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Title: Cryopreservation of citrus for long-term conservation

item Volk, Gayle
item Bonnart, Remi
item SHEPHERD, A - Colorado State University
item Krueger, Robert
item Lee, Richard

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2015
Publication Date: 1/20/2015
Citation: Volk, G.M., Bonnart, R.M., Shepherd, A., Krueger, R., Lee, R.F. 2015. Cryopreservation of citrus for long-term conservation. Acta Horticulturae. 1065:187-191.

Interpretive Summary: Diverse citrus cultivars are maintained in multiple collections throughout the U.S. Key cultivars in these field, greenhouse, and screenhouse collections could be lost to devastating diseases, abiotic stresses, or natural disasters. Collections can be successfully backed-up at secure secondary locations using cryopreservation technologies. Shoot tips excised from trees are sterilized, treated with preservation solutions, and then plunged into liquid nitrogen for long-term storage. For recovery, the 1-mm shoot tips are warmed, diluted, and micrografted onto seedling rootstocks. These plants can eventually be transferred to greenhouse conditions. In this work we demonstrate that shoot tips stored in liquid nitrogen remain viable for over one year. We also determined that shoot tips frozen on aluminum foil strips had higher viabilities than those frozen in cryovials. These data support the use of shoot tips to conserve diverse Citrus cultivars in genebank collections.

Technical Abstract: More than 850 varieties of Citrus, Fortunella, and Citrus-related species are maintained within the USDA-ARS National Plant Germplasm System and the University of California Citrus Variety Collection. These genetic resources are held within duplicated field, screenhouse, and greenhouse collections and are at risk of disease infestations, abiotic stresses, and natural disasters. We have developed a cryopreservation method by which we can back up Citrus genetic resources for the long term at liquid nitrogen temperatures. We excise shoot tips directly from greenhouse or screenhouse source plants, surface sterilize, and then treat with cryoprotectants. These solutions dehydrate and allow the shoot tips to survive liquid nitrogen exposure. Plants are recovered by micrografting thawed shoot tips onto ‘Carrizo’ seedling rootstocks. Experiments that compared survival after freezing in cryovials vs. on aluminum foil strips revealed higher levels and more uniform survival when foil strips were utilized. High viability was maintained for at least 1 year. These data further support the use of shoot tips to conserve diverse Citrus cultivars in genebank collections.