Australian Journal of Crop Science| Pages 60-68 | DOI: 10.21475/ajcs.21.15.09.sp-7 This article is published under a CC BY-IGO 3.0 licence as a fully Open Access paper, funded by the IAEA. All content is either published under licence or remains © IAEA 2021. Mutation breeding for heat and drought tolerance in tepary bean (Phaseolus acutifolius A. Gray) Ligia Carmenza Muñoz1*, Daniel G. Debouck2, Mariela Rivera2 , Jaime E. Muñoz 1, Deisy Alpala1, Fatma Sarsu3 and Idupulapati M. Rao2,4 1Universidad Nacional de Colombia- sede Palmira, Carrera 32, Chapinero, Palmira, Colombia 2Centro Internacional de Agricultura Tropical (CIAT), A.A 6713, Cali, Colombia 3International Atomic Energy Agency (IAEA), VIC, PO Box 100, 1400 Vienna, Austria 4Present address: Plant Polymer Research Unit, National Center for Agricultural Utilization Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA *Corresponding author: lcmunozf@unal.edu.co Abstract Tepary bean (Phaseolus acutifolius A. Gray) is more heat and drought tolerant than common bean (P. vulgaris L.). Four hundred mutant lines of two tepary accessions (G40068 and G40159) were generated by ethyl methane sulfonate (EMS) treatment. In preliminary studies of the M5 mutant lines under abiotic stress, three mutant lines (CMT 38, CMT 109, CMT 187) were selected from six mutated lines based on morpho-physiological traits and superior yield and advanced to the M6 generation. The M6 mutant lines were uniform and genetically stable. These mutant lines and their original (M0) parents were evaluated for heat and drought tolerance under greenhouse conditions. Their performance was evaluated for morpho-physiological attributes, seed yield and yield components. Under high temperature and drought conditions, the CMT 38 mutant (M6 line) and its original tepary (M0) accession (G40068) showed greater values of pod biomass, pod number and 100-seed biomass than the other lines tested. The CMT 109 and CMT 187 mutant lines and their G40159 original accession (M0) also showed the highest value of seed number under high temperature and drought conditions. This suggests that the previous screening performed during the population advancement of these mutant lines, based on morphological traits like growth habit, was not detrimental to the yield variables evaluated here. Under combined heat and drought conditions, different parameters could be incorporated into tepary breeding programmes, as selection criteria to screen genotypes for tolerance to heat and drought stress. These parameters included: chlorophyll (SPAD) readings, seed biomass, 100-seed biomass and seed number because they explain the observed variance in the principal component analysis. Two additional traits (root biomass and stem diameter) were also identified as useful attributes, based on univariate analysis. The mutant lines evaluated here offer potential for further improvement of tepary bean to high temperature and drought. Key Words: Abiotic stress; beans species; crop improvement; EMS mutagenesis; yield. Abbreviations: EMS, ethyl methane sulfonate; M6, Mutant line generation 6; PCA, principal component analysis; SPAD, Soil-Plant Analyses Development chlorophyll meter. Introduction more extensive and a thinner root system, better stomatal Global warming is responsible not only for global temperature control and more active para-heliotropism than common bean increase but also for region-specific increases or decreases in (Markhart, 1985; Bielenberg et al., 2003; Butare et al., 2012). It precipitation. This, in turn, has a negative impact on the is part of the tertiary gene pool of common bean and production systems of crops that are vital for improving food considered as a potential useful donor parent of drought and and nutritional security of people in developing countries. heat tolerance traits for common bean improvement, through Common bean, Phaseolus vulgaris L., is a valuable source of interspecific hybridization (Muñoz et al., 2004, Rao et al., protein, starch and other nutrients. Drought affects 60% of the 2013). Tepary could be a source of genes for the improvement dry bean production area worldwide (Beebe et al., 2008). of common bean through inter-specific crosses followed by Temperatures > 30 °C during the day or > 20°C during the backcrossing (Mejia et al., 1994). It could also serve as a night result in significant yield reduction of common bean valuable crop in itself, particularly for dryland environments (Rainey and Griffiths, 2005). where common bean is less adapted (Muñoz et al., 2006). An Tepary bean, Phaseolus acutifolius A. Gray, is a traditional crop of evaluation of tepary gene introgression showed that tepary desert and semi-arid regions of Mexico and southwestern USA DNA markers can be transferred to the interspecific progeny (Freeman, 1912; Nabhan and Felger, 1978). Renewed interest (Muñoz et al., 2004). However, success is limited to a lower- in tepary is due to its possession of many traits that enable it to than-expected percentage of genome contributed by tepary flourish in hot and dry regions: it is more heat tolerant at (Blair et al., 2012). Introgression of heat or drought tolerance biological tissue levels than common bean and it produces from tepary into common bean might be feasible through more leaves to compensate for reduced leaf size due to heat breeding, to generate elite lines that can tolerate up to stress (Lin and Markhardt, 1996). Tepary