CIMMYT 1996 Wheat Improvement Training Course.

R. L. Villareal and O. Banuelos.

Training is a major activity at the CIMMYT Wheat Program. The principal objective of this training program is to increase the professional expertise of wheat research personnel in developing countries. Our trainees are better equipped to meet the challenge of further improving their home country's capabilities for wheat research and food production after completing their course of study. The 1996 wheat improvement training course began on 26 February, at Ciudad Obregon, and ended on 18 August, at Toluca and El Batan near Mexico City.

Eighteen trainees from 14 countries participated in the course. Africa had the most representation with seven participants followed by Asia with six, Middle East with four, and one from Latin America. The trainees participated in plant selection, harvest, seed selection, assessment of diseases, agronomic management, and other hands-on activities of the Wheat Program. This year's distinguished course lecturers included Dr. Anne McKendry, University of Missouri; Dr. Greg Shaner, Purdue University; Dr. Mark Sorrells, Cornell University; Dr. Warren Kronstad, Oregon State University; and Dr. Adam Lukaszewski, University of California at Riverside.

CIMMYT can hope to train only a fraction of the thousands of wheat specialists needed by national programs. Therefore, CIMMYT attempts to reach those candidates who demonstrate leadership ability and who are potential future research leaders in national programs. A new group of trainees will arrive in Mexico during the last week of February 1997 for 6 months of training.

Canopy temperature depression as a selection criterion for wheat tolerance in wheat.

M.P. Reynolds ¹, O.A.A. Ageeb ², S. Nagarajan ³, L. Olugbemi ⁴, M.A. Razzaque ⁵, and S. Rajaram ¹.

CIMMYT¹, Sudan², India³, Nigeria⁴, Bangladesh⁵ Wheat Programs.

Site Collaborators:

India: Tandon JP, Hanchibal RR, Ruwali KN, Mohan D, Singh RP

Sudan: Rasoul O, Ibrahim A, Amani I, El-Sarag G, Mustafa M

Bangladesh: Barma N, Amin R, Rahman M, Meisner C

Nigeria: Abubakar I

Summary. The main objective of the study was to validate the use of canopy temperature depression (CTD) as a rapid, early generation, screening tool for heat tolerance in wheat. CTD was measured at hot sites in Mexico on F₂-derived bulks (BULKS) and on RILs derived from the same BULKS, using crosses of parents with different levels of heat tolerance. Results showed that (i) CTD measured on BULKS in Mexico were significantly correlated with their average performance at 11 international sites, (ii) CTD was an excellent indicator of which BULKS produced heat-tolerant inbred lines evaluated in Mexico, and (iii) CTD measured on RILs were highly significantly correlated with performance in Mexico. These results indicate the robustness of CTD as a selection trait, with potential application at early and intermediate stages of selection. In addition, the genetic link between CTD and heat tolerance was demonstrated by showing their association in RILs. Measurements of photosynthesis and leaf conductance on individual F₅ plants showed high heritabilities with measurements made on F₅-derived F₇ sister lines, as well as significant correlations with yield. Results suggest the potential of screening for quantitative traits on individual plants in early generation-derived bulks. The `genotype x environment' interaction among hot wheat-growing regions worldwide was tested by growing a set of 60 advanced lines (ALs), selected for heat tolerance in Mexico, at 15 international sites. The results showed Tlaltizapan to be the best site in Mexico for predicting yields in Bangladesh, northwest India, Sudan, and Nigeria, and northwest India to be a good site for heat tolerance screening. Correlation analysis corroborated these observations and indicated late sowing in Obregon as an additional site for heat tolerance screening. Average yield of the 60 ALs at 15 international sites was predicted equally well by CTD or yield, when both were measured in Mexico. Because a reliable yield estimate requires a plot approximately five times greater than that needed for an estimate of CTD, the use of CTD instead of yield estimates may be considerably more efficient. Alternatively, both yield and CTD could be combined in a selection index as a more powerful indicator of heat tolerance. Based on results from the current project, CIMMYT Wheat Breeding Program are currently evaluating the use of CTD as a selection criterion in preliminary yield trials for heat tolerance. Physiological and morphological data measured in the different experiments suggest that yields under heat stress are source (assimilate) limited. Wheat lines of diverse origin from the Indian and world wheat collections were screened for heat tolerance traits. Preliminary data indicated high levels of expression for CTD, chlorophyll content, rate of biomass accumulation, and kernel weight. A laboratory-based screening protocol for membrane thermostability demonstrated a high degree of genetic diversity for the trait in material from the Indian Bank.

