Acta agriculturae Slovenica, 117/2, 1–14, Ljubljana 2021 doi:10.14720/aas.2021.117.2.2079 Original research article / izvirni znanstveni članek Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Sintayehu ADMAS 1,2* , Teklehaimanot HAILESELASSIE 2 , Kassahun TESFAYE 2 , Eleni SHIFERAW 1 , and K. Colton FLYNN 3 Received February 04, 2021; accepted March 29, 2021. Delo je prispelo 4. februarja 2021, sprejeto 29. marca 2021. Evaluation of Ethiopian chickpea (Cicer arietinum L.) geno- types for frost tolerance Abstract: Frost stress is one of the most significant abio- tic factors affecting chickpea (Cicer arietinum L.) production in the Ethiopian highlands. To investigate the frost tolerance of chickpea, 673 genotypes were characterized using an augmen- ted design at Bakelo, Debre Berhan, Ethiopia for two years. A significant (p < 0.01) variability amongst genotypes was recor- ded for all agronomic traits considered. A considerable num- ber of accessions better performing over the frost susceptible genotypes were identified for agronomic traits. Stem/leaf pig- mented genotypes showed a better reaction for frost stress than non-pigmented genotypes. The majority of black seeded chic- kpea adapted well under frost stress when compared to with brown and white seeded genotypes. According to the freezing tolerance rate (FTR) and plant survival rate (SR), 83 (12.3 %) and 85 (12.6 %) genotypes were identified as frost tolerant. The- re was a strong correlation (p < 0.01) in grain yield with FTR, SR, seed shriveling score, stem/leaf pigmentation and seed co- lor. Based on our findings, Ethiopian chickpea landraces has a good genetic potential for frost resistance traits for use in bre- eding programs. Key words: chickpea; Ethiopian landraces; frost survival rate; frost tolerance; germplasm characterization 1 Ethiopian Biodiversity Institute, PO Box 30726 Addis Ababa, Ethiopia 2 College of Natural Sciences, Addis Ababa University, P .O. Box 3285, Addis Ababa, Ethiopia 3 USDA-ARS, PA, Grassland Soil and Water Research Laboratory, 808 East Blackland Road, Temple, TX 76502, USA: * Correspondence: sintayehu.admas@ebi.gov.et Ovrednotenje etiopskih genotipov čičerke (Cicer arietinum L.) za toleranco na mraz Izvleček: Mrazni stres je eden izmed najznačilnejših abiot- skih dejavnikov, ki vpliva na pridelavo čičerke (Cicer arietinum L.) v etiopskem višavju. Za preučevanje tolerance na mraz je bilo v izboljšanem poskusu analiziranih 673 genotipov čičerke v Debre Birhan, Etiopija, v obdobju dveh let. Med genotipi je bila ugotovljena značilna variabilnost (p < 0,01) za vse preučevane agronomske lastnosti. Prepoznano je bilo znatno število akcesij, ki so se izkazale boljše v preučevanih agronomskih lastnostih kot tiste občutljive na mraz. Genotipi z obarvanimi stebli ali listi so se boljše odzvali na mrazni stres kot neobarvani. Večina genotipov čičerke s črnimi semeni je bila bolje prilagojena na mrazni stres v primerjavi s tistimi z rjavimi ali belimi semeni. Glede na toleranco na mraz (FTR) in preživetje rastlin (SR), je bilo 83 (12,3 %) in 85 (12,6 %) genotipov na mraz toleratnih. Ugotovljena je bila močna povezava (p < 0,01) med pridelkom semena in FTR, SR, nagubanostjo semena, obarvanostjo steb- la in listov ter barvo semena. Na osnovi teh ugotovitev imajo etiopske tradicionalne sorte čičerke dober genetski potencial za odpornost na mraz in so lahko uporabne v žlahtniteljskih programih. Ključne besede: čičerka; tradicionalne etiopske sorte; lastnosti tolerance na mraz; ovrednotenje genotipov Acta agriculturae Slovenica, 117/2 – 2021 2 S. ADMAS et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) cultivation and uti- lization are profoundly notable within Ethiopian cul- ture and produced by smallholder farmers under rain- fed condition (Ferede et al., 2018). The cultivation is so profound that chickpea production in Ethiopia is one of the most widespread legume in terms of both area and volume. Across Ethiopia chickpea cultivation occupies ~1,620,497.30 hectares of land annually with an estima- ted production of 30,113,480570 kg (CSA, 2019). Both the land dedicated to chickpea production and the vo- lume of production itself has been increasing over the last decade in Ethiopia (Fikre & Bekele, 2020; Fikre et al., 2018). Ethiopia is thus the largest producer, consumer, and exporter of chickpea in Africa, and is among the top ten most vital chickpea producers in the world (FA- OSTAT , 2020). Chickpea production is suited to areas ha- ving vertisol-dominated soil with an altitudinal range of 1400 to 2300 meters above see level (Bejiga et al., 1996). Nevertheless, it is cultivated across a wide selection of zone (Fikre et al., 2018). Moreover, Ethiopia is conside- red to be the second greatest diversity hotspot of chick- pea amongst major chickpea growing countries (Van der Maesen, 1987). Taking into consideration both immense variability among the chickpea germplasm and many ag- roecological zones as well as the increased demand for animal feed and processed foods (Fikre et al., 2020; Mu- oni et al., 2019; Shiferaw &Hailemariam, 2007), Ethiopia features great potential to expand chickpea production within the highland areas if the chickpea varieties are re- sistance to frost stress. Chickpea is important for Ethiopian highland culti- vation and is preferably sown in early- to mid-September. Previously, mid-August was considered the appropriate sowing date, but due to the “belg” rainy season, chick- pea cultivation was heavily impacted by root rot. Root rot issues can be avoided by planting in mid-September, leading to higher yields. However, the later sowing date presents a new issue, due to the elevation of highlands, which is frost stress. The frost stress takes place late in the podding and flowering stages. Frost stress during these stages causes issues such as flower abortion, poor pod set, and impaired pod filling, leading to a drastic reduction in yield and quality (Croser et al., 2003). These stressors can be classified as chilling (0 o C to 12 o C) or freezing/frost (< 0 o C) temperatures (Gogoi et al., 2018; Toker et al., 2007). Moreover, temperatures lower than 10 °C at flowering can reduce grain yield by 15–20 % (Chaturvedi et al., 2009). Therefore, the need for improving frost-tolerance in chickpea has become evident which requires characte- rization of chickpea germplasm for frost tolerance. Determining the nature of genetic diversity and variability existing among chickpea genotypes for frost resistance is mandatory to identify promising genotypes that are productive in Ethiopian highlands with late sowing dates. However, few studies have been condu- cted so far in this regard. Hence, research is needed to further understand the optimal utilization of landraces as sources of novel traits for frost resistant chickpea va- riety development. Therefore, the aim of this research is to identify chickpea genotypes that are both highly pro- ductive and frost resistant through use of field screening of genotypes for frost-tolerance. The long-term goal is to establish highly productive and frost tolerant chickpea varieties supporting Ethiopian highland farmers by en- hancing food security and improving rural livelihoods. 