ARS | Publication request: Cryopreservation of Citrus shoot tips using micrografting for recovery

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2011
Publication Date: December 18, 2012
Citation: Volk, G.M., Bonnart, R.M., Krueger, R., Lee, R.F. 2012. Cryopreservation of Citrus shoot tips using micrografting for recovery. CryoLetters. 33(6): 418-426.

Interpretive Summary: The USDA-ARS National Plant Germplasm System and the University of California Citrus Variety Collection maintain more than 888 unique accessions of citrus trees in field and screenhouse collections. These collections are vulnerable to diseases and natural environmental threats. We have developed a method whereby Citrus cultivars that can be used for national and international breeding and selection programs can be backed-up for long-term preservation. Small (1 mm) shoot tips are excised from actively growing shoots and treated with cryoprotectant solutions. The shoot tips are then plunged into liquid nitrogen and can be recovered and micrografted onto sterile seedling rootstocks. For seven diverse Citrus species, we have obtained greater than 40% shoot tip regrowth after cryopreservation. This method has the advantage of using screenhouse plants as source materials, a recovery method that is not dependent upon developing culture media specific for each Citrus accession or species, and juvenility is avoided.

Technical Abstract: The USDA-ARS National Plant Germplasm System (NPGS) and the University of California Citrus Variety Collection maintain more than 888 unique accessions representing 132 taxa of Citrus, Fortunella, and Citrus wild species relatives within field, screenhouse, and greenhouse collections. We have identified a cryopreservation method by which Citrus genetic resources that are not maintained in vitro can be successfully conserved. Shoot tips are excised from actively growing vegetative flushes of plants grown in a screenhouse. Surface-sterilized shoot tips are treated with Plant Vitrification Solution 2 for 30 minutes at 0oC then plunged into liquid nitrogen. Thawed shoot tips are micrografted on ‘Carrizo’ seedling rootstocks after a 24 h recovery period. Micrografted shoot tips recover quickly and rooted plants can be transferred to the greenhouse within months. Regrowth rates average 50% for cryopreserved shoot tips of cultivars representing six Citrus species. This method has the advantage of using screenhouse plants as source materials, a recovery method that is not dependent upon developing culture media specific for each Citrus accession or species, and juvenility is avoided.