Introduction.

Background. Continual heat stress affects approximately 7 million ha of wheat in developing countries, whereas terminal heat stress is a problem in 40 % of the temperate environments that encompass 36 million ha. In a recent consulting with NARS representatives from the major wheat growing regions in the developing world, heat stress was identified as one of their top research priorities (CIMMYT 1995). The current project, evaluating physiological selection criteria that may help breeders select for more heat-tolerant wheat, arose from earlier collaborative work between CIMMYT's Wheat Program and NARS, namely the International Heat Stress Genotype Experiment (IHSGE). These studies indicated significant genetic diversity for heat tolerance in modern semidwarf spring wheat varieties and its association with a number of physiological traits (Reynolds et al. 1992; Reynolds et al. 1994; Amani et al. 1996; Reynolds et al. 1997). Based on these results, crosses between heat-tolerant and heat-sensitive parents were made to assess the potential genetic gain of applying physiological and morphological selection criteria in segregating generations. Subsequent work was incorporated into the current project supported by the U.K.'s Overseas Development Administration (ODA) holdback facility.

Canopy temperature depression - CTD. Although several physiological traits were tested for potential use as selection criteria for heat tolerance in this study, the principal focus was on the measurement of CTD using an infrared thermometer. CTD was believed to be the most promising technique for a number of reasons: (i) it is extremely rapid to integrate the CTD of scores of wheat plants in a plot in a few seconds; (ii) earlier work had shown CTD able to predict the heat tolerance of varieties in several wheat growing environments (Reynolds et al. 1994); and (iii) background studies at CIMMYT had indicated the optimal stages of development and environmental conditions to maximize genetic expression for the trait (Amani et al. 1996). In addition, although no causal relationship has been demonstrated between CTD and heat tolerance, both theory and experimentation show it be intimately associated with crop assimilation processes, lending it credibility as a worthwhile trait for crop improvement.

Selection for simple versus quantitative traits. There are several stages in the breeding process when selection pressure can be applied for a trait of interest. Traditional plant breeding attempts to maximize selection pressure in early generations for reasons of efficiency. Early generation selection (F₂-F₄) is practical when selecting for relatively simple traits such as disease resistance, phenology, and agronomic type, because a high proportion of progeny are genetically fixed in early generations. On the contrary, for quantitative traits such as CTD, segregation is still very likely in early generations, and later generation selection will be a more reliable strategy to fix the trait of interest. However, delaying selection pressure makes selecting for quantitative traits less attractive to most breeders, where selection for dominant traits still plays a big role in breeding methodology. For these reasons, this project focused on evaluating CTD as a selection criterion at different stages in the breeding process: (i) in early generation-derived BULKS, (ii) among intermediate generation F₅-derived RILs, and (iii) in ALs already selected empirically for heat tolerance. The objective was to evaluate the flexibility of CTD as a selection tool, given the multiple selection criteria that must be incorporated into a breeding methodology.

Collaboration between NARS and CIMMYT. A major strength of the current study was the partnership between CIMMYT and NARS working in hot, wheat-growing environments. By testing materials in representative environments worldwide, conclusions regarding the primary objective could be interpreted on a much broader basis than with a more narrowly focused study. At the same time, the partnership enabled the formation of other objectives. 1. By determining `G x E' in hot wheat environments and confirming reliable international testing sites, this information can assist a more strategic approach to germplasm and information exchange. Crucial feedback is provided to the centralized breeding strategy coordinated by CIMMYT. 2. Disseminating diverse genetic material for heat tolerance traits. Although integral to the projects primary objectives, materials distributed were of sufficient genetic diversity to avail themselves to reselection by NARS and CIMMYT breeding programs and provide genetic stocks for future strategic research on heat tolerance. 3. Enabling wheat scientists at collaborating NARS to gain experience in evaluating the use of physiological selection criteria to complement breeding objectives. In addition to sponsoring NARS scientists to visit CIMMYT and gain hands-on experience in evaluating the use of physiological selection criteria to complement breeding objectives, the ODA-funded project also provided for the purchase of equipment for use in future breeding and research activities of the participating NARS.

