Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 75-82, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-809 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: E. Ansari, M. Khosrowshahli, A. Ashraf Jafari, A. Etminan (2021) Induc- tion of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics. Caryolo- gia 74(1): 75-82. doi: 10.36253/caryolo- gia-809 Received: January 03, 2020 Accepted: April 26, 2021 Published: July 20, 2021 Copyright: © 2021 E. Ansari, M. Khos- rowshahli, A. Ashraf Jafari, A. Etminan. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Induction of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics Elham Ansari1, Mahmood Khosrowshahli2, Ali Ashraf Jafari3, Alireza Etminan4 1 PhD student, Department of plant breeding, Islamic Azad University, Science and Research Branch, Tehran, Iran 2 Department of plant breeding, Islamic Azad University, Science and Research Branch, Tehran, Iran 3 Research Institute of Forests and Rangelands, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran 4 Department o plant breeding, Islamic Azad University Kermanshah Branch, Kerman- shah, Iran *Corresponding author. E-mail: mkhosrowchahli@yahoo.com Abstract. In order to study of the effects of polyploidy on morph-physiological traits in some annual and perennial medics, five annual and three perennial diploid med- ics were subjected to different concentrations of colchicine solution (0.1%, 0.5%, 1% and 2%) in mitosis stage. The induced tetraploids were identified by counting stomata guard cells chloroplasts. The highest survival rate and tetraploidy induction with aver- age values of 80.2% and 74.3% were obtained using 0.1% colchicine concentration. All of the 21 entries (5 annual diploids and their induced tetraploids as M. lupulina, M. radiata, M. rijidula, M. truncatula, M. turbinata, and 3 populations of perennial dip- loid of M. sativa ssp. Caerulea (Karaj1, Karaj2 and Tehran) and their induced tetra- ploids and 5 commercial tetraploid alfalfa cultivars (Bami, Hamadani, Baghdadi, Ghareyongeh and Renger) grown in pots in a glasshouse experiment using completely randomized design with five replications in 2018 in Ahvaz, Iran. Data were collected for leaf length, leaf width, shoot and root length, seedling weight, shoot weight, root weight, branch number, leaf area index (LAI) and water use efficiency (WUE). Result of mean comparisons between three groups (2x, induced 4x and controls 4x), showed that both groups of induced and natural tetraploids had significantly higher mean val- ues for all the traits except leaf length, shoot length and root length than that for dip- loids. For the latter traits there were no significant differences between 2x and induced 4x. On the overall, the induced 4x had 60%, 62%, 68%, 65%, 22%, 109% and 47% high- er mean values than their parental 2x for seedling weight, shoot weight, root weight, LAI, WUE and branch number, respectively. It was concluded that increasing ploidy level provides plant breeders with a promising tool in the breeding improve new varie- ties suited for future climate scenarios. Keywords: ploidy level, colchicine, morphological traits, annual Medicago, perennial Medicago. 76 Elham Ansari, Mahmood Khosrowshahli, Ali Ashraf Jafari, Alireza Etminan INTRODUCTION Alfalfa (Medicago sativa) is a strategic and impor- tant forage species for animal feeding. A limited num- ber of Medicago species are cultivated for animal uses or for breeding improved varieties. Among these species, Medicago sativa is widespread in the most parts of the world and its wild forms are rarely available. Commer- cial varieties of M. sativa are perennial cross-pollinated autotetraploid (2n=4x=32), with Tetrasomic inheritance. M. sativa has both diploid and tetraploid forms (Lapiņa et al., 2011). The sub species of Caerulea is a perennial and diploid form (2n=2x=16). Theoretically, conventional alfalfa has been evolved by sexual polyploidy, which is equivalent to crossing non-reduced (2x) gametes present in diploid species (Bauchan and Hossain, 1997; Rosellini et al., 2016; Pfeiffer and Bingham, 1983). Caerulea’s germplasm has the unused potential of being selected as a perennial, sustainable, drought-resistant and soil improving and pasture