2 MATERIAL AND METHODS 2.1 EXPERIMENT SITE The experiment was conducted at Bakelo, Debre Berhan Agricultural Research Center experimental site (Debre Berhan, Ethiopia) for two consecutive growing seasons (2018/19 and 2019/20). The experimental site is located 147 km away from Addis Ababa at a N 09 o 41‘42‘‘ latitude and E 39 o 37‘20‘‘ longitude. Its altitude is 2,837 meter above sea level and receives an annual mean pre- cipitation of 965.25 mm. The temperature ranges from 6.5 o C to 20.1 o C with mean annual temperature of 13.3 o C. The dominant soil type of Bakelo is black verti- sol. The daily minimum and maximum temperature va- lues are indicated in Fig 1. 2.2 PLANT MATERIALS A total of 673 genotypes (559 Ethiopian genotypes from the Ethiopian Biodiversity Institute (EBI), 83 elite frost resistant genotypes from the International Center for Agricultural Research in the Dry Areas (ICARDA), three susceptible local checks and 28 improved chickpea varieties released from Ethiopian Agricultural Research Centers were screened for their tolerance against frost stress under field condition at Bakelo, Debre Brehan, Ethiopia, which is a frost prone area (see Supplementary Table S1 for further details) using freezing tolerance rate, plant survival rate and other frost resistant-related agro- nomic traits. The geographical origin of the Ethiopian chickpea germplasm used in the study is indicated in Fig. 2. Acta agriculturae Slovenica, 117/2 – 2021 3 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Figure 1: Daily maximum and minimum temperature of Bakelo, Debre Berhan during 2018/2019 (A) to 2019/2020 (B) growing seasons (Source: Debre Berhan Agricultural Research Center) Figure 2: Map showing the geographical distribution of Ethiopian chickpea germplasm 2.3 EXPERIMENTAL DESIGN Augmented design without replication was used. Each genotype was sown in two rows with 3 m row length and 0.2 m spacing between rows and 0.1 m be- tween plants. Diammonium phosphate fertilizer (100 kg ha -1 ) and other appropriate management practices were applied. Five individual plants were tagged ran- domly from each genotype per plot and they were used for morphological data collection. Recording agronomic characteristics were conducted following the procedure described by chickpea descriptor (IBPGR, ICRISAT and ICARDA 1993). 2.4 DATA COLLECTED Qualitative and quantitative morphological traits were recorded as per described in Table 1. Acta agriculturae Slovenica, 117/2 – 2021 4 S. ADMAS et al. Table 1: List of qualitative and quantitative characters recorded, their codes and descriptions *= Frost score was recorded when susceptible checks showed sign for frost damages or completely died. Characters Description Qualitative traits Stem/Foliage Pigmentation (SLM) 0 = No Anthocyanin, 1 = Low Anthocyanin 2 = Medium Anthocyanin, 3 = High Anthocyanin Seed Color (SC) 1 = Black, 2 = Brown, 3 = White Flower Color (FC) 0 = White, 1 = Pink Quantitative traits Plant Height (cm) (PLH) Average canopy height of five representative plants taken at maturity stage Days to 50 % Flowering (DTF) Number of days from sowing until 50 % of the plants have started to flower Days to 50 % Podding (DTP) Number of days from sowing until 50 % of the plants have started to podding Days to 90 % Maturity (DTM) Number of days from sowing until 90 % of the pods have matured and turned yellow Number of Primary Branches (NPB) Average number of basal primary branches per plant taken from five represen- tative plants Number Secondary Branches (NSB) Average number of secondary branches per plant taken from five representative plants Number of Fertile Pods per Plant (NIPPP) Average number of fertile pods taken from five representative plants taken at maturity stage Number of Infertile Pods per Plant (NIPPP) Average number of infertile pods taken from five representative plants taken at maturity stage Thousand Seed Mass (TSM) Thousand seeds were counted and weighted at 12 % moisture content on a 0.1 g sensitive balance in milligram Grain Yield (GY in kg ha -1 ) Dried mass (kg) of seed per plot at 12 % moisture content *Freezing tolerance rate (FTR) Scored on 1-9 scale bases (Singh et al., 1989): where, 1 = No visible symptoms of damage; 2 = Highly tolerant, up to 10 % leaflets show damage; 3 = Tolerant, 11-20 % leaflets show damage; 4 = Moderately tolerant, 21-30 % leaflets and up to 20 % branches show withering and drying, but no killing; 5 = Intermediate, 41-60 % of leaflets and 21-40 % branches show withering and drying, up to 5 % plant killing; 6 = Moderately susceptible, 61-80 % leaflets and from 41-60 % branches show withering and drying, 6-25 % plant killing; 7 = Susceptible, 81- 99 % leaflets and 41-80 % branches show withering and drying, 26-50 % plant killing; 8 = Highly susceptible, 100 % leaflets and 81-99 % branches show withe- ring and drying, 51-99 % plant killing; and 9 = 100 % plant killing Plant survival rate (SR) Calculated by dividing the number of surviving plants after the frost period by the number of emerged plants after sowing was calculated (Heidarvand et al., 2011) Seed shriveling score (SSS) Visual measurement and estimating the kernel’s condition (1 = plump, 3 = in- termediate and 5 = shriveled 2.5 DATA ANALYSIS The collected data for each trait were subjected to statistical analysis of variance using augmentedRCBD R Packages version 0.1.3 (Aravind et al., 2020). The analysis helps us to partition the variance into different sources (phenotypic, genotypic and environmental variance) and genetic parametrs to see if the difference among geno- types is statistically significant or not for each trait con- sidered (Singh & Chaudhary, 1977). Pearson correlation coefficients between variable was estimated and tested for significance using MINTAB 10 statistical package (Wild, 2005) Acta agriculturae Slovenica, 117/2 – 2021 5 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance 3 RESULTS AND DISCUSSION The performances of the chickpea genotypes in re- sponse to frost stress were assessed in natural condition and the results obtained are discussed. 