Grapevine Shoot Tip Cryopreservation (Droplet Vitrification and V-Cryoplate)
Grapevine Shoot Tip Cryopreservation (Droplet Vitrification and V-Cryoplate)
Jean-Carlos Bettoni, The New Zealand Institute for Plant & Food Research Limited, Private Bag 11 600, Palmerston North 4442, New Zealand.
Remi Bonnart, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521<EMAIL_ADDRESS>Outline
- Introduction
- Shoot tip excision
- Shoot tip processing and liquid nitrogen exposure
- Long-term storage
- Viability assessment
- References
- Additional information
- Acknowledgments
The Vitis shoot tip droplet vitrification cryopreservation procedure is available for download here.
The Vitis shoot tip regrowth procedure after droplet vitrification is available for download here.
1. Introduction
The USDA-ARS National Plant Germplasm System has two field collections of grapevine (Vitis). A total of 3649 unique cultivars of Vitis are maintained as vines at the National Clonal Germplasm Repository for Tree Fruits and Nut Crops and Grapes in Davis, California (Fig. 1) and a total of 802 unique cultivars of Vitis are maintained as vines at the Plant Genetic Resources Unit in Geneva, New York (Fig. 2). Some of the cultivars in these collections are backed-up in liquid nitrogen at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. Herein, we provide a method for cryopreserving shoot tips of Vitis that was developed at the NLGRP to preserve Vitis cultivars in liquid nitrogen.
Figure 1. Vitis in the NPGS field collection in Davis, California. Photo credit: Jean Bettoni.
Figure 2. Vitis in the NPGS field collection in Geneva, New York. Photo credit: Gayle Volk.
Vitis shoot tips can be successfully cryopreserved using several methods. One is the droplet vitrification technique (shown in Section 3.1) and the other uses a V-cryoplate (shown in Section 3.2). Both methods start with in vitro-grown Vitis plants. Nodal sections of the plants are pretreated and then shoot tips are excised and plated on Preculture Medium. They are then cryopreserved, placed into long-term storage, and then warmed and cultured for regrowth assessment.
2. Shoot tip excision
Nodal sections are cut from 2 to 3 month-old in vitro-grown Vitis plants (Fig. 3), grown on Shoot Maintenance Medium (MS + 30 g L-1 sucrose + 0.175 mg L-1 indole-3-acetic acid (IAA) + 2.5 g L-1 gellan gum at pH 5.7 (pH 6.0 prior to autoclaving)). Nodal sections are plated onto Pretreatment Medium (MS + 30 g L-1 sucrose + 0.2 mg L-1 6-benzylaminopurine (BAP) + 0.1 mM salicylic acid + 0.25 mM ascorbic acid + 0.25 mM glutathione (reduced form) + 3 g L-1 gellan gum at pH 5.7 (pH 6.0 prior to autoclaving)) in 100 x 25 mm Petri plates (50 mL of media per plate with 40 nodes each). Nodal sections are grown in a growth room at 25 oC with a 16 h photoperiod (40 μM m-2 s-1) and cultured for 2-3