rehabilitation (Li et al., 2010). Annual alfalfas are more resistant to plant pests than perennial alfalfa (Bauchan and Azhar, 1998) and can be used in breeding programs as a genetic source to fungal and pest diseases resistance (Yaege and Stute- ville, 2000), cold resistance and resistance to adverse and acidic soil conditions (Gillespie, 1989). Annual alfalfas through fertility and improved physical quality,  nitro- gen fixation, increased organic carbon of soils (Dalal et al., 1995) have a positive effect on grain yield of further grain crops. In addition, with the development of annu- al alfalfa cultivation, significant amounts of forage can be obtained. Annual alfalfa can grow in summer and fall and planted in rotation with wheat. They also pre- vent soil erosion (Biederbeck et al., 1993; Badaroddin and Meyer, 1990; Mirzaei Nodoushan, 2001). For many years, common alfalfa has been improved based on the classic plant breeding program. Alfalfa varieties are usu- ally synthetic varieties; Developed by crossing of selected heterozygous parents and their offspring have advanced over three or four generations of seed proliferation (Rowe and Hill, 1999). Duplication of the plant genomes, or polyploidy induction, which leads to changes in some traits, espe- cially in horticultural, pharmaceutical and agronomic plant species, is based on the application of different drugs such as colchicine, new dinitroanilines, phos- phorothioamidates oryzaline, and triflurarin etc. (Mel- nychuk et al, 2020; Niazian et al, 2020; Su-Jin et al, 2020; Touchell et al, 2020). Colchicine still remains the most efficient and clearly the most preferred and the most used anti mitotic agents because of its widely successful mitosis inhibition ability (Touchell et al, 2020), and for its advantages such as high percentage of viability (Mel- nychuk et al, 2020) and because of its widely successful mitosis inhibition ability, high solubility in water and ethyl alcohol and heat stability, ability to be autoclaved and easily applied to plant tissues solutions. Therefor reduce the use of additional solvents, it is heat-stable, and can be autoclaved and easily applied to plant tissues (Touchell et al, 2020). In the context of successful sexual autopolyploidy induction (by crossing (in alfalfa species, a comprehen- sive research was conducted by Rosellini et al. (2016). Osborn et al., (2003) in their review concluded that a novel variation in polyploids could involve changes in gene expression through increased variation in dosage- regulated gene expression, altered regulatory interac- tions, and rapid genetic and epigenetic changes. Mirzaei Nodushan, (2001) reported that induction of polyploidy in self-pollinated and annual species is likely to improve their morphological and physiological properties and probably can facilitate the adequate gene transfer. There are some reports on phylogeny of domestic and foreign populations of Medicago genus in Iran. Gha- navati et al. (2005) in study of Genetic diversity of 22 species of genus Medicago collected from Iranian natu- ral habitat using RAPD marker generated a phylogenet- ic tree with 5 main cluster. Populations of M. aculeala, M.constricta, M.rigiduloidos and M. rigidula with hard pod walls and spongy texture were classified in one clus- ter. Populations of M. sauvagei, M. laciniata and M. poly- morpha which had soft and flexible pod walls were classi- fied together in a separate cluster. In another experiment Ghanavat (2010) in phylogenetic analysis of 23 species of Medicago based on 90 morphological characteristics by maximum parsimony approach, observed the most rela- tionships between M. rugosa and M. scutellata, M. sativa and M. lupulina, M. coronata and M. minima, M. rigid- ula and M. rigiduloides, M. polymorpha and M. arabica, M. tornata and M. turbinata. Salimpour (2012) in phylo- genetic analysis of 23 species of alfalfa, the most phylo- genetical relationships was observed between M. rugosa and M. scutellata, M. sativa and M. lupulina, M. coro- nata and M. minima, M. rigidula and M. rigiduloides, M. polymorpha and M. arabica, M. tornata and M turbinata. This information can be used in determining the dgree of success of inter-specific hybridization