3.1 THE EFFECT OF FROST ON AGRONOMIC TRAITS ANOVA was performed for the two seasons sepa- rately because the intensity of frost stress was different for both years. There was a significant difference (p < 0.01) among genotypes for plant canopy height, number of primary branches, number of secondary branches, fertile pods per plant, infertile pods per plant, days to 50 % flowering, days to 50 % podding, days to 90 % ma- turity, thousand seed mass, and grain yield (Table 2) in 2018/2019 and 2019/2020 growing seasons. These diffe- rences in performance indicate the existences of variabi- lity among genotypes for frost tolerance. Similar finding was reported by Mir et al. (2019). Based on Fisher’s least significant difference (LSD) result indicated that there was a significant difference (p < 0.05) among genotypes for the mean value of agrono- mic traits examined in this study. A wide range value of the means was recorded for the traits recorded. The LSD means and range of values of the traits for chickpea geno- types examined is presented in Supplementary Tables S2 and S3 for further details. The LSD means value differen- ces and the mean rage value of the traits further confirms the existence of variable responses to frost stress among genotypes. The responses of genotypes to the effect of frost stresses at each crop stage are discussed below be- cause the genotypes responses to the frost damage were variable at each stage. 3.1.1 Seedling and vegetative stage The frost stress occurred in both seasons and ge- notypes had shown uniform germination and seedling establishment (Fig 3A). The lowest temperature recor- ded during this stage was -2.0 o C in Sept 2018 and -8.0 in Sept 2019 growing seasons. All genotypes did not show any symptoms or damage in response to frost stress, which means that these genotypes had shown good to- lerance to frost stress at seed germination and seedling development stages. However, most authors agreed that germination percentage and seedling development are sensitive to frost stress which results in poor crop stand establishment, and reduced seedling vigor with stun- ted seedlings (Croser et al., 2003; Maphosa et al., 2020; Srinivasan et al., 1998). During the vegetative stage, 43 (6.4 %) genotypes (One improved variety, one EBI ge- notypes and 54 ICARDA genotypes) were killed by frost (Fig 3B) in both growing seasons. The list of genotypes killed by frost is indicated in the Supplementary Table S4. Theses genotypes were identified as a highly susceptible to frost stress because they could not resist the frost stress when the minimum temperature of -6.0 o C and -12 o C were recorded in Oct 2018 and Oct 2019, respectively. These genotypes showed poor growth development, wil- ting, chlorosis, necrosis and finally death of the whole plant, which was the manifestation of frost injury. Similar observations were reported by Croser et al. (2003) and Mahajan & Tuteja (2005). The remaining genotypes had shown medium to good reactions to frost stress at vege- tative stage because the impact of frost stress at this stage was minimal in both growing seasons. 3.1.2 Number of branches and plant height The number of primary and secondary branches has been significantly affected by frost in both seasons where a wide range was recorded. The range of number of primary branches was 0 to 16.1 in 2018/2019 and 0 to 27.2 in 2019/2020 growing season and for number of se- condary branches it was 0 to 25.6 in 2018 and 0 to 46.5 in 2019. The majority of the accession scored below five for primary and secondary branch in both growing seasons. However, 69 (10.3 %) and 71 (10.6 %) genotypes produ- ced better number of primary branch (> 7) in 2018/2019 and 2019/2020 growing seasons, respectively. The res- ponse of genotypes to the effect of frost stress for plant height development was variable. A wide range of plant height was observed in both cropping seasons (20.3 to 58 cm in 2018/2019 and 17.2 to 57 cm in 2019/2020). One hundred two (15.2 %) and 89 (13.2 %) genotypes had a record of less than 35 cm plant height in 2018/2019 and 2019/2020 cropping season, respectively. Genotypes 132663 (58 cm) and 140294 (57.04 cm) had shown bet- ter plant height. In this experiment, most genotypes gave good positive reaction for plant height to the frost effect though frost significantly reduced plant height. This is probability because of the duration of time that frost oc- curred is not sufficient to have a negative impact to the plant development. 3.1.3 Reproductive stages Seventeen genotypes (2.5 %) (Seven EBI genotypes and 10 from ICARDA) were killed by frost stress during reproductive stages (Fig 3C and 3D). Moreover, the effect of frost was clearly examined in the delay of number of days to flower, pod and mature in the remaining geno- types with different degree. The range of 47.7 to 87.54, 54.2 to 89.6 and 118.7 to 160 days was recorded for days to flower, days to pod and mature for 2018/2019 growing season, respectively, while 48 to 77.7, 55 to 99.6 and 99.9 Acta agriculturae Slovenica, 117/2 – 2021 6 S. ADMAS et al. to 171.2 days for 2019/2020 cropping season, respecti- vely. The range of fertile pods per plant was 0 to 237.5 and 0 to 162.7 for 2018/12019 and 2019/2020 cropping seasons, respectively. The range of infertile pods per plant was 0 to 77.3 and 0 to 116 for 2018/2019 and 2019/2020 cropping seasons, respectively. The genotypes 227152-A (237.5) and 41301-B (162.7) produced the highest num- ber of fertile pods in 2018/2019 and 2019/2020 cropping seasons, respectively (Fig 3F). The minimum tempe- rature recordered during reparative stage especially at flowering and podding stages was below 5 o C in both seasons (Fig 1) which caused flower abortion and pod dropping for genotypes having poor response to frost stress. These frost symptoms were observed in most frost susceptible genotypes and they produced either empty pods or pods containing small shriveled seeds. Similar observation was reported by Gogoi et al. (2018) stating that temperature falls below 15 o C causes flower and pod abortions. Various authors agreed that the reproductive stage is more susceptible to frost stress than seedling sta- ges because frost stress negatively affects pollen fertility, pod set, number of aborted flowers, total number of pods per plant, seed number, size and shape, rate and duration of seed filling which consequently reduced biomass and grain yield (Berger et al., 2012; Croser et al., 2003; Gogoi et al., 2018; Kumar et al., 2010; Nayyar et al., 2007; Srini- vasan et al., 1999). Low temperature stress during repro- ductive development is responsible for the induction of flower abscission, pollen sterility, pollen tube distortion, ovule abortion and reduced fruit set leading to reduction in seed yield (Sharma & Nayyar, 2014). 