*6.2. Traits for Indirect Selection for Heat Tolerance*

Vegetative growth parameters should be avoided regarding heat tolerance since recent studies with a large number of tomato accessions showed that these parameters were not significantly correlated with fruit set [37] and fruit yield [56] in HT, which are key indicators for heat tolerance. Besides this, there was no association between seedling and reproductive growth stages in HT [56]. Therefore, the selection of heat-tolerant tomato genotypes based on seedling performance and vegetative growth parameters in HT should be conducted very carefully, although selection in the early growth stages can facilitate the breeding process.

Pollen traits may be promising candidates for indirect selection when screening tomato accessions in HS. In particular, pollen viability and germination may be promising candidates for indirect selection for heat tolerance since many of the studies discussed above reported an association between pollen traits and fruit set or yield. However, careful consideration must be taken when applying pollen traits for the selection of heat-tolerant lines in breeding programs since the results of these studies were largely based on a few genotypes and showed genotype-dependent responses. Studies using a large number of genotypes showed somewhat contrasting results. Pollen germination was not significantly correlated with fruit set [68] and fruit yield [56], when using 11 and 38 genotypes, respectively, and both fruit set and yield when using 43 genotypes (Table 2) in HS. In another study with 13 tomato genotypes, significantly positive correlations were observed between pollen viability and fruit set and fruit number in tomato plants [37]. In addition, no significant correlation was observed between pollen tube length and fruit set or number [56,68]. These contrasting results might be due to different HS regimes and environmental conditions (pollens receiving heat stress in a Petri dish [68], and plants in pots [37] and in soil in greenhouses [56]). The procedures for pollen tests were also different, such as pollen viability [37]

and germination [56,68]. A standardized procedure to screen for pollen tolerance to HS must be developed and applied in future studies and breeding programs.

Membrane thermostability may be a good candidate for indirect selection for heat tolerance and should generally be measured by ion or electrolyte leakage. In tomato plants, the level of ion leakage differed by tomato genotypes and significantly decreased under HS [37]. Heat-tolerant genotypes showed less electrolyte leakage than susceptible ones in tomato plants [26,36]. In a study using 13 tomato cultivars, fairly good correlations between ion leakage and fruit set (*r* = −0.444) or pollen viability (*r* = −0.294) were observed, although they were not significant [37]. In an experiment using a larger population (43 cultivars), a significantly negative correlation (*r* = −0.9) between electrolyte leakage and fruit yield was observed [57]. These correlation studies suggest the potential of ion or electrolyte leakage for heat tolerance screening in tomatoes.

The index of *Fv/Fm* has been utilized when screening large numbers of tomato genotypes in HS from seedlings to mature plant stages [82]. Specifically, *Fv/Fm* of 28 genotypes was measured under DAHS and MCHS conditions, following three different HS regimes, under conditions from a climate chamber to an open field. Tolerant genotypes that were selected by higher *Fv/Fm* in HS displayed a lower level of leaf heat injury, as well as a higher fruit yield [82]. Moreover, in a correlation experiment using four heat-tolerant and four susceptible cultivars, *Fv/Fm* was significantly correlated with plant dry weight under four days of HS (*r* = 0.974) in a climate chamber, which was significantly correlated with fruit dry weight (*r* = 1.00) and fruit set (*r* = 0.984) in field conditions [82]. The study may suggest that the parameters associated with photosynthetic apparatus can be applied to the rapid detection of heat-tolerant tomato plants during the early vegetative growth stage. However, correlation analyses were not performed with the same plant types or under the same environmental conditions; therefore, *Fv/Fm* is still far from sufficient as a predictor of HT tolerance. Further study is essential with a larger number of genotypes and trait investigations in the same plant type and environment.

#### **7. Conclusions and Future Perspectives**

According to a recent IPCC report, it is certainly a matter of major concern that climate change will be detrimental to food security. In particular, HS, an elevated temperature above a threshold level, can negatively influence plant behaviors during the entire growth and development cycle in both greenhouse and open-field conditions, thereby leading to the diminishing of the fruit set, quality, and yield of tomatoes. It is, therefore, essential to develop tomato cultivars that are tolerant of HS. Since the different organs and growth stages of tomato plants exhibit different sensitivities in response to HS, different HS regimes should be properly applied from seedlings to reproductive growth stages. In this review, we have discussed recent attempts in screening and breeding for heat tolerance in tomato plants under different HS regimes. In addition, we discussed the diverse selecting traits shown during the vegetative and reproductive growth stages and provided an association between the traits and the key biochemical factors, which can be effectively utilized to screen and/or select a large number of tomato genotypes under HS conditions. Finally, we proposed effective direct and indirect selection strategies to develop heat-tolerant tomato cultivars presenting promising candidate traits that are significantly correlated with fruit set and yield under HS.

Further studies are required with a large number of tomato genotypes to identify the key traits for indirect selection since there is still a lack of correlation studies for HS tolerance using a large population. In addition, further study is necessary to establish the effect of HS on the marketable yield of tomato plants. Seedling selection to accelerate the breeding process will apparently not be applied in the near future for the selection of heat-tolerant tomato genotypes due to the contrasting results of an association between the seedling and reproductive stages. To increase the efficiency of developing heat-tolerant tomato cultivars, it is also necessary to develop a marker-assisted selection system. Since the traits related to heat tolerance show quantitative inheritance, the development of molecular markers can be conducted with bi-parental quantitative trait loci mapping and genome-wide association studies, with plenty of sequence information being obtained from whole-genome sequencing, re-sequencing, and genotyping-by-sequencing. Emerging genome-editing techniques, including the CRISPR/Cas 9 system, can be adopted in the future to introduce or neutralize beneficial or deleterious genes, respectively, in elite tomato lines.

**Author Contributions:** Conceptualization, E.-Y.Y. and W.-B.C.; writing—original draft preparation, K.L. and W.-B.C.; writing—review and editing, K.L., S.N.R., H.-B.J., M.-C.C., O.-J.L., S.-G.K., E.-Y.Y. and W.-B.C.; supervision, E.-Y.Y. and W.-B.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by a grant (Project No: PJ01669601 "A study on the hightemperature tolerance mechanism of hot pepper and tomato using an evaluation population") from the National Institute of Horticultural and Herbal Science, Rural Development Administration.

**Data Availability Statement:** The datasets presented in this study are available upon request to the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
