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Title: Improved droplet-vitrification and histological studies of cryopreserved shoot tips of cultivated Jerusalem artichoke genotypes

item ZHANG, JIN-MEI - China National Genebank
item HAN, LI - China National Genebank
item LU, XIN-XIONG - China National Genebank
item Volk, Gayle
item XIN, XIA - China National Genebank
item YIN, GUANG-KUN - China National Genebank
item HE, JUAN-JUAN - China National Genebank
item WANG, LING - Northwest Agriculture And Forestry University
item CHEN, XIAO-LING - China National Genebank

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2016
Publication Date: 12/2/2016
Citation: Zhang, J., Han, L., Lu, X., Volk, G.M., Xin, X., Yin, G., He, J., Wang, L., Chen, X. 2016. Improved droplet-vitrification and histological studies of cryopreserved shoot tips of cultivated Jerusalem artichoke genotypes. Plant Cell Tissue and Organ Culture. 128:327-334.

Interpretive Summary: Jerusalem artichoke cryopreservation methods were first published by ARS and Canadian scientists in 2006. This work optimized the previously published procedures by altering the shoot tip preculture procedure. The procedural changes increased the regrowth percentage to levels as high as 83%. Histological observations revealed high levels of apparent cell survival within the shoot tip during and after the cryoprotectant and liquid nitrogen exposure. These improved methods will facilitate the implementation of cryopreservation technologies for conservation of vegetatively propagated cultivars of Jerusalem artichoke within genebanks.

Technical Abstract: Germplasm conservation of Jerusalem artichoke (Helianthus tuberosus L.) is crucial to preserve genetic diversity and to secure materials for genetic improvement. Long-term conservation is accomplished through cryopreservation, storing cells or tissues at an ultralow temperature in liquid nitrogen (-196 °C). This study optimized the factors for obtaining good survival and regrowth after droplet-vitrification cryopreservation of Jerusalem artichoke shoot tips, based on method developed in a previous report. The optimal cryopreservation procedure included preculturing excised shoot tips in liquid MS medium supplemented with 0.4M sucrose for 3 days, followed by osmoprotection in loading solution and dehydration with plant vitrification solution (PVS) 2 before rapid freezing in microdroplets of vitrification solution. Regrowth levels were highest when shoot tips were rapidly rewarmed in MS liquid medium containing 1.2M sucrose before being transferred to recovery medium and stored in the dark for several days prior to being cultured. The technique was highly efficient for the cryopreservation of meristem shoot tips of different Jerusalem artichoke cultivars. The highest survival (93%) and regrowth (83%) levels were obtained from cultivar ‘2009330128’. The other three cultivars, ‘M6’, ‘Stampede’, and ‘Relikt’, were cryopreserved with the optimized procedure, with survival levels of 44%, 72%, and 50%, respectively, and regrowth levels of 37%, 53%, and 37%, respectively. Differential scanning calorimetry was used to further investigate the cryopreservation procedures, which suggested that the vitrification solution treatment plays a critical role in successful cryopreservation. This is also the first report on the histological changes in Jerusalem artichoke shoot tips during the cryopreservation process.