3.1.4 Thousand seed mass and grain yield Seed development of all genotypes was severely affected by frost because the minimum temperature of -3.0 o C and -4.5 o C were recorded during seed dvelop- ment stage in Jan 2019 and Jan 2020, respectively (Fig 1). The majority of the genotypes produced shrived seed (Fig 4). Most genotypes that performed well till seed development became affected at seed development sta- ge. The range of 0 g to 300 g and 0 kg ha -1 to 2531 kg ha -1 were recorded for thousand seed mass and grain yield for 2018/2019 cropping season respectively, while for 2019/2020 cropping season the range was, 0 to 297 g and 0 kg ha -1 to 2604 kg ha -1 , respectively. Wu et al. (2014) indicated that the prolonged period of chilling range temperatures (0 o C to 12 o C) at any phenological stage of development in chickpea has detrimental effects on final seed yield. Low temperature has negative impact on yield and 15-20 % yield loss was estimated and temperature below 15 % during flowering leads to flower and pod abortion leading to poor yield (Croser et al., 2003). Frost stress affects the source-sink balance by markedly decre- asing the source of assimilates for grain filling which, in turn, reduces potential yield (Maphosa et al., 2020). Chaturvedi et al. (2009) estimated a yield loss of 15-20 % has been associated with low temperature. Low tempera- ture during vegetative stage leads to decreased vegetati- ve growth, biomass production and yield in north India (Mir et al., 2019; Singh et al., 1993). 3.1.5 Seed color The majority of the frost susceptible genotypes showed a seed color fade up. Some of the genotypes had shown plumped seed with fade up seed color. This indi- cates that frost causes seed size and seed discoloration in chickpea. Similar observation was made for faba bean (Sallam et al., 2015). This happened because frost affects the mobilization of plant resources in to seed setting (Croser et al., 2003). 3.2 PLANT SURVIV AL RATE (SR) Frost tolerance was assessed using plant survival rate (SR) for 673 diverse chickpea germplasms for two growing seasons under field condition (Table 3). It was observed that the SR values ranged from 0.0 (60 geno- types) to 0.86 (genotypes 16341-A, 24159-C and 30290- A) and 0.0 (60 genotypes) to 0.87 (genotype 41167-C) for 2018/2019 and 2019/2020 growing seasons, respectively. The value of SR score for the two growing seasons showed variation because of the different duration and intensity of frost stress occurred in the different seasons. The frost intensity and length of occurrence were more sever in 2019/2020 growing season than in 2018/2019. So, high value of SR was recorded in 2018/2019 than in 2019/2020 growing season (Fig 1). One hundred fifty seven and 87 chickpea genotypes had shown above 0.8 SR score, while the remaining 516 and 586 genotypes were below 0.8 SR score for 2018/2019 and 2019/2020 growing seasons, re- spectively. Eighty five genotypes were consistently given SR score value above 0.8 in both growing seasons. In the experimental site, frost occurred consistently throughout the life cycle of the crop’s development, and hence, the frost survival score were taken at the end of each crop stages. The fluctuation of minimum temperature of two different growing seasons exhibited a similar pattern of SR value change for all genotypes. Minimum tempera- ture of 2019/2020 growing seasons was lower than that of 2018/2019 growing season. It is clear that the SR of chickpea is closely associated to the temperature changes. Similar patterns were observed also in field pea (Liu et al., 2017). This approach has been employed to screen frost tolerance in rapeseed/canola (Fiebelkorn & Rah- man, 2016) and field pea (Liu et al., 2017). Acta agriculturae Slovenica, 117/2 – 2021 7 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Table 2: Mean square and mean for the tested traits of 673 (562 EBI genotypes, 83 exotic and 28 improved chickpea) genotypes grown at Bakelo, Debre Berhan, Ethiopia from 2018-2020 growing seasons (I for 2018/2019 and II for 2019/2020) Figure 3: Frost response in chickpea at different growing stages: chickpea genotypes seedling coverage (A), plant death during pre-flowering stage (B), reduced pod setting (C and D) and better pod setting (E and F) Symbols for level of significance:‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05, ns is none significant, PLH = Plant Canopy Height (cm), NPB = Number of primary branches, NSB = Number secondary branches, FPPP = Fertile pods per plant, IPPP = Infertile pods per plant, DTF = Days to 50 % flowering, DTP = Days to 50 % podding, DTM = Days to 90 % maturity, TSM = Thousand seed mass, and GY = Grain yield in kg ha -1 (I) Sources of Variation Degree of freedom Type III Mean Squares PLH NPB NSB FPPP IPPP DTF DTP DTM TSM GY Block 9 0.6 ns 0.3 ns 1.6 ns 168.3 ns 35.9 ns 4.55 ns 14.9 ns 3.51 ns 5.0 ns 7224 ns Treatment 612 23.5 *** 2.2 *** 4.9 *** 429.4 *** 47.6 * 8.15 * 17.4 * 28.57 * 814.8 *** 154550 *** Treatment: check 2 0.99 ns 0.3 ns 0.6 ns 35.3 ns 277 *** 41.6 ** 61.9 ** 21.43 ns 13.1 ns 174411 *** Treatment: test and test vs. Check 610 24*** 2.2 *** 4.9 * 430.7 *** 46.8 * 4.04 * 17.2 * 28.59 * 817.4 *** 154485 *** Residuals 18 0.78 0.43 0.53 84.8 21.6 4.82 7.8 11.59 6.8 8976 CV 2.32 13.32 20.06 22.88 41.34 4.16 4.57 2.56 3.07 8.72 Mean 38.35 4.89 3.65 40.31 10.9 52.72 61.22 133.0 87.11 1118.9 (II) Block 9 8.74 2.29 ns 0.9 ns 295 ns 59.6 ns 2.1 ns 34.4 ns 50 ns 10 ns 7435 ns Treatment 612 38.4 *** 2.2 * 3.6 * 513 ** 180 *** 22.5 *** 29.45 * 49.13 * 1177 *** 253659 *** Treatment: check 2 8.59 ns 0.8 ns 1.8 ns 211 ns 27 ns 7.03 ns 103. ** 65.1 ns 75.8 * 65909 ** Treatment: test and test