Cryopreservation of Dormant Apple Buds
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521<EMAIL_ADDRESS>Maria Jenderek, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Katheryn Chen, Department of Soil and Crop Sciences, Colorado State University, 307 University Ave., Fort Collins, Colorado 80523.
Outline
- Introduction
- Harvest dormant budwood
- Cut dormant budwood into sections
- Determine moisture content
- Desiccate and package
- Slow-cool and cryopreserve
- Long-term storage
- Warm cryopreserved budwood sections
- Graft buds onto rootstocks for viability assessment
- References
- Additional materials
- Acknowledgments
The apple dormant bud cryopreservation protocol is available for download here.
The apple dormant bud grafting protocol is available for download here.
1. INTRODUCTION
The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 3000 grafted apple (Malus) accessions in a field collection in Geneva, NY (Volk et al., 2015). Dormant bud cryopreservation methods have been used to cryopreserve the NPGS apple collection since 1988 (Volk et al. 2014). A total of 2052 Malus accessions are cryopreserved at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado (Volk et al. 2014). First, dormant twigs are harvested from field-grown trees and then shipped to the NLGRP. The twigs are then cut into nodal sections and dried to a moisture content of 25-30% (fresh weight basis, fwb). They are then packaged into polyolefin tubes and cooled at a rate of 1 oC hr-1 to -30 oC and held overnight. Then the tubes containing dormant buds are plunged into the vapor phase of liquid nitrogen and placed into cryotanks for long-term storage. Buds are warmed to 2 to 5 oC and then placed into moist peat moss for 14 to 21 days. They are then grafted onto rootstocks for viability assessment and plant recovery. The cryopreservation and regrowth assessment methods are described below.
2. Harvest dormant budwood
Dormant budwood twigs are harvested from the previous season’s growth of field-grown apple trees (Fig. 1; Video 1). In the USDA, budwood is derived from trees grown in Geneva, NY in January, ideally after temperatures have been below 0 oC for a few days. The harvested twigs are 5-7 mm in diameter, have about 10 nodal sections each, and should total 17-20 twigs (170-200 nodal sections) per accession; appropriate budwood is shown in Figure 2, in contrast to the material shown in Figure 3. The budwood is packaged in bundles and sent by overnight courier to NLGRP. It is kept at -5 oC in sealed plastic bags for up to a few weeks until it is used for cryopreservation.
Figure 1. Dormant budwood is harvested from field-grown apple trees. Photo credit: Reece Perrin.
Video 1. Video of Reece Perrin (Cornell University) explaining the process of harvesting apple dormant budwood