Screening genetically diverse wheat lines for heat tolerance traits. Trials were established in India and Mexico to screen material from the Indian and world wheat collections in an attempt to find new and better sources of heat tolerance traits, such as CTD, chlorophyll content, rate of biomass accumulation, and kernel weight. Previous studies have shown heat tolerance to be associated with membrane thermostability (Shanahan et al. 1990; Reynolds et al. 1994). This trait was evaluated on lines from the Indian collection by measuring expression in heat-acclimated seedlings. Seedling screening is a potentially attractive option in breeding, because large numbers of plants can be measured within a relatively short time frame.

Selecting for quantitative physiological traits in individual F₅ plants. Breeding for quantitative traits is more complex than selecting for dominant traits, as discussed earlier. Selections for traits such as photosynthetic rate or leaf chlorophyll could be made on individual plants as opposed to yield plots would considerably increase efficiency. Photosynthesis and related traits were measured on F₅ individual plants to assess whether the traits were expressed in lines deriving from those individuals, and their association with heat tolerance.

Materials and methods.

Experimental Sites. In total, up to 15 experimental environments were involved in five countries over 2 years: four environments in Mexico, Obregon (2 dates) and Tlaltizapan (2 dates); four sites in Sudan; three sites in Bangladesh; three sites in India; and one site in Nigeria. Summaries of locations (Table 1, p. 149) and meteorological data (Table 2, p. 149) are presented.

Breeding Materials. Three types of breeding material were used in order to test the potential utility of trait selection at different stages of the breeding program: F₂-derived BULKS from `Fang 60/Seri 82' (Cross 1), `Fang 60/Siete Cerros' (Cross 2), and 'Seri 82/Siete Cerros' (Cross 3) grown internationally in 1994-95 (IHSGE V) and from `Nesser/Pavon' grown in internationally in 1995-96 (IHSGE VIA); RILs derived from randomly-selected F₅ heads of the BULKS from crosses 1-3 (grown in 1995-96 at two or three locations in Mexico); and breeder-selected advanced lines (IHSGE VIB grown internationally in 1995-96).

Physiological Traits. Most physiological trait measurement was conducted in Mexico. NARS visiting scientists were actively involved in the 1994-95 cycle. Traits measured on field plots in the four environments in Mexico included CTD on yield plots (CTD-5) and 3-row plots (CTD-3) simulating the planting arrangement used commonly in breeding programs for early generation selection. Measurements were made with an IR thermometer (Telatemp/Everest) after anthesis, between noon and 4 p.m., when resolution for the trait is highest (Amani et al. 1996). Leaf conductance (COND), measured as leaf resistance using a viscous-flow porometer (CSIRO) and reported in relative units, was measured after anthesis in the afternoon on 6-10 flag leaves per plot. Flag leaf chlorophyll content was measured at 50 % anthesis (CHL-A) and 10 % physiological maturity (CHL-M) on 6-10 flag leaves per plot using a Minolta SPAD meter.
Table 1. Key features of the experiemental sites of the 5th and 6th International Heat Stress Genotype Experiments.

Table 2. Monthly maximum and minimum temperatures for Tlaltizapan and Obregon, 1994-1995 and 1995-1996 Ecycles, Mexico.

Other traits measured. Two other kinds of traits were measured in addition to physiological traits; parametric traits associated with yield components and traits typically evaluated visually. Parametric traits included grain yield, final biomass (above ground), grains/m², 1,000-kernel weight, grains/spike, harvest index, and spikelet sterility (upper and lower spike). Visual traits included days-to-anthesis, days-to-maturity, height, spikes/m², awn length, peduncle length, spike length, % ground cover at anthesis, and breeders scores (1-5). All traits were measured parametrically except for ground cover and breeders score, which were visually estimated.

Screening genetically diverse wheat lines for heat tolerance traits. Physiological traits in genetically diverse wheat lines were evaluated on 100 lines provided by the Indian Bank, the world collection at CIMMYT, and synthetics from the CIMMYT Wheat Program's wide-crosses group. These were evaluated in Tlaltizapan, Mexico, and Indore, India.

Screening for new sources of membrane thermostability using seedlings. Membrane thermostability was estimated through conductivity measurements of electrolyte leakage from leaf tissue of 10-day-old, heat-acclimated seedlings, after a heat shock of 49 C for 1 h. Work was in collaboration with Professor James Quick during his sabbatical leave at CIMMYT from Colorado State University.