vs. Check 610 38.5 *** 2.2 * 3.6 * 514 ** 180 *** 22.6 *** 29.21 * 49.08 * 1180 *** 254275 *** Residuals 18 2.57 1.38 2.56 184 26.3 5 14.49 49.36 16.7 9573 CV (%) 3.89 21.47 41.33 25.47 16.0 4.09 5.29 5.36 6.78 14.0 Mean 41.39 5.49 3.87 53.76 31.75 54.72 72.19 130.66 61.39 720.0 Acta agriculturae Slovenica, 117/2 – 2021 8 S. ADMAS et al. Table 3: Frost survival rate (SR) of 562 Ethiopian chickpea, 83 exotic and 28 improved chickpea genotypes tested at Bakelo, Debre Berhan, Ethiopia, 2018 to 2020 growing seasons Table 4: Freezing tolerance rate (FTR) of 673 (562 Ethiopian chickpea, 83 exotic and 28 improved) chickpea genotypes tested at Bakelo, Debre Berhan, Ethiopia from 2018 to 2020 growing seasons No SR Rating 2017/2018 2019/2020 Common genotypes for both years No of genotypes Percentage No of genotypes Percentage No of genotypes Percentage 1 ≥ 0.8 157 23.3 87 12.9 85 19.6 2 ≥ 0.6 to < 0.8 273 40.6 199 29.6 155 35.8 3 ≥ 0.4- < 0.6 136 20.2 213 31.7 96 22.2 4 ≥ 0.2- < 0.4 33 4.9 60 8.9 23 5.3 5 < 0.2 74 11.0 114 16.9 74 17.1 Total 673 673 433 3.3 FREEZING TOLERANCE RATE (FTR) Freezing tolerance rate with a rating scale of 1-9 has been used for measuring cold stress injury during early vegetative stage or seedling stage in earlier studies (Singh et al. 1989). Based on FTR, 169 (1-3 score) and 84 (2-3 score) genotypes were described as tolerant to highly tol- erant, while 504 (4-9) and 590 (4-9) were described as moderately tolerant to highly susceptible genotypes dur- ing 2018/2019 and 2019/2020 growing seasons, respec- tively (Table 4). Eighty three genotypes were rated within the score of 1-3 consistently in both growing season. In No FTR Rating 2017/2018 2019/2020 Common genotypes for both years No of genotypes Percentage No of genotypes Percentage No of genotypes Percentage 1 1 27 4.0 0 0 2 2 32 4.8 27 4.0 3 3 110 16.3 57 8.5 Sub Total 169 25.1 84 12.5 83 15.5 4 4 261 38.8 154 22.9 5 5 82 12.2 131 19.5 6 6 50 7.4 118 17.5 Sub Total 393 58.4 403 59.9 341 63.9 7 7 29 4.3 57 8.5 8 8 20 3.0 25 3.7 9 9 62 9.2 104 15.5 Sub Total   111 16.5 186 27.6 Grand Total   673   673   424 this experiment, it is observed that the majority of the genotypes that were resistant at seedling stages failed to resist frost that occured late at reproductive stage. From this result we can conclude that FTR score must be taken throughout the crop stages. Generally, single FTR score may work for areas where frost occurs once in the life cycle of the crop stages, however, in areas where, frost occurs consistently throughout the life cycle of the crop, FTR should be score frequently. In addition, genotypes that showed better FTR value gave either shriveled seeds or empty pods. So, FTR is not able to evaluate the capacity of frost resistance at reproductive stages and the suscep- Acta agriculturae Slovenica, 117/2 – 2021 9 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Table 5: Seed shriveling score (1-5) of 673 (562 Ethiopian chickpea, 83 exotic and 28 improved chickpea) genotypes tested at Bakelo, Debre Brehan, Ethiopia from 2018 to 2020 growing seasons Figure 4: Seeds of chickpea genotypes showing different reaction to frost stress (A and B are very shriveled (Score of 5), C is Shri- veled (Score of 4), D is intermediate (Score of 3), E is medium plumped (Score of 2) and F is plumped (Score of 1) No SSR Rate 2017/2018 2019/2020 Common genotypes for both years No of genotypes Percentage No of genotypes Percentage No of genotypes Percentage  1 1 145 21.6 47 7.0 33 12.6  2 2 128 19.0 83 12.3 33 12.6  3 3 126 18.7 154 22.9 42 16.0  4 4 177 26.3 194 28.8 78 29.8  5 5 97 14.4 195 29.0 76 29.0  Total 673 673 262 tible genotypes will be overlooked by this approach. FTR is the most important indices used for freezing screening for different crops tested at seedling stage (Badeck et al., 2015; Nezami et al., 2012; Srinivasan et al., 1998; Toker, 2005). 3.4 SEED SHRIVELING SCORE (SSS) Visual assessment of seed damage by frost stress was done for all the genotypes for both seasons (Table 5). One hundred forty five and 47 genotypes produced plumped seeds (Score of 1: Fig 4E and 4F) in 2018/2019 and 2019/2020 cropping seasons, respectively, while the remaining genotypes gave medium to shriveled seeds (Fig 4A to 4D). Genotypes that were rated as frost re- sistant based on SR and FTR indices failed to produce plumped seeds, which means that all genotypes that had a better SR and FTR score did not produce plumped seed. However, all genotypes that produced plumped seed had a better SR and FTR value. From this result, it is possible to conclude that SR and FTR indices can indicate frost re- sistances at seedling or vegetative stages alone. Therefore, SR and FTR indices will not be applicable to screen geno- types for frost resistance at reproductive stage. Visual as- sessment of frost damaged seed has been applicable also to screen faba bean genotypes for frost resistance vari- ability (Henriquez et al., 2018). In general to select the frost tolerant promising gen- otypes, it is advisable to consider frost tolerance related traits and agronomic traits together. Genotypes that are consistently selected by all the parameters are considered as a promising frost tolerant genotype which can be di- rectly taken by farmers or serve as a breeding material for further breeding activities. The selected frost toler- ant genotypes will help to stabilize yield and expand the chickpea production areas into Ethiopian highland where chickpea production is not a common practice because of frost damage. In this study, 94 (51 black, 29 brown and 14 white) genotypes were selected as frost tolerant, the remaining genotypes as intermediate to susceptible. The promising frost resistant genotypes were selected with the following criteria i.e. Frost survival rate (>0.75), seed shriveling score (1-2), and freezing tolerance rate (1-4). The selected genotypes are listed in table 6. Acta agriculturae Slovenica, 117/2 – 2021 10 S. ADMAS et al. Table 6: List of eighty two frost resistant chickpea genotypes selected based on SR (> 0.75), FTR (score of 1,2,3) and seed score (1 and 2) EBI = Ethiopian Biodiversity Institute, ICARDA is International International Center for Agricultural Research in the Dry Areas, EARCs = Ethiopian Agricultural Research Centers No Genotype Seed Color Source No Genotype Seed Color Source No Genotype Seed Color Source 1 16341-A Black EBI 33 208994-A Brown EBI 65 30293-A Brown EBI 2 207674 Black EBI 34 