Successful cryopreservation and histological observations of Vitis shoot tips isolated from growth chamber source plants

Authors: BETTONI, JEAN - University Of Santa Catarina; Bonnart, Remi; Shepherd, Ashley; KRETZSCHMAR, AIKE - University Of Santa Catarina; Volk, Gayle

Submitted to: Vitis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2019
Publication Date: 5/15/2019
Citation: Bettoni, J.C., Bonnart, R.M., Shepherd, A.N., Kretzschmar, A.A., Volk, G.M. 2019. Successful cryopreservation and histological observations of Vitis shoot tips isolated from growth chamber source plants. Vitis. 58(2):71-78. https://doi.org/10.5073/vitis.2019.58.71-78.
DOI: https://doi.org/10.5073/vitis.2019.58.71-78

Interpretive Summary: Field collections of grapes (Vitis) are maintained in Davis, CA and Geneva, NY as part of the USDA-ARS National Plant Germplasm System (NPGS). These field collections are vulnerable to biotic and abiotic threats and must be backed-up at a secure location such as the USDA-ARS National Laboratory for Genetic Resources Preservation, preferably using cryopreservation technologies. Vitis cryopreservation methods that use shoot tips excised from plants that are grown in tissue culture are available; however, these methods require that each of the Vitis cultivars in the NPGS collection is first introduced and then multiplied in tissue culture prior to proceeding with cryopreservation. This research demonstrates that shoot tips excised from nodal sections derived from Vitis growth chamber plants can be successfully cryopreserved. The high levels of regrowth observed in cryopreserved Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to increase the efficiency of the Vitis cryopreservation process by using growth chamber source plants instead of tissue cultured plants.

Technical Abstract: It is labor-intensive to establish in vitro cultures as source materials of shoot tips for the cryopreservation of field collections of fruit crops. We sought to determine if growth chamber Vitis plants could serve as the source of shoot tips for cryopreservation. Nodal sections from growth chamber plants were surface-disinfected and placed in tissue culture on pre-treatment medium for 2 weeks. Uniform shoot tips (1 mm) were first obtained from the nodal sections and then precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), ascorbic acid and plant preservative mixture. They were then treated with loading solution for 20 minutes at 22 degrees C followed by half-strength PVS2 for 30 minutes, and full-strength PVS2 for 30, 40 or 50 minutes at 0 degrees C. After liquid nitrogen (LN) exposure, shoot tips were warmed/diluted in unloading solution for 20 minutes and placed on recovery medium. This cryopreservation protocol achieved high levels of regrowth in V. vinifera ‘Chardonnay’ and ‘Riesling’ and V. hybrid ‘Oppenheim’. Histological observations revealed that shoot tips from growth chamber plants had apical as well as multiple lateral meristems that survived LN exposure. The preservation of multiple meristems in each shoot tip may increase the capacity of shoot tip regeneration in cryopreserved Vitis that originates from ex vitro sources. The high levels of regrowth after shoot tip cryopreservation using Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to cryopreserve Vitis shoot tips without first introducing each accession into tissue culture.