Selecting for quantitative physiological traits in individual F₅ plants. The heritability of photosynthetic rate between individual F₅ plants and F_5:7 lines was evaluated in collaboration with Colegio de Postgraduados, Montecillo, Mexico. The methodology was essentially as follows: in the F₅ generation, photosynthesis was measured on 200 random plants in eight F_2:5 BULKs from one cross; in the F₆ generation, divergent selection for photosynthesis, head rows were grown for F_5:7 lines; and in the F₇ generation, high and low PS lines were grown for yield and evaluation of photosynthesis.

Statistical Techniques. All field trials were laid out in lattice a design. In addition to ANOVA, the following statistics were calculated when appropriate: phenotypic correlations, genetic correlations, realized heritability, proportion of direct response (PDR), and canonical regression analysis.

Results.

Validation of CTD as selection criterion for heat tolerance.

F₂-derived bulks. Of the physiological traits measured, CTD measured on BULKS was generally best correlated with their yields in Mexico (Table 3). As expected, morphological traits such as above-ground biomass and grains/m² often showed better correlation with traits that are autocorrelated with yield itself.

Table 3. Phenotypic correlations between wheat yields and traits for F₂-derived bulks of three crosses, at Obregon (March sown), 1994-95.

^*significant at P <= 5 %.
¹ No trend for kernel weight, days to anthesis/maturity, or height.

To estimate the relative heritabilities of traits between F₄ and F₅ BULKS, traits measured at different locations in Mexico in the F₅ were compared with data collected on unreplicated plots in the F₄, from the cross between `Seri 82/Siete Cerros', which had the greatest contrast in heat tolerance. Phenotypic correlations indicated the highest association for days-to-flowering, intermediate levels for CTD and chlorophyll at anthesis, and poor association for yield (Table 4). These results were consistent with calculations of realized heritability (data not shown).

Table 4. Phenotypic correlation between traits measured on F_2:4 plots in Tlaltizapan, and trait expression in F_2:5 plots at three locations in Mexico for the cross `Seri 82/Siete Cerros'.

^* significant at P <= 0.05.
^** significant at P <= 0.01.

Table 5. Phenotypic correlation between wheat plant traits measured in Obregon (March sown), Mexico, and mean performance of F₂-derived bulks at 11 international, warm wheat-growing sites, 1994-95 cycle.

^* significant at P <= 5 %.
¹ No trend for biomass, spike number, days to anthesis/maturity, or chlorophyll maturity.

Phenotypic correlations between physiological traits measured in Mexico and yields measured at international locations were generally weaker than for within-site correlations. However, genetic correlations and values for proportion of direct response to selection for CTD indicated that environmental error was one factor reducing the strength of correlations. For BULKS of the three crosses, the phenotypic correlation between the average yield for all 11 international sites (X-YLD) and traits measured in Tlaltizapan and Obregon (Table 5) show a clear trend for CTD, chlorophyll at anthesis (CHL-A), and yield to be positively correlated with X-YLD, and combined analyses indicated statistical significance. For the cross `Nesser/Pavon', evaluation of traits was conducted in F₄ BULKS for comparison with international performance in the F₅. CTD measured on F₄ BULKS in three locations in Mexico was generally significantly associated with average performance of F₅ BULKS at 12 hot international locations, although yield and visual evaluations by breeders were not (Table 6).

Table 6. Phenotypic correlations between traits measured in F₄ bulks in Mexico and average performance of F₅ bulks at 12 hot, international sites for the cross `Nesser/Pavon', 1995-96.

^* significant at P <= 0.05.
^** significant at P <= 0.01.
¹ No trend for grains/m², kernel weight, or phenology.

Although these associations suggest that CTD may be useful in identifying heat tolerance of early generation bulks, a more interesting test is whether BULKS with high CTD produce a higher proportion of heat-tolerant lines. This was tested by taking random heads from BULKS to make RILs and evaluating their heat tolerance. At the same time, physiological traits were tested on the RILs to provide more definitive evidence of the genetic association between CTD and heat tolerance and of the potential value of selecting for quantitative traits in intermediate as opposed to early generations.

Performance of RILs predicted by CTD of original BULKS. RILs from all three crosses showed a clear association between superior heat tolerance and higher CTD in the BULKS from which they were derived (Table 7). These data suggest that CTD is a useful way to screen early generations for material likely to yield heat-tolerant lines despite the probability of further segregation. However, yield of the BULKS was not a reliable indicator of heat tolerance.

Selecting for quantitative physiological traits in individual F₅ plants. Measurements of photosynthesis, leaf conductance, and leaf chlorophyll in individual F₅ plants showed high heritabilities with measurements made on F₅-derived F₇ sisters, and significant correlations with yields under heat stress (Table 8). Full details of this study will be presented separately (Gutierez et al. 1997).

