Efficient Cryopreservation of Lilium spp. Shoot Tips using Droplet-vitrification

Abstract

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Newly developed shoot tips from adventitious buds induced by tissue cultured bulb-scale segments of five accessions of Lilium spp. were successfully cryopreserved by a droplet-vitrification method. Bulb-scale segments cultured on Murashige and Skoog (MS) medium with 0.1 mg·L -1 IAA and 0.1 mg·L -1 zeatin were then cold-hardened at 4℃ for 7 days. The excised shoot tips were pre-cultured on solidified MS medium containing 0.3 M sucrose for 1 day at 23℃ and then soaked in a mixture of 0.7 M sucrose for a day at 23℃. Pre-cultured shoot tips were cryoprotected with two loading solutions, LD1 and LD2, which included 35% and 40% plant vitrification solution (PVS3), respectively, for 40~60 min at 23℃. The cryoprotected shoot tips were then soaked in PVS2, modified PVS2 and PVS3 for 90~120 min at 23℃. The shoot tips, frozen in microdroplets of vitrification solution, were wrapped with aluminum foil strips, which were immersed rapidly in liquid nitrogen. The shoot tips were then rapidly warmed using unloading solution, transferred to a regeneration medium, stored in the dark for two weeks at 23℃, and then cultured under white fluorescent light at an intensity of 2000 lux with a 16-h photoperiod at 23℃. The average post-cryo regeneration rates of five accessions ranged from 52.7% to 87.5%.

Development, progress and future prospects in cryobiotechnology of Lilium spp.

“…Since then, various cryopreservation methods have been described for Lilium pollen [25], seeds [26–29] and shoot tips [4]. The major cryoprotocols available for Lilium include dehydration [26, 28, 29], encapsulation–dehydration [29, 30], encapsulation–vitrification [30], vitrification [30–33], and droplet-vitrification [34–38].…”

“…1Major steps of Lilium cryopreservation for shoot tips ( A ), pollen ( B ) and seeds ( C ). Shoot tip cryopreservation by droplet-vitrification (1 in A [35]; 2 in A [34, 43]), vitrification (3 in A [30, 31]), encapsulation–vitrification (4 in A [30]), and encapsulation–dehydration (5 in A [30]). Pollen cryopreservation by dehydration (B [53]).…”

“…× longiflorum and 60 for Oriental hybrid ‘Siberia’[36] L. callosum and Oriental hybrids5Droplet-vitrification78 for L . callosum and 53–88 for the four Oriental hybrids[34] Lilium spp, Asiatic hybrid and Oriental hybrid20 Lilium species, 1 Asiatic hybrid and 1 Oriental hybridDroplet-vitrification52 for L. amabile , 67 for L . auratum, 43 for L. bulbiferum, 67 for L. callosum, 62 for L. candidum, 42 for L. concolor, 85 for L. davidii, 41 for L. distichum, 48 for L. formolongi , 86 for L. hansonii , 38 for L. henryi ,41 for L. lancifolium, 40 for L. leichtlinii , 69 for L. leucanthum , 43 for L. longiflorum , 41 for L. pyrenaicum, 83 for L. regale , 43 for L. tsingtauense , 41 L. washingtonianum, 43 for Asiatic hybrid, 73 for Oriental hybrid[43] L. davidii var.…”

“…Droplet-vitrification combines the advantages of droplet freezing with vitrification, thus allowing samples to obtain rapid cooling and warming rates, increasing the survival of cryopreserved samples [41, 42]. Droplet-vitrification is considered to be a robust means for the cryopreservation of Lilium shoot tips [32, 34–36, 38, 43].…”

Genetic variation of maiden lily (Lilium rubellum Baker) within and among three natural populations in Fukushima Prefecture in Japan

“…Fifteen species are distributed across all prefectures of Japan, and Japanese Lilium species have played an important role in the development of major important cultivars of Eastern Lily, Asiatic Hybrids and Oriental Hybrids (Okasaki, Asano, & Oosawa, 1994). However, Lilium species are also under threat of genetic erosion, requiring increased attention and efforts to conserve Lilium germplasm (Yi, Lee, Chung, Lee, & Lim, 2013).…”