235036-A Brown EBI 66 207739-B Brown EBI 3 30336-A Black EBI 35 209016-B Black EBI 67 71875 Brown ICARDA 4 30336-B Black EBI 36 209022-A Black EBI 68 75095 Brown ICARDA 5 41004-C Black EBI 37 209026-A Black EBI 69 140941 Brown ICARDA 6 41036-B Black EBI 38 212589-B Black EBI 70 116451 Brown ICARDA 7 41051-A Black EBI 39 212914-B Black EBI 71 126302 Brown ICARDA 8 41081-A Black EBI 40 214731-B Black EBI 72 9427 Red ICARDA 9 41107-B Black EBI 41 214734-A Black EBI 73 128699 White ICARDA 10 41133-A Black EBI 42 215067-A Black EBI 74 9632 White ICARDA 11 41167-C Black EBI 43 215190-A Black EBI 75 10163 White ICARDA 12 41206-B Black EBI 44 215289-B Black EBI 76 140394 White ICARDA 13 207608 Black EBI 45 236196-B Black EBI 77 7339 White ICARDA 14 207622 Black EBI 46 236459-B Black EBI 78 70753 White ICARDA 15 207638 Black EBI 47 236479-C Black EBI 79 73395 White ICARDA 16 207640 Black EBI 48 237054-B Black EBI 80 69420 White ICARDA 17 207648 Black EBI 49 207686 Black EBI 81 132663 White ICARDA 18 207652 Black EBI 50 207664-A Black EBI 82 9415 White ICARDA 19 207668 Black EBI 51 30349-B Black EBI 83 Ye l e b e White EARCs 20 207670 Black EBI 52 30348-B Black EBI 84 Akaki Red EARCs 21 207684 Black EBI 53 41127-B Black EBI 85 mariye Red EARCs 22 207688-A Black EBI 54 207746 Black EBI 86 Natoli Red EARCs 23 207692 Black EBI 55 207173-C Black EBI 87 Teketay Red EARCs 24 207712 Black EBI 56 41075-C Brown EBI 88 kutaye Brown EARCs 25 207714 Black EBI 57 41093-B Brown EBI 89 Teji White EARCs 26 207728-A Black EBI 58 41255-B Brown EBI 90 Shola White EARCs 27 207730 Black EBI 59 207175-A Brown EBI 91 Worku Red EARCs 28 207748 Black EBI 60 207635-C Brown EBI 92 Harbu White EARCs 29 208988-A Red EBI 61 30350-B Red EBI 93 Dalota Brown EARCs 30 209026-B Red EBI 62 41301-B Red EBI 94 Mastewal Brown EARCs 31 227152-B Red EBI 63 207766 Black EBI 32 30334-C Red EBI 64 207770 Black EBI Acta agriculturae Slovenica, 117/2 – 2021 11 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Table 7:Phenotypic Pearson’s correlation matrix for 9 traits in chickpea 673 (562 Ethiopian chickpea, 83 exotic and 28 improved chickpea) genotypes tested at Bakelo, Debre Berhan, Ethiopia from 2018/2019 (above diagonal) to 2019/2020 (below diagonal) growing seasons ns = non significant; ** =Correlation is significant at the 0.01 level (2-tailed); * =Correlation is significant at the 0.05 level (2-tailed), PLH = Plant Canopy Height (cm), NPB = Number of primary branches, NSB = Number secondary branches, FPPP = Fertile pods per plant, IPPP = Infertile pods per plant, DTF = Days to 50 % flowering, DTP = Days to 50 % podding, DTM = Days to 90 % ma- turity, TSM = Thousand Seed Mass, GY = Grain yield in kg ha -1 , SR = Frost survival rate, FTR = Frost tolerance rate, SSS = Seed shriveling score, FC = Flower Color, SLP = Stem/leaf pigmentation, and SC = Seed color 3.5 PHENOTYPIC CORRELATION COEFFICIENT The phenotypic association of agronomic and frost tolerance related traits were analyzed for each genotype and the following result were obtained (Table 7). Most of the frost tolerance related traits have shown a strong significant relationship with agronomic traits. Grain yield was positively and significantly correlated (p < 0.01) with fertile pod per plant (0.33 and 0.21), thou- sand seed mass (0.69 and 0.72), SR (0.86 and 0.73), and stem/leaf pigmentation (0.59 and 0.48), while a strong negative correlation was seen for infertile pod per plant (-0.7 and -0.6), FTR (-0.70 and -0.6), SSS (-0.8 and -0.8), seed color (-0.52 and -0.30), and flower color (-0.43 and -0.21) for 2018/2019 and 2019/2020 growing seasons, re- spectively. It was observed that genotypes having strong stem/leaf pigmentation had shown a good performance in all agronomic traits and had also a better SR and FTR score. Similarly, flower and seed color had shown also a strong correlation with agronomic performances. Geno- types having pink flower and black seed color had bet- ter performances than the ones with white flower and white seed colored ones. From this result, the selection   PLH NPB NSB FPPP IPPP DTF DTM TSM GY SR FTR SSS FC SLP SC PLH 0 0.65 ** 0.43 ** 0.51 ** -0.3 ** 0.13 ** 0.13 ** 0.64 ** 0.59 ** 0.68 ** -0.66 ** -0.48 ** -0.52 ** 0.36 ** -044 ** NPB 0.64 ** 0 0.71 ** 0.68 ** 0.01 ** 0.12 ** 0.12 ** 0.35 ** 0.39 ** 0.43 ** -0.40** -0.31 ** 0.41 ** 0.28 ** -0.33 ** NSB 0.47 ** 0.68 ** 0 0.69 ** 0.04 ns 0.13 ** 0.19 ** 0.18 ** 0.23 ** 0.25 ** -0.25 ** -0.18 ** 0.20 ** 0.16 ** -0.17 ** FPPP 0.52 ** 0.69 ** 0.69 ** 0 -0.0 ns -0.1 ns 0.12 ** 0.28 ** 0.33 ** 0.37 ** -0.36 ** -0.3 ** 0.28 ns 0.22 ** -0.26 ** IPPP -0.2 ** 0.17 ** 0.19 ** 0.22 ** 0 0.08 * 0.06 ns -0.4 ** -0.7 ** -0.6 ** 0.56 ** 0.70 ** 0.1 ns -0.3 ** 0.11 ** DTF 0.01 ns 0.06 ns 0.15 ** -0.2 ** -0.1 ns 0.28 ** -0.0 ns -0.2 ** -0.2 ** 0.16 ** 0.15 ** -0.4 ** -0.2 ** 0.30 ** DTM 0.02 ns 0.00 ns 0.06 ns -0.2 ** -0.1 ns 0.30 ** 0 -0.0 ns -0.08 * -0.08 * 0.03 ns 0.01 ns -0.2 ** -0.1 ns 0.11 ** TSM 0.47 ** 0.24 ** 0.22 ** 0.20 ** -0.5 ** 0.14 ** 0.13 ** 0 0.69 ** 0.77 ** -0.76 ** -0.60 ** -0.27 ** 0.32 ** -0.27 ** GY 0.42 ** 0.20 ** 0.18 ** 0.21 ** -0.6 ** 0.0 ns 0.00 ns 0.72 ** 0 0.86 ** -0.84 ** -0.8 ** 0.43 ** 0.59 ** -0.52 ** SR 0.65 ** 0.4 ** 0.3 ** 0.38 ** -0.4 ** 0.0 ns -0.1 ** 0.66 ** 0.73 * 0 -0.90 ** -0.79 ** 0.41 ** 0.47 ** -0.47 ** FTR -0.6 ** -0.36 ** -0.3 ** -0.3 ** 0.44 ** -0.1 ns 0.09 * -0.6 ** -0.7 ** -0.9 ** 0 0.77 ** -0.37 ** -0.6 ** 0.53 ** SSS -0.5 ** -0.18 ** -0.2 ** -0.2 ** 0.54 ** -0.1 ** -0.1 ns -0.8 ** -0.8 ** -0.8 ** 0.75 ** 0 -0.32 ns -0.5 ** 0.44 ** FC 0.57 ** 0.46 ns 0.25 ** 0.41 ** 0.22 ** -0.4 ** -0.2 ** 0.11 * -0.21 * -0.3 ** -0.20 ** -0.11 ** 0 0.57 ** -0.79 ** SLP 0.43 ** 0.30 ** 0.18 ** 0.31 ** -0.4 ** -0.2 ** -0.1 ns 0.37 ** 0.48 ** 0.47 ** -0.49 ** -0.51 ** 0.60 ** 0 -0.80 ** SC -0.51 * -0.4 ** -0.2 ** -0.3 ** 0.11 ** 0.30 ** 0.18 ** -0.2 ns -0.3 ** -0.5 ** 0.40 ** 0.32 ** -0.79 ** -0.8 ** 0 of genotypes having strong stem/leaf pigmentations and genotypes with black seeded chickpea types and pink flower would greatly assist plant breeders to develop frost resistant variety to reduce the risk of frost damages. The majority of black seeded chickpea performed well in ag- ronomic traits and SR and FTR score was higher than brown and white seeded chickpea types. The majority of white seeded chickpea types were highly susceptible to frost stress. Brown seeded chickpea with strong stem/ leaf pigmentation exhibited better reaction to frost stress than the one with brown seeded with week stem/leaf pigmentation. The result agree with previous findings in faba bean where genotypes with white flower being susceptible to frost stress, while tannin-containing geno- types and wild relatives are more tolerant (Henriquez et al., 2018; Inci & Toker, 2011). Frost stress causes accu- mulation of anthocyanins in the basal part of the stem, branches and leaves (Croser et al., 2003). Bhasker et al. (2018) indicated that the accumulation of anthocyanin due to high temperature has a positive relation with high grain yield because of the induction of antioxidant de- fense system. Frost damage has strong correlation with lower yield (Henriquez et al., 2018; Kanouni et al., 2009). Acta agriculturae Slovenica, 117/2 – 2021 12 S. ADMAS et al. From this result it is possible to conclude that the pres- ence of pigmentation induced by frost stress can be a good indicator for frost tolerance mechanism. 