also has 4°C higher temperatures. However, most of the lines obtained come from a limited number of crosses between common and tepary beans (Muñoz et al., 2004). A diversity study of the 60 | Page tepary collection at the Genetic Resources Unit (GRU) of The 100-seed biomass/plant was higher under HT (16.5) as CIAT Colombia, using AFLP and microsatellite (SSR) markers compared to CT (15.7). Under HT, the value was higher for showed that the genetic base of the cultivated tepary accessions the tepary accession G40068 and mutant line CMT 38, and is narrow (Muñoz et al., 2006; Blair et al., 2012). A similar differences (P≤0.05) were observed between these genotypes conclusion was made after a study of variability of the seed and the others. Similar responses of these genotypes were storage proteins of wild and cultivated accessions of tepary observed under CT. The tepary accession G40068 and the (Schinkel and Gepts, 1988). A reason for this reduced genetic mutant line CMT 38 showed the highest values of 100-seed diversity might be the historic regression of tepary after the biomass. The differences were also significant (P≤0.05) when introduction of new watering technologies in Mesoamerica compared to the other genotypes (Table 1). Nodule formation after 1492 (Nabhan and Felger, 1978; Nabhan, 1985; Debouck, (nodules/plant) was lower under HT (3.0) as compared to CT 1992). Given this genetic extinction, future breeding rests on (12.5). The tepary accession G40068, showed a high mean exploiting the significant diversity provided by wild teparies value under HT. The mutant lines CMT 38 and CMT 109 and and related species (Muñoz et al., 2006), transformation (Dillen the tepary accession G40068 showed the highest values of et al., 1997) or by inducing variation via mutagenesis (explored nodule formation under CT (Table 1). The nodule biomass here). Traits that are particularly looked for in tepary are: (g/plant) was lower under HT (0.006) compared to CT (0.039). uniform red seed colour, erect growth habit and grouped pod Under HT conditions, the tepary accession G40068 showed maturity (Pratt and Nabhan, 1988). the highest nodule biomass, while the mutant line CMT 109 Mutagenesis has been used to broaden the genetic diversity of and the tepary accession G40159 showed the highest mean Phaseolus species (Ahloowalia et al., 2004; Blair et al., 2007; nodule biomass) under CT. This value was significantly Gwata et al., 2016). The results of chemical mutagenesis of different (P≤0.05) to the other genotypes evaluated (Table 1). common bean with ethyl methane sulfonate (EMS) in The results of the leaf biomass, stem biomass and roots morphological and physiological changes, as well as varietal biomass under HT (data not shown) showed significant development have been reported (Blair et al., 2007; Porch et differences (P≤0.001) between genotypes. The mutant line al., 2009). With the objective of genetic improvement of tepary CMT 38 (1.8 g/plant) and the G40068 (1.9 g/plant) tepary bean, a protocol was developed by Muñoz et al. (2013) for accession showed the lowest mean stem biomass compared to chemical mutation induction using EMS in two cultivated the CMT 187 (2.5 g/plant) and CMT 109 (2.2 g/plant) mutant tepary accessions (G40068 and G40159). lines and the G40159 (2.1 g/plant) tepary accession. Significant From the research discussed above, three further questions differences (P≤0.05) were observed between the two groups arise: (i) Why is it important to induce mutations in tepary? (ii) of genotypes. The highest mean values for root biomass were Which novel traits could be achieved by mutation induction in observed in the G40068 tepary accession (0.98 g/plant) and tepary? and (iii) How would the mutant lines be used in a the CMT 187 mutant line (0.95 g/plant). Significant differences breeding programme for tepary and/or common beans? The (P≤0.05) were observed between the means of these genotypes main objectives of the present study were to: i) evaluate tepary and the mean values of the mutant lines: CMT 38 (0.83 bean M6 mutant lines under conditions of high temperature g/plant), CMT 109 (0.76 g/plant) and the G40159 tepary and drought; and ii) identify heat and drought tolerant mutant accession (0.76 g/plant). No differences were observed lines that could serve as parents in breeding programmes that between genotype means for leaf biomass under HT (data not aim to improve heat and drought tolerance in common bean. shown). No difference between genotypes was observed for stem diameter (data not shown). Under CT, significant Results differences (P≤0.001) between genotypes were observed for leaf biomass and stem biomass, but not for stem diameter and Effect of high temperature (HT) on shoot and root root biomass. The CMT 187 mutant line showed the highest morpho-physiological characteristics means for leaf biomass and stem biomass and significant Table 1 presents the results of the effect of HT on genotypic differences (P≤0.05) were observed between this mutant line differences in yield components such as pod number, pod and the other genotypes (data not shown). In relation to biomass, seed number, and 100-seed biomass and also the physiological variables, under HT (results not shown), number and biomass of nodules (nodules variables). The significant differences (P≤0.001) were observed between the genotype parameter showed an effect (P≤0.01) on seed tepary parental (M0) accessions