between different species in Medicago genus. There are many comparative cytogenetic analyses between diploid and tetraploid perennial medics (Yu et al. 2017). But there are few published reports of comparative cytogenetic analysis between perennial and annual dip- loid medics and their induced tetraploids. The aim of this study was to generate the best populations of annual and 77Induction of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics perennial diploid medics using polyploidy breeding after induction by colchicine treatment and to compare chang- es of morpho-physiological traits between diploids 2x and induced 4x species and commerical 4x alfalfa cultivars. MATERIALS AND METHODS: In this study, seeds of five annual diploid alfalfa were provided from natural resource gene bank of the Institute of Forests and Rangelands, Tehran, Iran (Table 1). At the same time, seeds of three perennial diploid were provided from College of Agriculture, Tehran University and seeds of five commercial varieties were obtained from Pakan Seed Company, Isfahan, Iran. Prior to seed sowing, seeds were scarified mechani- cally with sand paper and then sterilized with a beno- myl fungicide. Seeds of five annual diploid medics (M. lupulina, M. radiate, M. rigidula, M. truncatula, M. tur- binate) and three perennial diploid Medicago sativa ssp. Caerulea populations (Karaj1, Karaj2 and Tehran) were sown in a mixture of peat moss and field soil at a ratio of 2: 1 in plastic pots with 17 cm diameter. Pots were kept in 20-25°C glasshouses and were irrigated regu- larly. About three months after sowing, 20 rooted single node cuttings from each of the young plants of diploid seedlings in mitosis stage subjected to three concentra- tions of colchicine solution (0.1, 0.5, and 1%) using drop- per method. A drop of colchicine in the lateral bud was put and was repeated for four days after drying or being absorbed and after 15 days, the viability of cuttings was evaluated. The survival rate of the plants was calculated based on the number of well-established plants on the total number of cuttings treated in all the species stud- ied. The induced tetraploids were identified using the usual method of chromosomes counting in the meta- phase cells of root tip meristems with having a mini- mum of 10 metaphase plate mitosis for each species/ populations. The highest induced tetraploidy (80.2%) was obtained using 0.1% colchicine concentration. Then, all of the 21 entries: 5 annual diploids and 5 induced tetra- ploids, 3 perennial diploids and 3 induced tetraploids of Medicago sativa ssp. Caerulea and 5 commercial tetraploid alfalfa cultivars as control (Baghdadi, Bami, Hmamadani, Renger, Ghareyounje) grown in pots in a glasshouse using completely randomized design with five replications in 2018 in Ahvaz, Iran. In diploid and induced tetraploids (using 0.1% col- chicine) and commercial tetraploids, data collected for leaf length (mm), leaf width (mm), shoot length (cm), root length (cm), shoot weight per plant (g), root weight (g), seedling weight (g) and, branches number per seedling, leaf area index (LAI) and water use efficiency (WUE). After about two months after growth of seed- lings and induction polyploidy plants, middle leaves of seedlings were selected for measurements. LAI meas- urements were performed manually using leaf plot and shadow measurements using mm checkerboard paper. The WUE was estimated as , where: Y= water benefi- cially used (seedling dry weight) and W=total amount of water that was estimated for each plant during the growth period. The data were analyzed by completely randomized design experiment and means were com- pared using Tukey’s test. The SAS9.1 software was used for analysis of variance and mean comparisons. RESULTS In the present study the effects of polyploidy induc- tion on morph-physiological traits in five annual and three perennial diploid medics were assessed and they were subjected to different concentrations of colchicine solution (0.1%, 0.5%, 1% and 2%) in mitosis stage. The highest survival rate (80.2%) and tetraploidy induction (74.3%) were obtained in 0.1% colchicine concentration. Result of analysis of variance showed a significant difference between all of the 21 