Abstract
Dioscorea deltoidea Wall. ex Griseb. is a critically endangered, commercially important crop of high medicinal value. Several accessions of this valuable plant are conserved as slow growth cultures in the in vitro gene bank of Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi, India. The purpose of this investigation was to develop a cryopreservation protocol that can be used for long-term conservation of 15 different D. deltoidea accessions. The vitrification technique of cryopreservation was compared to the droplet-vitrification technique. Shoot meristems excised from 4-wk-old in vitro plantlets were precultured on Murashige and Skoog (MS) medium containing 0.3 M sucrose for 16 h, treated with a loading solution for 20 min, dehydrated with plant vitrification solution (PVS2) for 90 min, and cryoconserved using both vitrification and droplet-vitrification protocols. Two-way ANOVA data subjected to post hoc Sidak’s multiple comparisons test (P ≤ 0.05) indicated that shoot regrowth after LN treatment, using vitrification (30 to 54%) and droplet-vitrification (21 to 51%) techniques, was statistically comparable in 66.7% accessions, while in 33.3% accessions vitrification gave significantly better results. Genetic stability of plants cryopreserved using both the protocols was confirmed using 39 Inter-Simple Sequence Repeat (ISSR) and 30 Expressed Sequence Tag-derived Simple Sequence Repeat (EST-SSR) markers. Results showed that there was no significant difference between mother plants and cryopreserved samples. The vitrification protocol has been implemented for long-term cryobanking of D. deltoidea germplasm (15 accessions), due to improved regrowth and ease of sample handling as compared to droplet-vitrification.
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References
Ahuja S, Mandal BB, Dixit S, Srivastava PS (2002) Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips. Plant Sci 163:971–977
Bi WL, Pan C, Liu J, Wang QC (2016) Greenhouse performance, genetic stability and biochemical compounds in Chrysanthemum morifolium ‘Hangju’ plants regenerated from cryopreserved shoot tips. Acta Physiol Plant 38:268. https://doi.org/10.1007/s11738-016-2288-2
Carmona-Martín E, Regalado JJ, Perán Quesada R (2018) Cryopreservation of rhizome buds of Asparagus officinalis L. (cv. Morado de Huétor) and evaluation of their genetic stability. Plant Cell Tiss Org Cult 133:395–403. https://doi.org/10.1007/s11240-018-1392-y
CITES (2017) The CITES species. https://www.cites.org/eng/disc/species.php. Accessed 29 Nov 2021
Dixit S, Mandal BB, Ahuja S, Srivastava PS (2003) Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscoreabulbifera L. using RAPD, biochemical and morphological analysis. CryoLetters 24:77–84
Doyle JJ, Doyle JJ (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem Bull 19:11–15
Dumet D, Diebiru E, Adeyemi A, Akinyemi O, Gueye B, Franco J (2013) Cryopreservation for the ‘in perpetuity’ conservation of yam and cassava genetic resources. CryoLetters 34:107–118
Fedorovsky DN, Popov AS (1992) Plasmalemma injuries in different strains of Dioscoreadeltoidea during cryopreservation. Soviet Plant Physiol 39:381–386
Gong G, Qin Y, Huang W (2011) Anti-thrombosis effect of diosgenin extract from Dioscoreazingiberensis C.H. Wright in vitro and in vivo. Phytomedicine 18:458–463. https://doi.org/10.1016/j.phymed.2010.08.015
Gowthami R, Sharma N, Pandey R, Agrawal A (2021) Status and consolidated list of threatened medicinal plants of India. Genet Resour Crop Evol 68:2235–2263. https://doi.org/10.1007/s10722-021-01199-0
Hong S, Yin M, Shao X, Wang A, Xu W (2009) Cryopreservation of embryogenic callus of Dioscoreabulbifera by vitrification. CryoLetters 30:64–75
Jaccard P (1908) Nouvelles Recherches sur la distribution florale. Bull De La Soc Vaud Des Sci Nat 44:223–270