and the mutant lines for the number, pod number and nodule biomass under both efficiency of the photosystem II (QY) and the stomatal conditions of temperature HT and CT (control temperature). conductance. The CMT 187 (46.6) and CMT 109 (58.8) mutant Under HT conditions, the effect of genotype was significant lines and the G40159 (58.1) tepary accession showed the for pod biomass, seed biomass and nodule number. lowest mean stomatal conductance values as compared to the There was no difference between the mean values of pod CMT 38 (79.8) mutant line and the G40068 (66.7) tepary numbers for the genotypes under HT and CT conditions, but accession. Significant differences (P≤0.05) in stomatal the value was higher under CT (24.7 pods/plant) compared to conductance were only observed between CMT 187 and HT (20.5 pods/plant). The mutant line CMT 38 showed a high CMT38 mutant lines and G40068. No differences were mean value for pod number under HT and CT conditions. For observed between genotypes for the SPAD and leaf pod biomass, a similar trend was observed: the mean value was temperature variables. Under CT, significant differences higher under CT (17.2 g /plant) compared to HT (14.0 g (P≤0.001) were observed between genotypes for the SPAD /plant). The pod biomass of the CMT 38 mutant line was and the stomatal conductance tests. The CMT109 mutant line higher under both conditions of temperature and differences showed a SPAD mean higher (45.0, P≤0.05) than the CMT 38 (P≤0.05) were observed between the CMT 38 mutant line and mutant line (39.7). The G40159 tepary accession, the mutant the other mutant lines or parental accessions G40068 and line CMT 38 and the tepary M0 accession G40068 showed G40159 (Table 1). higher means (121.9, 118.5 and 107.6) for stomatal The seed number/plant was also higher under CT (88.9 conductance, respectively, as compared to the CMT 187 and seeds/plant) compared to HT (69.3 seeds/plant). Under both CMT 109 mutant lines (95.9 and 75.9), respectively. conditions of temperature, the CMT 109 mutant line showed Effect of temperature under drought and irrigated soil the highest seed number/plant. Differences (P≤0.05) were conditions on shoot and root morpho-physiological observed between this mutant line (CMT 109) and the other characteristics two mutant lines (CMT 38 and CMT 187) and differences were The results on the effect of HT and drought as compared to also observed between the mutant line CMT 109 and the tepary CT and irrigated conditions of soil on pod number, pod parental accession G40068 (Table 1). biomass, seed number, 100-seed biomass, nodule number and 61 | Page nodule biomass are shown in Tables 2 and 3. Soil conditions values under irrigated conditions, but there was no difference (drought or irrigated) showed significant effects (P≤0.01) on between the genotypes. Under drought conditions, no pod number /plant, seed number /plant, pod biomass /plant, difference was observed between the genotypes, for this 100-seed biomass/plant and nodule biomass /plant under variable (Table 3). The pod biomass (g/plant) was also lower both temperature conditions. The effect of soil condition was under drought (11.2) as compared to irrigated conditions (23.1) also significant (P≤0.01) for the nodule number/plant, under for all genotypes. Under irrigation, the mutant line CMT 38 HT treatment. and the tepary accession G40068 showed the highest values. There were significant differences (P≤0.05) between the Effect of HT under drought and irrigated conditions of mutant lines CMT 38, CMT 109 and CMT 187 and the tepary soil accession G40159. The mutant line CMT 38 also showed the Under HT the pod number/plant was lower under drought highest value under drought conditions. There were significant (14.5) as compared to the irrigated treatment (26.6) for all differences (P≤0.05) between this mutant line and the other genotypes (Table 2). Under drought conditions the mutant line genotypes (Table 3). The seed number/plant was lower under CMT 38 showed the highest value as compared to other drought as compared to irrigated conditions (56.8 vs 121) for genotypes. Significant differences (P≤0.05) were observed all genotypes (Table 3). Under drought treatment, the mutant between this mutant line, the mutant line CMT 109 and the line CMT 109 and the tepary accession G40159 showed higher tepary accession G40068 (Table 2). Under irrigated conditions, values. Significant differences were observed between these two mutant lines (CMT 38 and CMT 109) showed the highest genotypes and the others (Table 3). Under irrigated conditions, values. Significant differences (P≤0.05) were observed the mutant line CMT 109 showed the highest value. There between these mutant lines and the mutant line CMT 187 that were significant differences (P≤0.05) between this mutant line, showed the lowest pod number (23.4) (Table 2). For pod the mutant line CMT 38 and the tepary accession G40068. In biomass (g/plant), the value was also lower under drought (9.6) relation to the 100-seed biomass (g/plant) variable, there was than the irrigated treatment (18.4) for all genotypes. The a modest difference between drought (16.2) and irrigated mutant line CMT 38 showed the highest value under drought treatments (15.5) for all genotypes. Under drought and and irrigated conditions. Under drought conditions, significant irrigated conditions, the mutant line CMT 38 and the G40068 differences (P≤0.05) were