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Title: An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

Author
item YIN, ZHEN-FANG - Northwest Agricultural University
item BI, WEN-LU - Northwest Agricultural University
item CHEN, LONG - Northwest Agricultural University
item ZHAO, BING - Northwest Agricultural University
item Volk, Gayle
item WANG, QIAO-CHUN - Northwest Agricultural University

Submitted to: Acta Physiologiae Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2014
Publication Date: 4/30/2014
Citation: Yin, Z., Bi, W., Chen, L., Zhao, B., Volk, G.M., Wang, Q. 2014. An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification. Acta Physiologiae Plantarum. 36(7):1683-1692. DOI: 10.1007/s11738-014-1543-7.

Interpretive Summary: Diverse varieties of lily are economically important to the horticulture industry. They are expensive to maintain in genebanks since they must be maintained as plants rather than as seeds. We have developed a method to conserve lily genetic resources in liquid nitrogen for their long term conservation in gene banks. The method uses 2 mm shoot tips excised from in vitro cultures. The shoot tips are then dehydrated and treated with cryoprotectants before plunging into liquid nitrogen. The method was successfully applied to cultivars that represent five diverse lily species. Genetic tests that compared the shoot tips of the initial plants to those that were recovered after cryopreservation did not identify any changes that occurred as a result of the liquid nitrogen exposure.

Technical Abstract: We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg L-1 a-naphthaleneacetic acid (NAA) and 0.5 mg L-1 thidiazuron (TDZ). Shoot tips (1.5-2 mm in length) including 2-3 leaf primordia, were precultured on MS medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were rewarmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-culture for shoot regrowth. Shoot regrowth levels ranged from 42.5% for L. longiflorum × Oriental ‘Triumphator’ to 87.5% for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1% across the six diverse Lilium genotypes tested. Intersimple sequence repeat (ISSR) markers using eight primer pairs revealed no differences in genotypes between the original source plants and those recovered after cryoexposure. This Lilium droplet-vitrification cryopreservation method is straightforward and widely applicable for the long-term conservation of lily genetic resources.