Development of Cryotherapy as an Improved Method of Eliminating Graft Transmissible Pathogens in Citrus
National Clonal Germplasm Repository for Citrus & Dates
Project Number: 5310-21000-010-06
Start Date: Oct 01, 2011
End Date: Sep 30, 2013
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 (CTlV). 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. In cryotherapy, a meristem with up to three leaf primodia (about 1 mm long) are excised, in contrast to the meristem with a single leaf primordium (about 0.1 mm long) excised in traditional STD. Use of larger shoot tips increases the survival rate and makes the procedures more reproducible.
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. Validation of method to eliminate exotic pathogens will be done.