observed between the mutant lines showed the highest value for 100-seed biomass. Significant CMT 38 and CMT 109, which showed the lowest value. Under differences (P≤0.05) were observed between these two irrigated conditions, significant differences (P≤0.05) were genotypes and the others under both conditions (Table 3). The observed between the mutant line CMT 38 and CMT 187 and number of nodules/plant increased considerably under CT and the tepary accession G40159. The seed number/plant was irrigated conditions as compared to results obtained under HT lower under drought (45.9) compared to irrigated conditions (Table 1). The value was 23.3 under irrigated conditions as (92.7) for all genotypes (Table 2). The tepary parental accession compared to 1.62 under drought for all genotypes. The mutant G40159 showed the highest value under drought conditions. lines CMT 38 and CMT 109 and the G40068 tepary accession Significant differences were observed between this accession, showed the highest values (Table 3). The differences between the mutant line CMT 38 and the tepary accession G40068. all genotypes were not significant. Under irrigated conditions, Under irrigated conditions, the mutant line CMT 109 showed the CMT 109 mutant line showed the highest nodule biomass, the highest value. Significant differences (P≤0.05) were and there were significant differences (P≤0.05) between this observed between this mutant line, the mutant line CMT 187 mutant line and the other genotypes tested (Table 3). Under and the tepary accession G40068. There was a small difference both temperature conditions and considering drought and in 100-seed biomass (g/plant), between drought (17.3) and irrigation treatments for all variables and genotypes, the values irrigated conditions (16.1) for all genotypes. Under drought were lower under drought, except for the 100-seed biomass. In conditions, the tepary accession G40068 and the mutant line this case, the value was higher under drought (Table 2). Under CMT 38 showed the highest value of 100-seed biomass. There HT and CT, significant differences (P≤0.05) were observed were significant differences (P≤0.05) between these genotypes between the values obtained under drought and irrigation for and the mutant lines (CMT 109, CMT 187) and the tepary all evaluated variables and genotypes. In relation to 100-seed accession G40159 (Table 2). Under irrigated conditions, the biomass, under CT conditions, significant differences (P≤0.05) same genotypes also showed the highest 100-seed biomass were observed between drought and irrigated conditions for values. Significant differences (P≤0.05) were observed the mutant lines and G40159 tepary accession. Under CT between all genotypes. The nodule number/plant was very low conditions, significant differences (P≤0.05) were observed under drought and irrigated conditions (1.01 and 5.10, only with the mutant lines (Table 3). respectively), for all genotypes evaluated. Under irrigated A significant strain effect (Rhizobium tropici or Bradyrhizobium conditions, the G40068 tepary parental accession showed the spp.) was observed only for the number of nodules /plant (data highest number. There were significant differences (P≤0.05) not shown). between this tepary accession, the mutant line CMT 38 and the other genotypes (Table 2). Under drought conditions, all Multivariate analysis of shoot and root morpho- genotypes showed a lower level of nodule formation. With physological variables respect to nodule biomass (g/plant), only the G40068 tepary accession showed a higher mean (0.024) under irrigated Effect of temperature conditions, and significant differences (P≤0.05) were observed Principal component analysis was performed to identify the between this accession and the other genotypes (Table 2). major components (i.e. principal components) that could explain much of the total variation observed in the data. The Effect of CT under drought and irrigated conditions of PCA showed that under HT and CT, the first four components soil represented 73 and the 75 % of the total variance, respectively The results obtained with the five genotypes under normal (Table 4). Under HT, the first component accounted for 46% (control) conditions of temperature (CT) in a greenhouse are of the variance, the second 11%, the third 9% and the fourth shown in Table 3. 7%, while under CT, the first component accounted for 53% For each treatment (drought or irrigated), all variables were of the variance, the second 8%, the third 7 %, and the fourth higher under CT as compared to HT. The pod number/plant 7%. The dominance of these four components suggests that was lower under drought (18.3) compared to irrigated they contained the main variables that discriminate between conditions (31.2) under CT for all genotypes. The mutant line the genotypes evaluated under HT and CT conditions (Table CMT 38 and the tepary accession G40068 showed the highest 4). 