Table 7. Phenotypic correlations between yields of recombinant inbred lines averaged over three sites in Mexico (1995-96), and traits measured on original Bulks in Obregon (March sown), 1994-95.

^* significant at P <= 0.05.
^** significant at P <= 0.01.

Table 8. Phenotypic correlation between traits measured in individual F₅ plants at Tlaltizapan, 1994-95, and agronomic traits in F₅-derived F₇ lines (averaged for two sowing dates at Tlaltizapan, Mexico, 1995-96).

^* significant at P <= 0.05 when r >= 0.47.
¹ Flag leaf chlorophyll content at maturity.

RILs. RILs from the cross `Seri 82/Siete Cerros', with the greatest contrast in heat tolerance, showed a very strong relationship between CTD and yield, giving strong evidence for a genetic basis for the association between CTD and heat tolerance (Table 9). Chlorophyll at anthesis also was strongly correlated with yield, as was height. In the `Seri 82/Fang 60' cross, breeders selection pressure was applied in the F₆ (for agronomic type and disease resistance in a temperate environment). In these lines, CTD was correlated with yield (Table 10), but not as strongly as in the previous cross, although height was not. For the cross `Fang 60/Siete Cerros', similar and highly significant trends were observed (data not shown), though associations were generally weaker, perhaps because of the use of augmented single rep designs.

Table 9. Recombinant inbred lines from the cross `Seri 82/Siete Cerros' (n = 40); correlation between average yield and traits measured at two dates in Tlaltizapan, Mexico, 1995-96.

^* significant at P <= 0.05.
^** significant at P <= 0.01.

Table 10. Recombinant inbred lines from the cross `Seri 82/Fang 60' (n = 33); correlation between average yield and traits measured at 2 sites in Mexico, 1995-96.

^* significant at P <= 0.05.
^** significant at P <= 0.01.

Advanced lines. Phenotypic correlations between traits measured in Mexico and the average performance of the 60 lines across 15 international locations indicate the power of CTD to predict performance and yield itself in some cases (Table 11). Using data from ALs, it is possible to quantify the potential effect of selecting for CTD on performance at different international sites and compare with selection for other traits. Selection for CTD is very favorable in most cases.

Table 11. Phenotypic correlations between average yields of 60 advanced lines at 15 and 11 international sites and traits measured in Mexico, 1995-96.

¹ 11 locations with least `G x E' as determined by cluster analysis for crossover interactions.
^* significant at P <= 0.05.
^** significant at P <= 0.01.

Table 12. Lines exhibiting high values of heat tolerance traits in comparison to Seri 82, Tlaltizapan, Mexico, 1995-96.

Genotype by environment interaction among hot wheat growing regions.

The `G x E' was examined using data from the 60 advanced lines grown at all 15 international sites. Cluster analysis grouped environments so that rank changes among lines were minimal (Crossa et al. 1995). The analysis indicated two main groups. One large group included Nigeria, three sites in Sudan, all three sites in Bangladesh, three sites in Mexico, and the site in northwest India. The other cluster included two sites in south and central India, one site in Mexico, and one in Sudan. The results confirm Tlaltizapan to be the best site in Mexico for predicting yields in Bangladesh, northwest India, Sudan, and Nigeria, and indicated northwest India as a good site for heat-tolerance screening. Correlation analysis corroborated these observations and indicated March sowing in Obregon as an additional site for heat-tolerance screening.

Screening genetically diverse wheat lines for heat-tolerance traits.

Several lines were identified that had heat-tolerance traits with values higher than for the check (Seri 82), including canopy temperature depression, chlorophyll at anthesis, chlorophyll at maturity, and growth factors (Table 12). The laboratory-based test for membrane thermostability used on heat-acclimated seedlings demonstrated a high degree of genetic variability for this trait in genetically diverse wheats from the Indian collection (Quick and Reynolds 1997).

Discussion.

Validating the use of early generation selection criteria for heat tolerance.

