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United States Department of Agriculture

Agricultural Research Service

Research Project: RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS

Location: Plant Germplasm Preservation Research Unit

Title: Cryopreservation and Cryotherapy of Citrus Cultivars

Authors
item Volk, Gayle
item Krueger, Robert
item Lee, Richard

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 8, 2010
Publication Date: June 14, 2010
Citation: Volk, G.M., Krueger, R., Lee, R.F. 2010. Cryopreservation and Cryotherapy of Citrus Cultivars. Meeting Abstract National Citrus Research Coordination Symposium, Denver, Colorado, June 16-18, 2010.

Interpretive Summary: Long-term conservation of Citrus clones can be accomplished by cryopreservation. Shoot tips will survive liquid nitrogen exposure and storage when appropriately desiccated and treated with cryoprotectant solutions. In our research, vegetative Citrus budwood is shipped from Riverside to Fort Collins, Colorado and kept in 4oC coolers until use. Excised shoot tips (1mm3) are treated with solutions of sucrose, sucrose+glycerol, and PVS2 (sucrose, glycerol, dimethyl sulfoxide, and ethylene glycol) prior to rapid cooling in liquid nitrogen. Shoot tips are thawed quickly, diluted in a sucrose solution, and then micrografted onto in vitro cultures of 2-week old ‘Carrizo’ seedling rootstocks. Preliminary results reveal high levels of shoot-tip survival after liquid nitrogen exposure. This research will be continued to identify methods that ensure survival of diverse Citrus varieties. The process of cryopreservation has been shown to eradicate diseases such as Huanglongbing from citrange shoot tips (Ding et al. 2008). This eradication, termed “cryotherapy”, is believed to occur because differentiated, disease-infected cells in cryopreserved shoot tips do not survive liquid nitrogen exposure and only non-infected, intact meristems regenerate after cryopreservation. We have planned collaborative research to determine the feasibility of cryotherapy for key Citrus genetic resources.

Technical Abstract: Long-term conservation of Citrus clones can be accomplished by cryopreservation. Shoot tips will survive liquid nitrogen exposure and storage when appropriately desiccated and treated with cryoprotectant solutions. In our research, vegetative Citrus budwood is shipped from Riverside to Fort Collins, Colorado and kept in 4oC coolers until use. Excised shoot tips (1mm3) are treated with solutions of sucrose, sucrose+glycerol, and PVS2 (sucrose, glycerol, dimethyl sulfoxide, and ethylene glycol) prior to rapid cooling in liquid nitrogen. Shoot tips are thawed quickly, diluted in a sucrose solution, and then micrografted onto in vitro cultures of 2-week old ‘Carrizo’ seedling rootstocks. Preliminary results reveal high levels of shoot-tip survival after liquid nitrogen exposure. This research will be continued to identify methods that ensure survival of diverse Citrus varieties. The process of cryopreservation has been shown to eradicate diseases such as Huanglongbing from citrange shoot tips (Ding et al. 2008). This eradication, termed “cryotherapy”, is believed to occur because differentiated, disease-infected cells in cryopreserved shoot tips do not survive liquid nitrogen exposure and only non-infected, intact meristems regenerate after cryopreservation. We have planned collaborative research to determine the feasibility of cryotherapy for key Citrus genetic resources.

Last Modified: 10/1/2014