62 | Page Table 1. Mean values of pod number, pod biomass, seed number, 100 seed biomass, nodules number and nodule biomass for the M6 mutant lines (CMT 38, CMT 109 and CMT 187) and their original M0 tepary accessions (G40068 and G40159), grown in greenhouses under high temperature (HT) and controlled temperature (CT) conditions. Pods/plant Pod biomass, Seeds/plant 100 seeds Nodules/plant Nodule biomass, g/plant biomass g/plant HT CT HT CT HT CT HT CT HT CT HT CT Genotype CMT 38 22.2 a 25.6 a 15.2 a 18.2 a 69.9bc 83.5c 18.0 a 18.0 a 3.6 a 15.3 a 0.006 ab 0.029 b CMT 109 21.2 a 23.9 a 13.8 b 16.6 b 75.2 a 95.8 a 14.4 d 13.4 d 2.7 a 14.4 a 0.005 ab 0.054 a CMT 187 18.9 a 24.7 a 13.4 b 16.9 b 68.3 c 91.1 b 15.9 b 15.2 b 2.8 ab 9.4 a 0.005 ab 0.029 b G40068 20.1 a 24.9 a 14.0 b 17.3 b 60.2 d 78.0 d 18.9 a 17.3 a 5.1 a 14.1a 0.013 a 0.031 b G40159 20.3 a 24.7 a 13.5 b 16.9 b 73.1ab 96.3 a 15.2 c 14.8 c 1.1 b 9.1a 0.003 a 0.054 a Mean 20.5 24.8 14.0 17.2 69.3 88.9 16.5 15.7 3.0 12.5 0.006 0.0394 *Means between a yield component and treatment not followed by the same letters are significantly different at P≤ 0.05 according to Duncan's multiple rang test. Table 2. Mean values of pod number, pod biomass, seed number, 100 seed biomass, nodules number and nodules biomass for the M6 mutant lines (CMT 38, CMT 109 and CMT 187) and their original M0 tepary accessions (G40068 and G40159), grown in a greenhouse under high temperature (HT) and irrigated and drought conditions. Pods/plant Pod biomass, Seeds /plant 100 seed biomass, Nodules/plant Nodule biomass, g/plant g/plant g/plant Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Genotype CMT 38 28.8 a 15.6 a 20.3 a 10.1 a 95.5 ab 44.3 b 17.5 b 19.0 a 5.9 ab 1.3 a 0.0117 b 0.0017 a CMT 109 28.5 a 13.9 b 18.5 ab 9.1 b 103.0 a 47.3 a 13.9 e 15.5 c 4.6 bc 0.9 a 0.0083 b 0.0018 a CMT 187 23.4 b 14.5 ab 17.4 a 9.4 ab 88.8 bc 47.7 a 15.8 c 16.4 b 4.1 bc 1.5 a 0.0078 b 0.0017 a G40068 26.5 ab 13.8 b 18.5 ab 9.6 ab 80.2 c 40.2 c 18.7 a 19.4 a 9.4 a 0.8 a 0.0233 a 0.0009 a G40159 25.7 ab 14. 9 ab 17.4 b 9.7 ab 96.0 ab 50.2 a 14.7 d 16.1 b 1.6 c 0.6 a 0.0033 b 0.0008 a Mean 26.6 14.5 18.4 9.6 92.7 45.9 16.1 17.3 5.1 1.02 0.0108 0.00138 *Means between a yield component and treatment not followed by the same letters are significantly different at P≤ 0.05 according to Duncan's multiple rang test. Table 3. Mean values of pod number, pod biomass, seed number, 100 seed biomass, nodule number and nodules biomass and number of pods for the M6 mutant lines (CMT 38, CMT 109 and CMT 187) and their original M0 tepary accessions (G40068 and G40159), grown in a greenhouse under controlled temperature (CT) and drought and irrigated conditions. Pods/plant Pod biomass, Seeds /plant 100 seeds biomass Nodules/plant Nodule biomass g/plant g/plant Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Irrigated Drought Genotype CMT38 32.4 a 18.7 a 24.4 a 11.9 a 113.6 b 53.4 c 17.7 a 18.5 a 28.9 a 1.6 a 0.053 b 0.007 a CMT109 30.2 a 17.7 a 22.1 c 11.0 b 128.8 a 62.7 a 13.4 c 13.7 c 27.0 a 1.9 a 0.103 a 0.008 a CMT187 30.9 a 18.4 a 22.6 bc 11.2 b 123.4 b 58.8 b 15.2 b 15.3 b 17.7 a 1.1 a 0.054 b 0.004 a G40068 31.6 a 18.3 a 23.7 ab 10.9 b 108.9 b 47.1 d 17.5 a 18.7 a 25.9 a 2.3 a 0.056 b 0.005 a G40159 31.0 a 18.4 a 22.6 bc 11.1 b 130.6 a 61.9 a 13.7 c 14.7 b 16.9 a 1.2 a 0.055 b 0.004 a Mean 31.2 18.3 23.1 11.2 121.1 56.8 15.5 16.2 23.3 1.62 0.064 0.005 *Means between a yield component and treatment not followed by the same letters are significantly different at P≤ 0.05 according to Duncan's multiple rang test. 63 | Page Table 4. Eigen values and per cent of total variation and component matrix for the principal component axes - high temperature (HT) and control temperature (CT)under greenhouse conditions. Principal components CP1 CP2 CP3 CP4 HT Eigen value 7.30 1.79 1.41 1.05 Variance proportion 0.46 0.11 0.09 0.07 Cumulative proportion variance 0.46 0.57 0.66 0.73 Shoot biomass (g/plant) 0.365 -0.395 0.027 0.019 Pods biomass (g/plant) 0.358 -0.160 0.079 -0.032 Stem biomass (g/plant) 0.296 -0.154 -0.179 0.131 Stem diameter (mm) 0.150 -0.024 0.025 0.368 Leaf biomass (g/plant) 0.322 -0.037 -0.038 0.126 Pod number 0.336 -0.067 0.003 -0.132 Seed total biomass (g/plant) 0.354 -0.008 0.086 -0.054 100-seed biomass -0.103 0.361 0.323 -0.234 Seed number 0.352 -0.138 -0.029 0.032 Root biomass (g/plant) 0.221 0.224 -0.187 0.128 Nodule biomass 0.173 0.553 0.086 0.007 Nodule number 0.174 0.508 0.172 0.105 SPAD chlorophyll content -0.176 -0.009 0.068 0.652 Leaf stomatal conductance 0.009 0.035 0.577 -0.101 Efficiency of photosystem II 0.108 -0.399 0.369 -0.317 Leaf temperature (°C) 0.002 0.204 -0.549 -0.435 CT Eigen value 8.48 1.26 1.19 1.08 Variance proportion 0.53 0.08 0.07 0.07 Cumulative proportion variance 0.53 0.61 0.68 0.75 Shoot biomass 0.337 -0.078 -0.061 -0.019 Pod biomass 0.329 -0.127 -0.031 -0.036 Stem biomass (g/plant) 0.299 0.060 -0.223 0.049 Stem diameter (mm) 0.188 0.034 -0.200 0.302 Leaf biomass (g/plant) 0.311 0.037 -0.049 0.003 Pods number 0.308 -0.103 -0.086 -0.065 Seed total biomass 0.326 0.183 0.001 -0.045 100-seed biomass -0.056 -0.660 0.431 0.132 Seeds number 0.328 0.069 -0.164 -0.078 Roots biomass 0.253 -0.040 -0.120 -0.101 Nodules biomass 0.223 0.271 0.306 0.389 Nodules number 0.168 0.297 0.513 0.454 SPAD chlorophyll content -0.259 0.217 -0.145 0.022 Leaf stomatal conductance 0.11 -0.152 0.392 0.357 Efficiency of photosystem II 0.046 0.493 0.255 -0.405 Leaf temperature (°C) -0.135 -0.098 -0.267 0.463 Values in bold indicate the traits that were decisive in genotype differentiation. The traits that separated genotypes in the first component first component accounted for 39% of the variance, the second included shoot biomass, pod biomass, total seed biomass and 14 %, the third 10%, the fourth 9% and the fifth 7%; while seed number under HT and CT. Under HT, only pod under irrigated conditions, the first component accounted for number/plant differed