Canopy temperature depression. CTD was the physiological trait best associated with heat tolerance in these studies. The results indicated the robustness of CTD as a selection trait, with potential application at early (BULKS), intermediate (RILs), and late (ALs) stages of selection. Although the CTD measured on the BULKS was generally significantly, though not highly, correlated with performance of the BULKS at other locations (Tables 5-6), their CTDs were highly significantly correlated with yields of RILs deriving from the BULKS (Table 7). In contrast to CTD, yields of the BULKS were not good indicators of heat tolerance in the RILs. The fact that CTD was highly correlated with heat tolerance, in both unselected RILs and advanced lines that had already undergone selection for heat tolerance, is a clear indication that CTD is a powerful screening tool with potentially wide application in early and subsequent generations.

F₂-derived bulks. The main problem with working with early generation-derived BULKS, especially when left largely unselected so as not to cause genetic bias, is that they are highly heterogeneous and not necessarily well adapted to the environments. In many cases, the high values for proportion of direct response to selection for CTD (data not shown) reflected the poor relationship between yields in Mexico and those of many of the international sites where they were tested. The relationships between traits and international performance are perhaps difficult to interpret, because most breeding programs do not handle their material in this way. Nonetheless, the value of measuring CTD in early generations was clearly demonstrated by the good performance of RILs deriving from BULKS with favorable CTDs. A logical follow-up is to compare CTD of individual F₂ plants with performance in subsequent generations.

RILs. The high correlations between yield and CTD for the RILs suggest a role for CTD in screening intermediate generations for heat tolerance. A potential strategy would be to select in early generations for agronomic type and disease resistance under temperate conditions, followed by selection for CTD under heat stress on F₄- or F₅-derived head rows, when a greater proportion of lines are genetically fixed. RILs from the cross `Fang 60/Seri 82' were subjected to selection for diseases and agronomic type in F<sub>6</sub> head rows. Although their association between CTD and yield (Table 10) was not as strong as for the unselected RILs from the `Seri 82/Siete Cerros' cross (Table 9), the results for the former were more convincing from a breeding point of view, because heat tolerance was not confounded by undesirable characteristics such as height. An interesting pattern emerged in this material, where the relationship between yield and CTD measured prior to heading was weakly negative, in contrast to the positive correlation observed postheading (Table 13). This observation on largely unselected materials was not seen in varieties (Amani et al. 1996) and needs to be considered when establishing selection protocols. Further work will help understand the physiological basis of the response.

Table 13. Genetic correlations between yield and the traits canopy temperature depression (CTD), and chlorophyll content (CHL), for the recombinant inbred lines of two crosses, Tlaltizapan, Mexico, 1995-96.

Selecting quantitative traits in individual F₅ plants. The fact that measurements of several physiological traits in individual F₅ plants showed high heritabilities with measurements made on F₅-derived F₇ sister lines and significant correlations with yields under heat stress (Table 8) indicates the potential of screening for quantitative traits on individual plants in early generation derived bulks. The data complement the results showing the association between CTD measured in F₅ bulks and performance in RILs (Table 7).

Potential genetic gains of selecting for CTD. For an idea of how selection for CTD might improve heat tolerance in terms of performance, mean yields were calculated for high and low CTD groups, where high and low CTD represented the lines in the top and bottom 50 % of specific germplasm groups with respect to their CTD (Table 14). For the NILs (cross `Seri 82/Siete Cerros'), yield for the high CTD group averaged 4.1 T/ha and the low averaged 3.3 t/ha, representing a difference of 1.0 C in average CTD between the two groups. For the ALs using data averaged across both sowing dates in Tlaltizapan, the high CTD lines had an average yield of 5.2 T/ha in comparison to 4.7 T/ha for the low CTD group, representing a difference of 0.5 C in average CTD. Selection for CTD gave an apparently greater resolution for yield in the NILs than the Als, as one might expect from unselected material.

Table 14. Average yields of high and low canopy temperature depression groups, for two sets of germplasm, 40 F_5:7 inbred lines from the cross `Seri 82/Siete Cerros', and 60 heat tolerant advanced lines, measured in Tlaltizapan, Mexico, 1995-96.

Comparing CTD with other physiological selection criteria. An important part of this project was to compare CTD as a selection criterion with other physiological selection traits. The results of this study indicate that CTD generally gives a more reliable association with yield than the other two physiological traits investigated. In some ways this is not surprising, because CTD is measured on a plot basis, unlike leaf conductance (L-COND) or CHL-A. These traits are measured on individual leaves, thus increasing experimental error, because fewer plants are incorporated into an average reading. CHL-A and L-COND seem to be a useful selection criterion for heat tolerance, but canonical correlation analysis suggests that they would not improve selection efficiency very much where CTD is already measured. Correlation analysis also indicated a degree of auto-correlation among CTD, L-COND, and CHL-A. However, they may be useful techniques for the selection of individual plants, where CTD measurements are difficult with current technology. L-COND and CHL-A also may be an alternative selection criterion to CTD in humid environments where a low vapor pressure deficit does not permit good expression of CTD (Amani et al. 1996).

