# New Insights in Potato Leaf Freezing by Infrared Thermography
## 1 Introduction  
After the first employments of infrared thermography to study freezing processes in plants [1; 2; 3], several advances in high-resolution infrared thermography (HRIT), brought significant new insights about ice nucleation and ice propagation in plant samples and whole individuals [4; 5; 6; 7; 8; 9]. Especially, when HRIT is used in the differential imaging mode (infrared differential thermal analysis IDTA; [10; 11; 12]) ice growth in plant tissues can be visualized at such high resolution that in some cases allows for the tracing of the freezing signal to single cells in the xylem [13]. The method additionally allows the identification of plant parts or tissues that supercool and stay free of ice and usually a second freezing process upon breakdown of supercooling can be traced to intracellular lethal freezing [5; 14; 15; 16; 17; 18]. Still, rare attempts have been made to study plant freezing under natural frost conditions by infrared thermography [6; 19]. Freezing signals measured by IDTA in plant samples can originate from both extracellular or extra-tissue [20] ice formation that can be tolerated in freezing resistant plants down to a species-specific freezing temperature threshold and intracellular freezing that is usually lethal [21]. A combination of IDTA with viability tests under controlled freezing treatments permits an evaluation of the consequence that ice formation inside the tissue exhibits on cell vigor. In freezing resistant plant samples, several freezing processes at different freezing temperatures can be detected. Usually one matching frost injury is suggested to be intracellular [13; 15; 16; 17; 18]. While the overwhelming majority ofstudies on the freezing process in plants by infrared thermography focuses on freezing resistant plants [3; 4; 6; 7; 9; 10; 11; 12; 13; 15; 16; 17; 18], comparatively little information exists about plants that tolerate little or no ice [22]. Particularly, in freezing susceptible leaves the question whether ice formation per se or rather its consequences (freeze dehydration and freezing cytorhysis) are lethal to the cells should be answerable by employment of IDTA. _Solanum tuberosum_ is a common and important field crop in the temperate regions and at high elevations in the tropics where frost is a chronic problem [23]. Unfortunately potatoes possess very little freezing resistance [24]. _S. tuberosum_ leaves get frost killed around \(-3\)\({}^{\circ}\)C and are unable to frost harden [25]. Under field conditions, leaf and stem tissue of potato nucleates at \(-0.5\) to \(-3\)\({}^{\circ}\)C [21; 26] and plants freeze at relatively slow cooling rates, i.e., 1-3 K\(\cdot\)h\({}^{-1}\)[21]. The basic nature of frost injury at the cellular level in leaves of potatoes, and how it relates to ice formation, is still not well understood. However, important mechanisms for survival appeared to be the tolerance of freeze-induced dehydration and preservation of membrane integrity, a greater capacity to tolerate more frozen water and the avoidance of intracellular freezing [25]. In an early pioneer thermal imaging study, whole potato plants revealed a relatively high deep supercooling capacity in extreme down to \(-8\)\({}^{\circ}\)C during cooling at 15 K\(\cdot\)h\({}^{-1}\) down to \(-10\)\({}^{\circ}\)C [27]. However, for the investigation of how freezing processes in potato leaves relate to freezing injury, it may be advisable to keep the freezing treatment as close as possible to natural field freezing conditions. This includes control of ice nucleation, slow cooling rates (1-3 K\(\cdot\)h\({}^{-1}\)) and the final target freezing temperature should not exceed the frost killing temperature of \(-3\)\({}^{\circ}\)C.  
We aimed to study the freezing process in potato leaves by IDTA in combination with a viability assessment in order to find out whether _S. tuberosum_ leaves tolerate ice or get immediately killed upon ice formation. This should allow addressing the question of the basic nature of frost injury in _S. tuberosum_ leaves.