**4. Screening methodologies for heat tolerance breeding**

Efficient screening procedures and identification of key traits in diverse donor or tolerant lines are very much essential toward breeding for heat tolerance. Screening for heat tolerance in the field is very challenging due to interactions with other environmental factors, but a wide variety of relevant traits are available that allows successful selection in the field conditions [47]. Tolerant genotypes may also be selected in controlled environments provided validated screening tools are in place. However, very often the more expensive controlled environments do not allow natural selection for other factors that interact with the heat stress tolerance mechanisms under field conditions, thus limiting its potential of wider applications in any trait screening [48]. Heat tolerance can be evaluated by a variety of viability assays, measurements, visual assessment, and testing under hotspot locations as described below.


as a function of temperature. It is simple, quick, and inexpensive and holds promise for the rapid screening in a large number of crops, e.g., wheat [58] and legumes (pigeon pea, chickpea, groundnut, and soybean) [59].

HS responses among plants are mainly due to their inherent ability to survive and also to acquire thermotolerance to lethal temperatures. Genetic variability among crops for HT is mainly due to expression of different stress-responsive genes [44], acquisition of thermotolerance, and synthesis and accumulation of HSPs that are well correlated with the antioxidant defense system [45]. The maintenance of high membrane thermostability (MTS) is related to thermotolerance [46] and an important selection criterion which is determined by measuring the electrical conductivity. MTS has been successfully employed to assess thermotolerance in many food crops worldwide. The role of thermoprotectants such as HSPs, proline, glycine betaine, trehalose, brassinosteroids, salicylic acid, abscisic acid, polyamines, and nitric oxide in offering heat tolerance through endogenous synthesis or by exogenous application in different crops has been discussed in detail by Kaushal et al. [17]. Future pioneering studies in model plants can pave the way to identify key regulators as target for gene manipulation of stress tolerance in crop plants. It has also been envisaged that metabolic fingerprinting can be used as breeding tool for development of plants with the best potential to tolerate abiotic stresses.

Efficient screening procedures and identification of key traits in diverse donor or tolerant lines are very much essential toward breeding for heat tolerance. Screening for heat tolerance in the field is very challenging due to interactions with other environmental factors, but a wide variety of relevant traits are available that allows successful selection in the field conditions [47]. Tolerant genotypes may also be selected in controlled environments provided validated screening tools are in place. However, very often the more expensive controlled environments do not allow natural selection for other factors that interact with the heat stress tolerance mechanisms under field conditions, thus limiting its potential of wider applications in any trait screening [48]. Heat tolerance can be evaluated by a variety of viability assays, measurements, visual assessment, and testing under hotspot locations as described below.

**i.** *Cell membrane thermo-stability test*: Cellular membrane dysfunction due to stress leads to increased permeability and leakage of ions, which can be readily measured by the efflux of electrolytes from affected leaf tissue into an aqueous medium. This method was initially developed by the C.Y. Sullivan (University of Nebraska) in the late 1960s for assessing sorghum and maize heat tolerance. This has been used to study cellular thermostability for heat in wheat [49, 50], soybean [51], maize [52], and chickpea [53]. A positive correlation between membrane injury and grain weight was observed in wheat suggesting that membrane thermostability (MTS) may be better indicator of heat tolerance [54]. The membrane thermostability (MTS) can be measured as follows: MTS = (1 − 1/2) × 100, where 1 is conductivity reading after heat treatment and 2 is conductivity reading after autoclaving [55]. This has been tested in pearl millet and found effective under field condition and thus can be used for screening large number of genotypes. **ii.** *Chlorophyll fluorescence measurement*: Heat damage in photosynthetic tissue can be measured by chlorophyll fluorescence [56]. Chlorophyll fluorescence has been linked to a thermal kinetic window established by enzymatic assays [57]. In this approach, leaf discs are exposed to a brief illumination period and the time of dark recovery of the fluorescence

(ratio of variable to minimum chlorophyll fluorescence) is determined

**4. Screening methodologies for heat tolerance breeding**

parameter Fv

50 Next Generation Plant Breeding

/Fo


All these techniques need to be validated for a large number of crops for their applicability in future. Regardless of the screening method, a key objective for plant breeders is to develop an effective set of thermotolerance markers which can be used for further implementation of breeding for heat tolerance in various crop species.