Comparing CTD with morphological selection criteria. Although a number of the traits measured appeared to be associated with yield in different studies, CTD was generally the most consistent. Where data from several locations permitted yield to be evaluated as a selection criterion itself, it sometimes predicted average yields better than CTD (Tables 5 and 11), sometimes equal to CTD (Tables 10 and 11), and other times not at all (Tables 6 and 7). Canonical regression analysis was used to compare physiological and visual traits. For example, for the `Fang 60/Siete Cerros' cross of the BULKS grown in Obregon (March sown), CTD, L-COND, and CHL-M together explained 53 % of the variability with yield. The easily observed visual traits of spikes/m<sup>2</sup>, anthesis and maturity dates, and height explained 28 % of the variability. For RILs of' Seri 82/Siete Cerros', 57 % of the variability in yield could be explained by physiological traits and 59 % by visual traits, with a combination explaining 72 % of the total variability. For the `Seri 82/Fang 60' cross, CTD and CHL-A alone explained 50 % of the variability in yield, visual traits (spikes/m², height, and days-to-anthesis and maturity) explained 46 % and the combination of visual and physiological traits explained 58 %. For canonical regression analysis with ALs, where traits measured in Mexico were compared with average yield across 15 international environments, physiological traits explained almost 40 % of the variability with yield, whereas visual traits explained 20 %, and in combination they explained 43 %. Yield and all other morphological traits combined explained 46 % of the variability (Table 15). Where more than one trait may be associated with yield, the most practical approach is to develop a selection index. Canonical regression analysis was done to see which traits could best explain yield of the 15 international environments when used in an index. The combination of yield and CTD appears to be the best combination, CTD explaining 37% and yield 35 % with the combination explaining 46 %. Adding spikes/m² and anthesis to the model explain no further variability in the yield of the 15 international environments. Even when all traits are added to the model, only a further 16 % of the variability is explained. In conclusion, although CTD may not be the only trait associated with heat tolerance, it is one of the most reliable and has the advantage of being easier to measure than most other traits.

Determining `G x E' in hot wheat environments confirms reliable international testing sites.

Cluster analysis for the advanced lines confirmed Tlaltizapan to be the best site in Mexico for predicting yields in most of the wheat regions studied, with the notable exception of central and peninsular India. This information can assist CIMMYT and NARS in strategic approaches to germplasm exchange and indicated additional screening environments to CIMMYT's centralized breeding program based in northwest Mexico. Because temperatures are similar between Tlaltizapan and late sowing in northwest Mexico, differences in genotypic ranking may be related to photoperiod effects associated with sowing times.

A better understanding of physiological basis of variation in heat tolerance traits.

In terms of improved understanding of physiological mechanisms, the main achievement of this work has been to prove a genetic link between CTD and heat tolerance by demonstrating their association in recombinant inbred lines (Table 9). Physiological and morphological data measured in the different experiments demonstrated an association between the following: grain yield under heat stress, aboveground biomass, canopy temperature depression, stomatal conductance, and leaf chlorophyll content. Together, these suggest that heat tolerance is source (assimilate) limited. Further studies are needed to establish whether the primary limitation is related to photoassimilation or assimilate utilization. Other work suggests that dark respiration and membrane thermostability are associated with heat tolerance (Reynolds et al. 1997), and in vitro work on starch synthase has shown the heat sensitivity of this enzyme to be a possible rate-limiting step to grain filling at temperatures above 25 C (Singletary et al. 1994).

Table 15. Canonical correlation analysis between yield of 60 advanced lines averaged across 15 international sites and traits measured in Obregon (March), Mexico, 1995-96.

Using CTD as an efficient method for evaluating heat tolerance in yield trials.

For the 60 advanced lines, average yields at 15 international sites was predicted equally well by either CTD or yield, measured at the Bread Wheat Breeding Station in Obregon with late sowing (Table 11). Data also indicated that CTD measured on 3-row plots was an equally good predictor of yield as those measured in yield plots, suggesting that the technique would be amenable to selections on small plots. Because a reliable yield estimate requires a plot about five times bigger than that needed for an estimate of CTD, the use of CTD instead of yield estimates should be considered as an efficient alternative. The CIMMYT Bread Wheat Program is considering using CTD as a selection criterion in preliminary yield trials for heat tolerance. Alternatively, both yield and CTD could be combined in a selection index as a more powerful indicator of heat tolerance, based on results from canonical regression analysis of this data.