4 CONCLUSIONS This experiment has shown that the degree of frost damage varied at different crop stages. The effect of frost was not seen on seed germination and seedling establish- ment. However, considerable frost damage was observed at vegetative and reproductive stages for most genotypes. The capacities of genotypes for frost tolerance were esti- mated using freezing tolerance rate (FTR) and frost sur- vival rate (SR) and their agronomic performances. Eighty three and 85 genotypes were selected based on FTR and SR respectively. However, both indices are not able to evaluate frost resistance of the genotypes at reproduc- tive stage, if the frost occurs consistently throughout the crop stages. Genotypes having good SR and FTR value produced shriveled seed and empty pods due to frost stress that occurred later at flowering and seed develop- ment stages. Therefore, to select the frost tolerant po- tential genotypes, it is advisable to consider SR and FTR values, pod setting, seed shriveling score, and grain yield together. Genotypes that are consistently selected by all these parameters are considered as promising frost toler- ant genotypes. In addition, in areas where frost occurs consistently during the seedling and vegetative stages of the crop only, the selection of frost resistance at these stages by considering less FTR and high SR values are enough to select the frost resistant promising genotypes. The effect of frost stress to chickpea genotypes are vari- able depending on seed color type, presence and absence of stem/leaf pigmentation and different level of stem/leaf pigmentation. Chickpea genotypes with black seeded and/or having strong stem/leaf pigmentation performed well for frost stress reaction. From these observations, it is possible to conclude that stem/leaf pigmentation and black seeded color might be linked to a gene that con- fers frost resistance in chickpea. From this experiment, 94 genotypes were identified to be frost tolerant geno- types which can be taken by plant breeder for frost toler- ant chickpea variety development program attesting that Ethiopian checkpea genotypes have a potential source for frost tolerance trait. Identification of the mechanism of stem/leaf pigmentation and black seed color for frost resistance is required. Also, identification of quantitative trait loci (QTLs) associated with gene controlling frost tolerances in chickpea is equally important. ACKNOWLEDGMENT Ethiopian Biodiversity Institute and Addis Ababa University for financial support. Ethiopian Biodiversity Institute, Debre Ziet Agricultural Research Center and International Center for Agricultural Research in the Dry Areas for providing the chickpea genotypes. Debre Berhan Agricultural Research Center for allowing us to use the research station for field work and to access me- treological data. 5 REFERENCES Aravind, J., Sankar, S. M., Wankhede, D. P ., & Kaur, V . (2020). AugmentedRCBD: analysis of augmented randomised com- plete block designs. R package version 0.1. 2. Badeck, F.W., & Rizza, F. (2015). A combined field/laboratory method for assessment of frost tolerance with freezing tests and chlorophyll fluorescence. Agronomy, 5(1), 71-88. htt- ps://doi.org/10.3390/agronomy5010071 Bejiga, G., Eshete, M., & Anbessa, Y. (1996). Improved cultivars and production technology of chickpea in Ethiopia. Berger, J. D., Kumar, S., Nayyar, H., Street, K. A., Sandhu, J. S., Henzell, J. M., ... & Clarke, H. C. (2012). Temperature-stra- tified screening of chickpea (Cicer arietinum L.) genetic resource collections reveals very limited reproductive chil- ling tolerance compared to its annual wild relatives. Field Crops Research, 126, 119-129. https://doi.org/10.1016/j. fcr.2011.09.020 Bhasker, P ., Nandwal, A. S., Kumar, N., Chand, G., Yadav, S. P ., Devi, S., & Singh, S. (2017). High temperature significance of anthocyanins accumulation stress responses in chickpea (Cicer arientinum L.). International Journal of Agricuture Innovation and Research, 6, 2319-2473. Chaturvedi, S. K., Mishra, D. K., Vyas, P ., & Mishra, N. (2009). Breeding for cold tolerance in chickpea. Trends in Bioscien- ces, 2(2), 1-4. Croser, J. S., Clarke, H. J., Siddique, K. H. M., & Khan, T. N. (2003). Low-temperature stress: implications for chickpea (Cicer arietinum L.) improvement. Critical Reviews in Plant Sciences, 22(2), 185-219. https://doi.org/10.1080/713610855 CSA (Central Statistical Agency) .(2019). Agricultural sample survey report on area and production of crops private peasant holdings, Meher season. Central Statistical Agency, statisti- cal bulletin 589, Addis Ababa FAO. (2020). FAO in Ethiopia: Ethiopia at a glance. Retrie- ved from http://www.fao.org/ethiopia/fao-in-ethiopia/ ethiopia-at-a-glance/en/ on 07 Dec 2020 Ferede, S., Fikre, A., & Ahmed, S. (2018). Assessing the compe- titiveness of smallholders Chickpea production in the cen- tral highlands of Ethiopia. Ethiopian Journal of Crop Scien- ce, 6(2), 51-65. URL: http://oar.icrisat.org/id/eprint/10635 Fiebelkorn, D., & Rahman, M. (2016). Development of a proto- col for frost-tolerance evaluation in rapeseed/canola (Bras- sica napus L.). The Crop Journal, 4(2), 147-152. https://doi. org/10.1016/j.cj.2015.11.004 Acta agriculturae Slovenica, 117/2 – 2021 13 Evaluation of Ethiopian chickpea (Cicer arietinum L.) genotypes for frost tolerance Fikre, A., & Bekele, D. (2020). Chickpea Breeding and Crop Im- provement in Ethiopia: Past, Present and the Future. Uni- versal Journal of Agricultural Research, 