between genotypes. The traits that 30% of the variance, the second 21%, the third 14 %, the contributed most to the discrimination in the second fourth 9% and the fifth 6%. The dominance of these five component were: nodule biomass, nodule number under HT components suggests that they contained the main variables and 100-seed biomass and the efficiency of photosystem II that discriminate the genotypes evaluated under drought and under CT. In the third component, the separation of genotypes irrigation (Table 5). was mainly due to leaf stomatal conductance under HT and The traits that discriminated genotypes in the first component CT, leaf temperature under HT and nodule number under CT. included shoot biomass, pod biomass, pod number and seed In the fourth component, the main traits were: SPAD readings total biomass under drought and irrigated treatments. The under HT and nodule number and leaf temperature under CT traits that contributed most to the discrimination in the second (Table 4). component are 100-seed biomass under drought conditions and leaf biomass, and seed number and nodule biomass under Effect of HT and CT under drought and irrigated irrigated conditions. In the third component, the differences conditions between genotypes were mainly due to the root biomass under drought and irrigated conditions; and the nodule biomass and Effect of HT under drought and irrigated conditions nodules number under drought conditions. Under irrigated The PCA showed that, under drought and irrigation, the first conditions, differences were due to the efficiency of five components represented 79% and 80 % of the total photosystem II and leaf temperature. variance, respectively (Table 5). Under drought conditions, the 64 | Page Table 6. Eigen values and per cent of total variation and component matrix for the principal component axes - control temperature (CT) under drought and irrigated conditions in a greenhouse. Principal components CP1 CP2 CP3 CP4 CP5 CT -Drought condition Eigen value 6.67 2.10 1.75 1.41 1.18 Variance proportion 0.42 0.13 0.11 0.09 0.07 Cumulative proportion variance 0.42 0.55 0.66 0.75 0.82 Shoot biomass (g/plant) 0.368 0.179 -0.020 0.079 -0.059 Pods biomass (g/plant) 0.348 0.238 -0.070 0.143 -0.038 Stem biomass (g/plant) 0.312 -0.077 0.227 0.057 -0.093 Stem diameter (mm) 0.162 -0.062 -0.061 -0.506 -0.040 Leaves biomass (g/plant) 0.302 0.033 0.012 -0.223 -0.091 Pods number 0.343 0.120 -0.150 0.154 -0.040 Seed total biomass (g/plant) 0.332 0.288 -0.103 0.130 -0.023 100-seed biomass -0.045 0.573 -0.314 0.168 0.057 Seeds number 0.319 -0.234 0.209 -0.021 -0.059 Roots biomass (g/plant) 0.136 0.170 0.210 -0.334 0.532 Nodules biomass -0.189 0.334 0.459 0.181 0.195 Nodules number -0.149 0.270 0.549 0.104 -0.190 SPAD chlorophyll content 0.172 -0.293 0.371 0.317 0.228 Leaf stomatal conductance -0.033 0.316 0.228 -0.340 0.439 Efficiency of photosystem II 0.292 -0.042 0.229 -0.080 -0.175 Leaf temperature (°C) 0.053 -0.129 -0.021 0.465 0.579 CT-irrigated conditions Eigen value 4.85 3.43 1.76 1.64 1.01 Variance proportion 0.30 0.22 0.11 0.10 0.06 Cumulative proportion variance 0.30 0.52 0.63 0.73 0.79 Shoot biomass 0.439 -0.091 0.021 0.019 0.100 Pods biomass 0.365 -0.279 0.083 0.061 0.008 Stem biomass (g/plant) 0.280 0.346 -0.139 -0.063 0.029 Stem diameter (mm) 0.250 0.100 -0.107 -0.099 -0.517 Leaves biomass (g/plant) 0.330 0.166 -0.036 -0.050 0.349 Pods number 0.401 -0.182 0.045 0.011 0.049 Seed total biomass 0.307 -0.353 0.011 0.052 -0.113 100-seed biomass -0.029 -0.492 0.027 -0.067 -0.179 Seeds number 0.319 0.284 -0.306 0.146 0.113 Roots biomass -0.108 0.272 -0.114 0.413 -0.353 Nodules biomass 0.092 0.244 0.506 0.229 -0.014 Nodules number 0.021 0.022 0.701 0.028 -0.090 SPAD chlorophyll content 0.128 0.291 -0.196 -0.195 0.081 Leaf stomatal conductance -0.135 -0.188 -0.115 0.387 0.331 Efficiency of photosystem II -0.103 0.144 0.363 -0.421 0.508 Leaf temperature (°C) 0.005 -0.007 0.110 0.625 0.117 Values in bold indicate the traits that were decisive in genotype differentiation. In the fourth component, the main traits were: stem diameter number under drought and irrigated conditions, and seed total under drought and irrigated conditions and leaf temperature biomass under drought conditions. The traits that contributed under drought, stem diameter and nodule number under most to the discrimination in the second component were: irrigated conditions. In the fifth component, the main traits 100-seed biomass under both drought and irrigation, stem were the SPAD readings and stem diameter under drought and biomass; and total seed biomass under irrigated conditions. In irrigated conditions, respectively (Table 5). the third component, the separation of genotypes was mainly due to the nodule biomass and nodule number under drought Effect of CT under drought and irrigated conditions and irrigated conditions. In the fourth component, the main The PCA showed that under drought and irrigated conditions, traits were: leaf temperature under drought and irrigated the first five components represented 82% and the 79% of the conditions and the stem diameter under drought conditions. total variance, respectively (Table 6). Under drought Under irrigated conditions, the main traits were root biomass, conditions, the first component accounted for 42% of the leaf stomatal conductance and the efficiency of photosystem variance, the second 13 %, the third 11%, the fourth 9% and II. In the fifth component the main traits were: root biomass the fifth 7%, while under irrigated conditions, the first and stem diameter under drought and irrigated conditions, component accounted for 30% of the variance, the second respectively (Table 6). 