Kanchan DM, Somani GS, Peshattiwar VV, Kaikini AA, Sathaye S (2016) Renoprotective effect of diosgenin in streptozotocin induced diabetic rats. Pharmacol Rep 68:370–377. https://doi.org/10.1016/j.pharep.2015.10.011
Kaya E, Alves A, Rodrigues L, Jenderek M, Hernandez-Ellis M, Ozudogru A, Ellis D (2013) Cryopreservation of Eucalyptus genetic resources. CryoLetters 34:608–618
Kaya E, Souza FVD (2017) Comparison of two PVS2-based procedures for cryopreservation of commercial sugarcane (Saccharum spp.) germplasm and confirmation of genetic stability after cryopreservation using ISSR markers. In Vitro Cell Dev Biol - Plant 53:410–417. https://doi.org/10.1007/s11627-017-9837-2
Le Bras C, Le Besnerais PH, Hamama L, Grapin A (2014) Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration. Plant Cell Tiss Org Cult 116:235–242. https://doi.org/10.1007/s11240-013-0400-5
Leunufna S, Keller ERJ (2003) Investigating a new cryopreservation protocol for yams (Dioscorea spp.). Plant Cell Rep 21:1159–1166. https://doi.org/10.1007/s00299-003-0652-3
Leunufna S, Keller ERJ (2005) Cryopreservation of yams using vitrification modified by including droplet method: effects of cold acclimation and sucrose. CryoLetters 26:93–102
Li M, Zhao X, Hong S, Zhang X, Li P, Liu J, Xie C (2009) Cryopreservation of plantlet nodes of Dioscoreaopposita Thunb. using a vitrification method. CryoLetters 30:19–28
Malaurie B, Pungu O, Dumont R, Trouslot MF (1992) The creation of an in vitro gemplasm collection of yam (Dioscorea spp.) for genetic resources preservation. Euphytica 65:113–122. https://doi.org/10.1007/BF00022573
Mandal BB, Ahuja-Ghosh S (2007) Regeneration of Dioscorea floribunda plants from cryopreserved encapsulated shoot tips: effect of plant growth regulators. CryoLetters 28:329–336
Mandal BB, Dixit-Sharma S (2007) Cryopreservation of in vitro shoot tips of Dioscoreadeltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration. CryoLetters 28:461–470
Mandal BB, Ahuja-Ghosh S, Srivastava PS (2008) Cryopreservation of Dioscorearotundata Poir.: a comparative study with two cryogenic procedures and assessment of true-to-type of regenerants by RAPD analysis. CryoLetters 29:399–408
Mandal BB, Chandel KP, Dwivedi S (1996) Cryopreservation of yam (Dioscorea spp.) shoot apices by encapsulation-dehydration. CryoLetters 17:165–174
Mandal BB, Dixit-Sharma S, Srivastava PS (2009) Cryopreservation of embryogenic cultures of Dioscoreabulbifera L. by encapsulation - dehydration. CryoLetters 30:440–448
Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13:442–446
Mukherjee P, Mandal BB, Bhat KV, Biswas AK (2009) Cryopreservation of Asian Dioscoreabulbifera L. and D. alata L. by vitrification: importance of plant growth regulators. CryoLetters 30:100–111
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Ng SYC, Ng NQ (1997) Germplasm conservation in food yams (Dioscorea spp): constraints, application and future prospects. In: Razdan MK, Cocking EC (eds) Conservation of plant genetic resources in vitro, vol 1. General aspects. Science Publishers Inc, USA, pp 257–286
Olsen CS, Larsen HO (2003) Alpine medicinal plant trade and Himalayan Mountain livelihood strategies. Geogr J 169:243–254. https://doi.org/10.1111/1475-4959.00088
Panis B, Lambardi M (2006) Status of cryopreservation technologies in plants (crops and forest trees). In: Ruane J, Sonnino A (eds) The role of biotechnology for the characterisation and conservation of crop, forestry, animal and fishery genetic resources. Food and Agriculture Organization of the United Nations, Roma 61–78
Raju J, Mehta R (2008) Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin. Nutr Cancer 61:27–35. https://doi.org/10.1080/01635580802357352
Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Obs. Var brasilliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33. https://doi.org/10.1007/BF00232130
