# New Insights in Potato Leaf Freezing by Infrared Thermography
## 4 Discussion  
The use of IDTA under controlled freezing conditions gave new insights into the freezing process of potato leaves and revealed two distinct freezing events. The first freezing event was also detectable by DTA. The average rise in temperature during this first freezing event was 2.5 K. The freezing pattern with initial spread through the vascular system and from there in to the leaf blade and the duration of the overall freezing process were all quite similar to the earlier findings for potato by thermal imaging [27]. Infrared equipment has significantly improved since this early thermal imaging study on potato freezing which might explain why the second exotherm found in the current IDTA study was not detected [27]. Technical advancements in infrared thermography during the last decades nowadays provide much more detailed insights [19]. Leaves removed from the freezing test being frozen or supercooled at \(-2.5\)\({}^{\circ}\)C \(\pm\) 0.2 SE, respectively, showed differential damage symptoms with the frozen leaves becoming completely water soaked and having a darkened appearance and the unfrozen leaves remaining quite healthy. This would suggest ice susceptibility of the leaves. Similar observations have been reported earlier [27]. This clearly demonstrates that frost injury has something to do with the formation of ice in the leaves and as suggested earlier might be an important point for the development of crop frost protection strategies [27]. Strikingly, when leaves were rewarmed after the first freezing exotherm, they did not show any symptoms of frost injury. This suggests that the first freezing event is not lethal. There is some indication in the literature that potato leaves might tolerate freezing [25]. In a thermal imaging study, it was observed that if potato plants were removed from the freezing chamber within 5 min of freezing then injury was limited while irreversible frost damage occurred to plants within 10 min of shoot freezing [27]. This already indicated that the after effects of this ice formation causes frost injuries. A dramatic increase in frost injury of potato leaves (_S. acaule_, _S. commersonii_) was observed when ice was initiated at about \(-4\)\({}^{\circ}\)C as compared to similar leaf tissue frozen to the same freezing temperature  
Figure 4: A leaf of _S. tuberosum_ (**a**) before and (**b**) after a controlled freezing treatment down to \(-2.7\)\({}^{\circ}\)C. The leaf was rewarmed immediately after the first ice wave (as monitored by IDTA) and hence the second freezing was prevented. The digital image after freezing was taken 4 d after the low temperature treatment. but with ice, which was initiated at about \(-1\)\({}^{\circ}\)C [29]. This may also indicate that freezing may be tolerated when ice nucleation occurs at a moderate freezing temperature. Our findings support that _S. tuberosum_ leaves tolerate ice, even though only for a short time period (10-78 min). IDTA reveals that ice initially propagates via the vascular tissue, but at the same time ice captures the whole leaf blade. By IDTA it is not possible to localize where exactly ice forms and propagates in the mesophyll during this initial freezing event. The observed freezing pattern could be extra-tissue freezing [20] where ice accumulates primarily in spaces outside the tissue, and a little in the intercellular space within the tissue. Unfortunately, by microscopic inspection of dissected frozen leaves extracellular/extra-tissue ice masses could not be localized. In this respect, precise localization of ice in leaves after the first freezing event could yield valuable insights about freezing resistance mechanisms. When leaves remained exposed after the first freezing exotherm under the experimental conditions a second freezing event could be detected. This freezing process had a diffuse pattern and occurred all over the leaf blade. No distinct starting point of this freezing could be depicted.