestrial orchids. Aust J Bot 56:609–615

Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot 104:543–556

Thuiller W, Richardson DM, Pysek P, Midgley GF, Hughes GO, Rouget M (2005) Niche-based modelling as a tool for predicting the risk of alien plant invasions at a global scale. Glob Change Biol 11:2234–2250

Towill LE (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep 9:178–180

Towill LE, Bonnart R, Volk GM (2006) Cryopreservation of Arabidopsis thaliana shoot tips. Cryog Lett 27:353–360

Urugami A, Sakai A, Nagai M, Takahashi T (1989) Survival of cultured-cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep 8:418–421

Watanawikkit P, Tantiwiwat S, Bunn E, Dixon KW, Chayanarit K (2012) Cryopreservation of in vitro-propagated protocorms of Caladenia for terrestrial orchid conservation. Bot J Linn Soc 170:277–282

Wright M, Cross R, Dixon K, Huynh T, Lawrie A, Nesbitt L, Pritchard A, Swarts N, Thomson R (2009) Propagation and reintroduction of Caladenia. Aust J Bot 57:373–387

Yin M, Hong S (2009) Cryopreservation of Dendrobium candidum Wall. ex Lindl. Protocorm like bodies by encapsulation-vitrification. Plant Cell Tiss Organ Cult 98:179–185

Yin LL, Poobathy R, James J, Julkifle AL, Subramaniam S (2011) Preliminary investigation of cryopreservation by encapsulation-dehydration technique on Brassidium shooting star orchid hybrid. African J Biotech 10:4665–4672

Yin ZT, Bi WL, Chen L, Zhao B, Volk GM, Wang QC (2014) An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification. Acta Physiol Plant 36:1683–1692

Zettler LW (1997) Terrestrial orchid conservation by symbiotic seed germination: techniques and perspectives. Selbyana 18:188–194

Thông tin
Thông tin xuất bản

In Vitro Cellular & Developmental Biology - Plant

Thông tin tác giả