Schafer-Menuhr A, Schumacher HM, Mix-Wagner G (1997) Long-term storage of old potato varieties by cryopreservation of shoot-tips in liquid nitrogen. Plant Genet Resources News 111:19–24
Sharma N, Pandey R, Gowthami R (2020) In vitro conservation and cryopreservation of threatened medicinal plants of India. In: Rajasekharan PE, Wani SH (eds) Conservation and utilization of threatened medicinal plants. Springer, India, pp 181–228. https://doi.org/10.1007/978-3-030-39793-7_8
Shin JH, Kang DK, Bae JS, Lee BH, Sohn JK (2006) Development of cryopreservation system using shoot-apex in yam (Dioscoreabatatas). J Plant Biotechnology 8:43–50
Tanaka D, Nishiuchi A, Niino T, Matsumoto T (2011) A comparison of vitrification and droplet vitrification procedures for the cryopreservation of in vitro grown black chokeberry shoot tips. Acta Hortic 908:325–330. https://doi.org/10.17660/ActaHortic.2011.908.43
Thankappan SS, Morawala-Patell V (2021) Germplasm conservation techniques and assessment of physiological, biochemical and molecular integrity of indigenous, endangered Dioscoreaprazeri. CryoLetters 42:168–177
Uchendu EE, Keller ERJ (2016) Melatonin-loaded alginate beads improve cryopreservation of yam (Dioscoreaalata and D. cayenensis). CryoLetters 37:77–87
Wang LY, Li YD, Sun HY, Liu HG, Tang XD, Wang QC, Zhang ZD (2017) An efficient droplet-vitrification cryopreservation for valuable blueberry germplasm. Sci Hortic 219:60–69. https://doi.org/10.1016/j.scienta.2017.03.007
Wang MR, Lambardi M, Engelmann F, Pathirana R, Panis B, Volk GM, Wang QC (2021) Advances in cryopreservation of in vitro-derived propagules: technologies and explant sources. Plant Cell Tiss Org Cult 144:7–20. https://doi.org/10.1007/s11240-020-01770-0
Wilms H, Sleziak NF, Van der Auweraer M, Brands M, Verleije M, Hardeman D, Andre E, Panis B (2020) Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources. Sci Rep 10:14674. https://doi.org/10.1038/s41598-020-70869-3
Zhang Y, Tang L, An X, Fu E, Ma C (2009) Modification of cellulase and its application to extraction of diosgenin from Dioscoreazingiberensis CH Wright. Biochem Eng J 47:80–86. https://doi.org/10.1016/j.bej.2009.07.006
Zhang Z, Skjeseth G, Elameen A, Haugslien S, Sivertsen A, Clarke J, Wang QC, Blystad DR (2015) Field performance evaluation and genetic integrity assessment in Argyranthemum ‘Yellow Empire’ plants recovered from cryopreserved shoot tips. In Vitro Cell Dev Biol - Plant 51:505–513. https://doi.org/10.1007/s11627-015-9707-8
Acknowledgements
The authors thank the Indian Council of Agricultural Research (ICAR) and Director, ICAR-NBPGR, New Delhi, India, for providing the funding and research facilities under an in-house project to carry out this work. The assistance provided by Mr. Sanjeev Thankur is gratefully acknowledged.
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NS and EVM equally contributed and share first authorship. NS conceived, planned, and managed the experiments; RC and GR prepared explants; EVM conducted genetic stability experiments and prepared and edited the manuscript; SB performed genetic stability experiments; and SS collected the germplasm, maintained the field gene bank, and provided germplasm for experimentation. AA provided overall supervision for the work; AA and MS carried out statistical analysis and critically edited the manuscript.
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Sharma, N., Malhotra, E.V., Chandra, R. et al. Cryopreservation and genetic stability assessment of regenerants of the critically endangered medicinal plant Dioscorea deltoidea Wall. ex Griseb. for cryobanking of germplasm. In Vitro Cell.Dev.Biol.-Plant 58, 521–529 (2022). https://doi.org/10.1007/s11627-022-10267-8
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DOI: https://doi.org/10.1007/s11627-022-10267-8