Project Number: 2032-21000-024-06-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 15, 2017
End Date: Oct 31, 2018
The objective of this project is to perform reliable and efficient phenotyping for freeze tolerance in developing fruitlets of peach accessions from the National Clonal Germplasm Repository. This phenotypic information will subsequently be available via GRIN and Clemson University cultivar evaluation web site (www.clemsonpeach.org) to Prunus CGC and allied scientist for use in breeding and in genetics research.
Freeze tolerance in peach fruitlets will be determined using the electrolyte leakage method developed by Lerew and Melgar (2016). Fruitlets will be sampled at the NCGR in the afternoon, immediately taken to the mail courier, and sent overnight to the Clemson lab. Upon arrival, fruitlets from the same accession (<13 mm in diameter) will be placed in test tubes (five fruitlets per tube). One mililiter of deionized water will be added to each test tube and tubes will be immersed in an ethylene glycol–water refrigerated bath (AP 20R-30, VWR, Radnor, PA, USA), thus preventing freezing of the medium in which tubes are immersed. Tubes will be exposed for 1 h to temperatures ranging from 0 to -10 °C, decreasing the temperature at intervals of 2 °C h-1. Three test tubes (replicates) per cultivar and temperature will be used. The replicates corresponding to each temperature will be removed from the bath every hour, and then the bath temperature will be reset to the next lower temperature. Every time the test tubes are removed from the bath, they will be covered with Parafilm® (Bemis, Neenah, WI, USA) and placed in a refrigerator (4°C) to thaw gradually. After 1 h, 10 ml of deionized water will be added to each tube and tubes will be left at room temperature. Then, covered test tubes will be shaken at 200 rpm overnight, for a minimum of 12 h. The following morning, each tube will be vortexed for a few seconds and electrical conductivity (EC1) will be measured with a conductivity meter (Fisher Scientific Accumet AP85, Thermo Fisher Scientific, Waltham, MA, USA) to determine the amount of solutes from the fruitlets released into the solution. Test tubes will be autoclaved for 20 min at 121°C to completely disrupt the cell membranes so that all solutes inside the cell leak into the solution. Samples will be left at room temperature until they are cooled down and then, electrical conductivity will be measured again (EC2). Electrolyte leakage will be calculated as the ratio of ion leakage from freeze injury: EL = EC1 x 100/EC2 (Barranco et al., 2005). Tree response to freezing temperatures is characterized by an asymmetric sigmoid function (von Fircks and Verwijst, 1993). The inflection point will be selected as the temperature at which 50% of ion leakage occurs. The temperature giving an index of 50% ion leakage (or injury) is usually termed as LT50 (Burr et al., 1990); however, when working with woody plants, most authors utilize the inflection point of the sigmoid curve to predict the lethal freezing temperature or in which temperatures slightly below zero already causes more than 50% ion leakage (Barranco et al., 2005). We will use the inflection point to determine LT50 of each of the accessions. Once a curve is plotted, there are four parameters that will be recorded: x0, temperature at the inflection point (LT50); y0, lowest electrolyte leakage value (at 0 °C); a, difference in electrolyte leakage between minimum and maximum temperature; and b, slope of the curve (steepness of the curve at x0). We will be provided to GRIN-Global and communicated with allied scientists through national horticultural symposia and research publications.