8(2), 33-40. https:// doi.org/10.13189/ujar.2020.080202 Fikre, A., Desmae, H., & Ahmed, S. (2020). Tapping the econo- mic potential of chickpea in Sub-Saharan Africa. Agronomy, 10(11), 1707. https://doi.org/10.3390/agronomy10111707 Fikre, A., Funga, A., Korbu, L., Eshete, M., Girma, N., Zewdie, A., ... & Ojiewo, C. O. (2018). Stability analysis in chickpea genotype sets as tool for breeding germplasm structuring strategy and adaptability scoping. Ethiopian Journal of Crop Science, 6(2), 19-37. Gogoi, N., Farooq, M., Barthakur, S., Baroowa, B., Paul, S., Bha- radwaj, N., & Ramanjulu, S. (2018). Thermal stress impacts on reproductive development and grain yield in grain le- gumes. Journal of Plant Biology, 61(5), 265-291. https://doi. org/10.1007/s12374-018-0130-7 Heidarvand, L., Amiri, R. M., Naghavi, M. R., Farayedi, Y., Sadeghzadeh, B., & Alizadeh, K. (2011). Physiological and morphological characteristics of chickpea acces- sions under low temperature stress. Russian Journal of Plant Physiology, 58(1), 157-163. https://doi.org/10.1134/ S1021443711010080 Henriquez, B., Olson, M., Hoy, C., Jackson, M., & Wouda, T. (2017). Frost tolerance of faba bean cultivars (Vicia faba L.) in central Alberta. Canadian Journal of Plant Science, 98(2), 509-514. https://doi.org/10.1139/cjps-2017-0078 Inci, N. E., & Toker, C. (2011). Screening and selection of faba beans (Vicia faba L.) for cold tolerance and comparison to wild relatives. Genetic Resources and Crop Evolution, 58(8), 1169-1175. https://doi.org/10.1007/s10722-010-9649-2 Kanouni, H., Khalily, M., & Malhotra, R. S. (2009). Assessment of cold tolerance of chickpea at rainfed highlands of Iran. American-Eurasian Journal Agriculture & Environment Science, 5, 250-254. Kumar, S., Nayyar, H., Bhanwara, R.K., & Upadhyaya, H.D. (2010). Chilling stress effects on reproductive biology of chickpea. Journal of SAT Agricultural Research, 8, 1-14. Corpus ID: 32128576 Liu, R., Fang, L., Yang, T., Zhang, X., Hu, J., Zhang, H., ... & Zong, X. (2017). Marker-trait association analysis of frost tolerance of 672 worldwide pea (Pisum sativum L.) collec- tions. Scientific Reports, 7(1), 1-10. https://doi.org/10.1038/ s41598-017-06222-y Mahajan, S., & Tuteja, N. (2005). Cold, salinity and drou- ght stresses: an overview. Archives of Biochemistry and Biophysics, 444(2), 139-158. https://doi.org/10.1016/j. abb.2005.10.018 Maphosa, L., Richards, M. F., Norton, S. L., & Nguyen, G. N. (2020). Breeding for abiotic stress adaptation in chickpea (Cicer arietinum L.): A comprehensive review. Crop Bree- ding, Genetics and Genomics, 4(3). https://doi.org/10.20900/ cbgg20200015 Mir, A. H., Bhat, M. A., Fayaz, H., Dar, S. A., Maqbool, S., Bhat, N. A., ... & Mir, R. R. (2019). Assessment of cold toleran- ce in chickpea accessions in North-Western Himalayas of Jammu and Kashmir, India. Journal of Pharmacognosy and Phytochemistry, 8(4), 2268-2274. Muoni, T., Barnes, A. P., Öborn, I., Watson, C. A., Bergkvist, G., Shiluli, M., & Duncan, A. J. (2019). Farmer perceptions of legumes and their functions in smallholder farming sys- tems in east Africa. International Journal of Agricultural Sustainability, 17(3), 205-218. https://doi.org/10.1080/147 35903.2019.1609166 Nayyar, H., Kaur, G., Kumar, S., & Upadhyaya, H. D. (2007). Low temperature effects during seed filling on chickpea genotypes (Cicer arietinum L.): probing mechanisms affec- ting seed reserves and yield. Journal of Agronomy and Crop Science, 193(5), 336-344. https://doi.org/10.1111/j.1439- 037X.2007.00269.x Nezami, A., Bandara, M. S., & Gusta, L. V. (2012). An evalu- ation of freezing tolerance of winter chickpea (Cicer arie- tinum L.) using controlled freeze tests. Canadian Journal of Plant Science, 92(1), 155-161. https://doi.org/10.4141/ cjps2011-057 Sallam, A., Martsch, R., Moursi, Y.S. (2015). Genetic variation in morpho-physiological traits associated with frost tole- rance in faba bean (Vicia faba L.). Euphytica, 205(2), 395- 408. https://doi.org/10.1007/s10681-015-1395-2 Sharma, K. D., & Nayyar, H. (2014). Cold stress alters tran- scription in meiotic anthers of cold tolerant chickpea (Cicer arietinum L.). BMC Research Notes, 7(1), 1-13. https://doi. org/10.1186/1756-0500-7-717 Shiferaw, B., & Teklewold, H. (2007). Structure and functioning of chickpea markets in Ethiopia: Evidence based on analy- ses of value chains linking smallholders and markets. IPMS Working Paper 6, ILRI, Nairobi, Kenya. 55 pp. Singh RK, Chaudhary BD. (1977). Biometrical methods in quantitative genetices analysis. Kalyanin Puplishers, New Delhi. Singh, K. B., Malhotra, R. S., & Saxena, M. C. (1989). Chick- pea evaluation for cold tolerance under field conditions. Crop science, 29(2), 282-285. https://doi.org/10.2135/cro- psci1989.0011183X002900020009x Singh, K. B., Malhotra, R. S., & Saxena, M. C. (1993). Rela- tionship between cold severity and yield loss in chickpea (Cicer arietinum L.). Journal of Agronomy and Crop Science, 170(2), 121-127. https://doi.org/10.1111/j.1439-037X.1993. tb01065.x Srinivasan, A., Johansen, C., & Saxena, N.P. (1998). Cold tole- rance during early reproductive growth of chickpea (Cicer arietinum L.): characterization of stress and genetic varia- tion in pod set. Field Crops Research, 57(2), 181-193. htt- ps://doi.org/10.1016/S0378-4290(97)00118-4 Srinivasan, A., Saxena, N. P ., & Johansen, C. (1999). Cold tole- rance during early reproductive growth of chickpea (Cicer arietinum L.): genetic variation in gamete development and function. Field Crops Research, 60(3), 209-222. https://doi. org/10.1016/S0378-4290(98)00126-9 Toker, C. (2005). Preliminary screening and selection for cold tolerance in annual wild Cicer species. Genetic Resources and Crop Evolution, 52(1), 1-5. https://doi.org/10.1007/ s10722-005-1743-5 Toker, C., Lluch, C., Tejera, N. A., Serraj, R., & Siddique, K. H. M. (2007). 23 Abiotic stresses. Chickpea breeding and ma- nagement, 474. https://doi.org/10.1079/9781845932138.023 Van der Maesen, L. J. G. (1987). Origin, history and taxonomy of chickpea. In The chickpea (pp. 11-34). Acta agriculturae Slovenica, 117/2 – 2021 14 S. ADMAS et al. Wild, D. J. (2005). MINITAB release 14. https://doi.org/10.1021/ ci040130h Wu, Y . F., Zhong, X. L., Hu, X., Ren, D. C., Lv, G. H., Wei, C. Y ., & Song, J. Q. (2014). Frost affects grain yield components in winter wheat. New Zealand Journal of Crop and Horticul- tural Science, 42(3), 194-204. https://doi.org/10.1080/01140 671.2014.887588