Identification of the superior germplasm for heat tolerance is essential for effective genetic manipulation through breeding process. However, identification of reliable and effective heat screening methods is a major challenge in conventional breeding to facilitate detection of heat tolerance lines [6]. Although a number of screening methods and selection criteria that have been developed/proposed by different researchers are briefly discussed above, however, the primary field screening methods also include seedling thermo-tolerance index (STI) [73], seed to seedling thermo-tolerance index (SSTI) in pearl millet [74], and heat tolerance index (HTI) as growth recovery after heat exposure in sorghum [75]. Thermo-tolerance screening at germination and early vegetative stage is found effective for pearl millet and maize [76]. These field techniques would help in preliminary identification of heat tolerance lines and thus proceed with minimum number of lines for further screening and validation. At the same time, breeder should ensure the quality of individual line data by comparing with tolerant check at all the times. This will facilitate the more reliable way of advancing the heat tolerance genotypes in any afore-discussed screening tools.

**Crop Heat tolerance sources\* HT associated trait/index References**

*Aegilops tauschii* Coss. Cell membrane stability and TTC-based cell viability

Moomal-2000, Mehran-89 Germination-related traits [82]

CB # 367, 333, 335 Grain development and survival [84] WH # 1021, 730 Grain yield [85] SYN # 11, 36, 44 1000-grain weight [86]

N22, NH219 Spikelet fertility and pollen viability [89]

— Spikelet fertility [91] *Oryza meridionalis* Growth rate and photosynthesis [92] N-22 Spikelet fertility [93] Nipponbare, Akitakomachi Spikelet fertility [94]

— Grain filling duration, kernel dry weight, starch,

protein, and oil contents

Jimai-22 Photosynthesis, PS II, carboxylation, and grain yield

temperature depression (CTD)

1000-grain weight (TGW) [78]

Breeding Cultivars for Heat Stress Tolerance in Staple Food Crops

http://dx.doi.org/10.5772/intechopen.76480

Spikelet fertility [80]

Leaf chlorophyll, grain weight, and grain yield [81]

Spikelet fertility and seed set [87]

Spikelet fertility and seed set [88]

Spikelet fertility [90]

Heat shock protein (HSP) [95]

Leaf firing and tassel blast [96]

Yield [98]

Seed set [21]

STI and SSTI [100]

Seedling thermotolerance index (STI), seed to seedling thermotolerance index (SSTI), and membrane thermostability (MTS)

[77]

53

[79]

[83]

[97]

[99]

Wheat CWI # 59788, 60155, 60391 Leaf chlorophyll content (LCC) and canopy

Raj 4014 × WH730 (HT) RIL

*A. speltoides* Tausch; *A. geniculata*

ALTAR 84/AO'S′; ALTAR 84/*A.* 

population (113)

Roth

*tauschii*

Rice Dular, Todorokiwase, Milyang23, IR2006-P12-12-2-2, Giza178

(*O. glaberrima*)

population

Maize ZPBL 1304 (HT); (ZPBL 1304 ×

Pearl millet 9444, Nandi 32, ICMB 05666,

CP 1

ZPL 389) F2 population (160)

B76, Tx205, C273A, BR1, B105C, C32B, S1W, C2A554-4

Hybrids: YH-1898, KJ.Surabhi, FH-793, ND-6339, NK-64017

F1's: H77/29-2 × CVJ-2-5-3-1-3; H77/833-2 × 96 AC-93

CVJ-2-5-3-1-3; 77/371 × BSECT

ICMB 92777; ICMB 02333

N22, Bala, Co 39; CG14

Bala (HT) × Azucena RIL