Identifying genetic diversity for heat tolerance traits.

Field trials to screen material from the Indian and World wheat collections for new sources of heat-tolerance traits were encouraging (Table 12). Follow-up studies already underway offer the hope of providing suitable new sources of heat tolerance to breeding programs. If that becomes a reality, it may open the window of opportunity for extending the cropping cycle of wheat in vast areas of the world, such as south Asia, where high temperatures at the end of the winter cycle currently limit higher yield potential. Previous studies have shown membrane thermostability to be associated with heat tolerance (Shanahan et al. 1990; Reynolds et al. 1994). Material from the Indian collection expressed considerable genetic diversity for membrane thermostability when measured in heat acclimated seedlings. Seedling screening is an attractive option for breeders, because large numbers of plants can be tested within a short time frame. Using controlled environments for screening and offering greater reliability and repeatability in comparison to the more variable field environment also are possible.

Future use of germplasm and technology.

The germplasm distributed by the project can be used by breeders looking for new sources of heat tolerance and for strategic research. The RILs from two crosses can be used to test the genetic basis of heat tolerance for essentially any trait for which genetic diversity is detectable. Information generated by this project is, or shortly will be, available on a public database called the International Wheat Information System (Fox et al. 1996). The Indian Wheat Program is planning a project to evaluate physiological trait selection on germplasm from three major wheat growing regions in India where yields are reduced by heat stress: the northwest zone where approximately 4.5 million ha of wheat experience late heat stress; the northeast zone where 4 million ha experience hot and humid conditions; and approximately 2 million ha in the central and peninsular zones, which is continually hot.

Conclusions.

Recommendations to breeders on CTD as early generation selection criterion. Most of the data generated by the project point to CTD as a reliable indicator of heat tolerance that is robust enough to be used at different stages of selection. The recommendation has been delivered to breeders to compare methodologies involving CTD with conventional approaches using current breeding material.

Confirming reliable international testing sites for heat tolerance. Cluster analysis and correlation among sites indicate timely sowing in Tlaltizapan to be the best environment in Mexico for predicting yields in Bangladesh, northwest India, Sudan, and Nigeria. Northwest India also was indicated as a good site for heat-tolerance screening, and the current CIMMYT site for heat screening (Obregon, March sown) was confirmed to be reasonably representative.

CTD an efficient method for evaluating heat tolerance. International yield testing of 60 advanced lines suggest CTD to be a powerful predictor of heat tolerance in elite breeding material. CTD, which can be evaluated on relatively small plots, should be considered as a complementary and efficient means of assessing heat tolerance.

New genetic diversity for heat tolerance traits. Identification of genetic diversity for physiological traits associated with heat tolerance in germplasm collections has already led to follow-up work at CIMMYT. Heritability will be tested on lines expressing extremes of favorable traits, and evaluation for CTD is already underway on a much broader set of accessions from CIMMYT's seed bank. An economics group also has requested data from the project to help quantify the value of germplasm collection.

NARS-CIMMYT Heat-Tolerance Network strengthened. The NARS-CIMMYT heat-tolerance experimental network (IHSGE) originally established in 1990, has been able to improve its capability through human resource development and the establishment of research models. Research approaches developed in the project can be applied to almost any breeding objective where identification and selection of quantitative physiological traits may enable progress when empirical approaches are not meeting demands, such as in marginal environments where problems of breeding for abiotic stress have met with limited success to date.

Literature cited.

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Reynolds MP, Singh RP, Ibrahim A, Ageeb OAA, and Larque-Saavedra A. 1997. Evaluating Physiological Traits to Compliment Empirical Selection for Wheat in Warm Environments. Proc 5th Inter Wheat Conf, 10-14 June, 1996, Ankara, Turkey.

Reynolds MP, Acevedo, Ageeb OAA, Ahmed, Balota, Carvalho, Fischer RA, Ghanem, Hanchibal RR, Mann, Okuyama, Olugbemi L, Ortiz-Ferrara, Razzaque MA, and Tandon JP. 1992. Results of the 1st International Heat Stress Genotype Experiment. Wheat Special Report. CIMMYT, Mexico DF.

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