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United States Department of Agriculture

Agricultural Research Service

Research Project: Citrus Cryopreservation to Increase Security of Critical Collections

Location: Plant Germplasm Preservation Research Unit

2013 Annual Report


1a.Objectives (from AD-416):
The primary goal of this research is to provide a security back-up of critical California Citrus genetic resources.

Objectives 1) Improve upon the existing cryopreservation procedure (developed in the Volk Laboratory) for clones representing diverse Citrus species that use budwood directly from the screenhouse as source materials. 2) Perform long-term cryopreservation exposure experiments to confirm viability. 3) Evaluate a subset of recovered shoot tips to ensure materials are true-to-type.


1b.Approach (from AD-416):
This research project will develop and implement technologies to cryopreserve critical clones included in the Citrus Clonal Protection Program in Riverside, California. Preliminary data suggests that diverse Citrus clones can be placed into long-term liquid nitrogen storage and then recovered on-demand using modified shoot tip micrografting techniques. Cryopreserved shoot tips can remain viable for dozens of years. This research project will establish a long-term cryoexposure experiment to confirm viability after extended lengths of LN exposure. The project will also identify optimal cryoprotectant exposure times and source plant materials for improved cryopreservation success. These experiments are critical for the implementation of Citrus cryopreservation methods as a collection conservation strategy. It is critical that materials be backed-up for security purposes before California Citrus genetic resources are threatened by Huanglongbing and other diseases or natural threats.


3.Progress Report:

The objectives of this project are to.
1)Improve upon the existing cryopreservation procedure (developed by ARS) for clones representing diverse Citrus species that use budwood directly from the screenhouse as source materials,.
2)Perform long-term cryopreservation exposure experiments to confirm viability,.
3)Evaluate a subset of recovered shoot tips to ensure materials are true-to-type,.
4)Perform Cryopreservation procedures on 50 accessions of Citrus received from the NCGR. In the spring and fall of 2012, 15 Citrus accessions were processed with an average viability of 29% after LN exposure using our standard procedures. In the Fall of 2012 an experiment was performed to determine if freezing on foils would result in higher survival than freezing in vials. For the 7 cultivars included in this experiment, the average cryovial viability was 53% and the viability on foil strips was 83%. All subsequent experiments have been performed using foil strips. It also appears that fall processing is preferable to winter, spring, and summer processing for cryopreservation. We have hosted a visiting student from China, Zhenfang Yin, and she has documented the recovery process as cryopreserved shoot tips are micrografted onto seedling rootstock. The long-term storage experiment initiated in 2011 has been highly successful. Cryopreserved vials of shoot tips that have been thawed at the designated time intervals and then micrografted onto seedling rootstocks have viabilities comparable to the 0 timepoint, indicating that we do not have a decline in viability in storage over the first 18 months.


Last Modified: 7/22/2014
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