22%, the third 11%, the fourth 10% and the fifth 6%. The dominance of these five components suggests that they contain the main variables that discriminate the genotypes evaluated under drought and irrigated conditions (Table 6). The traits that separate genotypes in the first component included shoot biomass, pod biomass, pod number and seed 65 | Page Discussion demonstrating the advantages that this species has over P. vulgaris under terminal drought stress (Rao et al., 2013, 2017). In all experiments the stress treatments (high temperature and The PCA analysis under heat and drought conditions of all drought) were effective as all genotypes performed less well traits evaluated showed that the four first components (CP1, under these stresses compared to control conditions. The CP2 CP3 CP4) represented 73% of the total variance under HT treatments were also effective in discriminating between good conditions (Table 4). The SPAD readings appeared to explain performing and poor performing lines in stress treatments, the variance in the PCA under HT but not under CT. Three with the mutant line CMT 38 showing superior characteristics variables: root biomass, stem diameter and biomass do not in pods/plant and pod biomass/plant compared to its parental explain the variance in the analysis performed under HT or CT. line G40068 and other mutant lines. B u t t h e s e v a r i a b l e s a p p e a r t o e x p lain the variance in the PCA There are three main points for discussion: (i) Why is it analysis, when the drought or irrigated conditions of the important to induce mutations in tepary? (ii) Which novel traits experiment were considered under HT and CT conditions are sought from mutagenesis in tepary bean? and (iii) How (Tables 5 and 6). This indicates that these traits can be used to would the mutant lines be used in a breeding programme for select better adapted genotypes under drought conditions. A tepary and / or common bean improvement? On the first greater capacity to develop roots that go deep into the soil can point, although genetic variability among tepary wild provide a better adaptation to conditions of water accessions is high (Muñoz et al., 2006; Blair et al., 2012), these stress (White and Castillo, 1992; Polania et al., 2009, 2016). are in general more heat and drought tolerant, they also show There is a direct correlation between drought and heat stresses, agronomic disadvantages such as since during heat stress water availability can be at a deficit indeterminate growth habit and very small seeds. The chemical caused by the high temperature (Omae et al., 2012). It is mutagen (EMS) was used in this study to obtain variability in necessary to identify specific morpho-physiological traits that the cultivated accessions (Muñoz et al., 2013). The contribute to improved resistance to combined stresses of heat introduction of characteristics, such as an indeterminate erect and drought in beans, and that could be useful as selection growth habit, is necessary in the case of large-scale production, criteria in breeding. to facilitate mechanical harvesting and mechanical weed On the last point, how would the mutant lines be used in a removal. This growth habit was also obtained in common breeding programme for tepary and/or common beans, using breeding and screening, because it does not exist beans? These genetically stable mutant lines, which were in traditional varieties. In terms of seed colour, it would selected for their phenotypic characters, and/or for their probably be necessary to introduce a uniform red seed colour, tolerance to HT and drought, could have two possible uses in the colour preferred by the consumers of Central America. On a bean breeding programme. First, these mutant lines could be the second point, in the first generations of the mutant included in interspecific crosses, between P. vulgaris and P. populations, lines with deleterious phenotypic variations were acutifolius, to try to introgress these physiological characteristics observed: dwarf plants, plants with apparent virosis, yellowing, to common bean. Second, they could be used for the or sterile plants. The selection of mutant lines, presenting improvement per se of the species P. acutifolius. desirable characteristics: plants with a determinate growth habit and/or a larger seed size, was carried out, through the Materials and methods generational development of the mutant lines (data not shown). In the present study, the CMT 38 and CMT 187 tepary Plant materials mutant lines had larger seed size, as reflected by their higher We evaluated two accessions of tepary bean, P. acutifolius A. values of 100-seed biomass, compared to the original Gray (G40068 from Arizona, USA and G40159 from Sonora, accessions (G40068 and G40159) under CT conditions (Table Mexico, and three mutant M6 lines (CMT 38, CMT 109 and 1). Gwata et al., 2016 showed that genotype does not affect CMT 187), which were uniform and genetically stable. The seed size of three mutant tepary bean genotypes. The seed size mutant line CMT 38 was obtained from the G40068 accession, was smaller, as compared to that reported for tepary in other while CMT 109 and CMT 187 