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United States Department of Agriculture

Agricultural Research Service

Research Project: Development of Cryotherapy As An Improved Method of Eliminating Graft Transmissable Pathogens in Citrus

Location: Plant Germplasm Preservation Research Unit

2013 Annual Report


1a.Objectives (from AD-416):
There are currently two methods available for therapy of Citrus: thermotherapy and shoot tip grafting (STG). Thermotherapy does not eliminate viroids, and is inefficient at eliminating HLB and Citrus Tatterleaf virus (CT1V). Although STG has been shown to eliminate all graft transmissible pathogens, pathogens such as viroids and Citrus Tatterleaf virus are still difficult. However, the major drawback to STG is the requirement for a high level of expertise since the technique is mastered only after many months of practice and it requires a very steady hand. A relatively new technology, cryotherapy, has been successfully implemented in potato, sweet potato, grapevine, raspberry, and Prunus to eliminate pathogens that have been challenging when using traditional methods. Recently HLB was shown to be eliminated from Citrus by cryotherapy. In cryotherapy, cells containing pathogens do not survive the exposure to liquid nitrogen, thus eliminating the pathogen. Meristems up to 1 mm long (including about three leaf primordia, about 1 mm long) are excised, in contrast to the meristem with a single leaf primordium (about 0.1 mm long excised in traditional STG. In cryotherapy, a meristem with up to three leaf primordial (about 1 mm long) use of larger shoot tips increases the survival rate and makes the procedures more reproducible.


1b.Approach (from AD-416):
A cryotherapy protocol will be developed by the Volk lab and then applied to tissue infected with stock virus isolates obtained from the USDA-ARS Citrus repository. Experimental variables will include shoot tip size, antibiotic treatments, and liquid nitrogen exposure. Recovering shoots will be micrografted to Carrizo seedlings and then tested for the presence of pathogens by an ARS Scientist.


3.Progress Report:

The primary goal of this project is to develop cryotherapy as a practical, reliable method of eliminating graft transmissible pathogens from Citrus and citrus relatives without inducing juvenility. Permits have been obtained to ship pathogen infected citrus material from Riverside, CA and from the Exotic Disease Quarantine Laboratory, Beltsville, MD to Ft. Collins, CO. Initial research was directed to elimination of viroids and Citrus tatterleaf virus. ARS personnel have visited the Repository facilities and learned the traditional shoot tip grafting protocol for elimination of graft transmissible pathogens. The protocol is presently being developed using several approaches. Cryotherapy and shoot tip grafting are being compared to determine the efficiency of pathogen eliminationWe have demonstrated the efficient elimination of viroids and Citrus tatter leaf virus.


Last Modified: 12/22/2014
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