mutant lines were obtained from studies. The analysis of a common bean variety and its 34 the G40159 accession. . These mutant lines were selected based NaN3-induced mutants (M6 generation) showed that the seed on two key traits: large seed size and/or a determinate growth yield and yield components differed among the 34 common habit and superior yield from previous experiments, where M5 bean mutants (Wang et al., 2010). mutant lines were evaluated under drought and high Heat and drought reduce yield and quality and restrict the temperature conditions in greenhouse tests (data not shown). geographic adaptation of common beans (Rainey and The evaluated tepary mutant lines were obtained from a Griffiths, 2005; Beebe et al., 2008). The HT treatment applied protocol established by Muñoz et al. (2013), using ethyl to common bean genotypes reduced the yield components: methane sulfonate (EMS). seed number, pod number, mean seed weight and seeds/pod (Rainey and Griffiths, 2005b). In contrast, tepary accessions Experimental conditions that produce substantial numbers of pods and seeds under very The experiments were conducted at the International Center HT conditions or drought were reported (Rainey and Griffiths, for Tropical Agriculture (CIAT) in Palmira, Colombia, located 2005; Rao et al., 2013; Polania et al., 2016). The mutant lines at latitude 3° 29' N, longitude 76° 21' W and 965m above sea evaluated here under HT and drought conditions, showed a level. yield higher or comparable to the original accessions G40068 Two experiments were conducted simultaneously in two and G40159. This indicates that screening based on separate greenhouses, to evaluate the M6 mutant lines and the morphological traits is useful and not detrimental to seed yield two tepary parental (M0) accessions (G40068 and G40159) to and yield components. G40068 and G40159 were outstanding high temperature and drought stress conditions. Experiment 1 in their adaptation to terminal drought stress. The superior was carried out with a high temperature treatment (HT) in a performance of these accessions was associated with their greenhouse. Experiment 2 was carried out at normal (control ability to mobilize photosynthates from leaves and stems to temperature) conditions (CT) in another greenhouse. Both developing grains. Tepary was superior to common bean in experiments included three replicates and were conducted combining several desirable traits that contribute to adaptation using pots with a Mollisol soil from Palmira. The seeds were to terminal drought stress (Rao et al., 2013). Under rainfed germinated in wet paper towels and uniform seedlings were conditions, these two accessions yielded more than any elite selected for transplanting into pots. The plants of each line or accession of P. vulgaris under terminal drought, thus accession and of the mutant tepary lines from the two experiments were inoculated at 10 days after sowing with 6668 || PPaaggee Rhizobium tropici (strain CIAT 899) or Bradyrhizobim spp. (strain (SPAD readings, seed biomass, 100-seed biomass and seeds CIAT461) as is normal practice. To obtain high temperature number) explained a major part of the variance under heat and conditions and simulate the changes in temperature between drought conditions, and suggests that these traits and two day and night, conditions in the greenhouse were modified others (root biomass and stem diameter, identified from the using heaters, ventilation and thermostats. The HT treatment univariate analysis) could be incorporated into tepary breeding was set at 29±5 ⁰C during the day and >24 ⁰C during the night, programmes as selection criteria to screen the tepary with an average relative humidity of 65%. The maximum accessions and their mutant lines, for combined tolerance to day/night temperatures of the greenhouse for normal heat and drought stresses. conditions (CT) were set at day/night of 30°C /20°C. Data on Mutation breeding has potential to generate phenotypic and relative humidity and temperature were monitored with genotypic variations in tepary bean that can be exploited by thermo-hygrometers that registered the parameters every 15 plant breeders in the development of new cultivars with minutes. The mean and minimal/maximal temperatures were improved adaptation to heat and drought stress. calculated per day. Acknowledgements Plants were grown in optimal conditions of soil moisture (80% field capacity) for 10 days and were then submitted to their We acknowledge the partial support from the International respective treatment with soil moisture, either at 80% field Atomic Energy Agency (IAEA) under the CRP23029 for the capacity (irrigated) or 40% (drought). In both cases, the pots project on “Heat effects in tepary beans and its relatives”. We were weighed twice a week and water was added to bring back thank the late Mr. Orlando Toro at the Genetic Resources Unit the required moisture level. of CIAT, for his technical support during the process of selection of the accessions and mutant lines of tepary bean. Measurement of shoot and root morpho-physiological characteristics References Plants were harvested between 80 to 86 days under drought and at 100 days under irrigated conditions. At the mid-pod Ahloowalia BS, Maluszynski K, Nichterlein K (2004) Global filling growth stage, the following non-destructive impact of mutation derived varieties. 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