entries for all of traits (P<0.01). Such differences were predictable due to the origin and genome differences and ploidy levels between populations (Table 2). The Maximum of coefficient of variation (CV%) was 21.77% for root length and mini- mum was 11.87% for seedling weight, indicating that good accuracy of the experiment in evaluating traits. Table 1. Name, Specifications and Origin of Annual Diploid alfalfa. Scientific name Code Origin of seed Province Altitude m Latitude Longitude Medicago turbinate 24646 Kermanshah Kermanshah 1200 34°08’00” 46°10’00” Medicago rigidula 45078 Dallahou Kermanshah 1213 34°23’34” 46°03’31” Medicago radiate 44137 Izzeh Khozestan 767 35°36’07” 39°03’78” Medicago lupulina 20307 Unknown Medicago truncatula 20587 Unknown 78 Elham Ansari, Mahmood Khosrowshahli, Ali Ashraf Jafari, Alireza Etminan Mean comparisons were made between average over three groups of 2x, induced 4x and controls cv. 4x (Table 3). Result showed that the higher values of all traits were observed in control (cv. 4x) followed by induced tetraploid in terms of leaf width, shoot length, shoot weight, seedling weight, WUE and branches number. In the other word, there were no significant differences between control cv. 4x and induced tetra- ploids for the latter traits. However, for leaf length, root length, root weight and LAI, the means of induced tetraploids were significantly lower than that for con- trols (cv. 4x). The overall means of diploids were ranked as third place, however, for leaf length and root length. There was no significant differences between 2x and induced 4x. for other traits as: leaf width, shoot weight, root weight, seedling weight, LAI, WUE and branch number, the means of induced tetraploids were signifi- cantly higher than diploids (Table 3). A separate mean comparisons were made among only naturally tetraploids varieties (Table 4). Result showed Baghdadi cv. had higher mean values for all of traits except root length and root weight. For these two traits, the higher values were observed in Ghareyounja cv. and for other traits, the Hamadani cv. ranked in the third place (Table 4). Mean comparison were made between populations separately for diploids and induced tetraploids (Tables 5 and 6). For diploid populations, the higher values of WUE, LAI, root and shoot weight, seedling weight, root and shoot length, leaf size were obtained in M. trunca- tula 2x followed by M lupulina 2x as the second place. Both species are annual (Table 6). In comparisons between perennial diploids, there was no significant differences between Tehran Karaj1 and Karaj2 2x for WUE, seedling, shoot weight, root and shoot length and leaf width. For branch number the higher value was obtained in Tehran 2x (Table 5). Simi- larly, in comparisons between perennial induced tetra- ploids Tehran 4x, ranked in the first class than two other induced perennial populations in terms of the morpho- logical traits (Table 5 and 6). In comparisons between annual induced tetra- ploids, the highest values of all of traits except of LAI and branches number were observed in M trancatula 4x. Similarly, M. lupulina 4x in terms of leaf width, root weight, LAI, branches number was ranked in the first class and for the shoot and root length, shoot weight and seedling weight it was ranked as the second class. In comparisons of total average of 2x and total aver- age of induced 4x groups, result showed in M. radiate by increasing ploidy level,  leaf length and leaf width decreased by 12% and 15%, respectively. In contrast, for M. rigidula the leaf length and leaf width of the 4x, increased by 15 and 42% than 2x, respectively. In the other diploid species, the increase in ploidy level showed no significant effect on leaf size, but a decrease in root and shoot length was observed in all 2x species rang- ing from 7 to 30% for shoot length and between 15 and 51% for roots length. Thus, root weight and WUE value in 4x were higher than twice that of 2x. In contrast, low increase was observed for LAI (Table 5 and 6). In overall, there were no significant differences between 2x and 4x for leaf size and LAI. The induced 4x had 25% and 38% lower values than 2x for Shoot and Table 2. Analysis of variance studied traits in 23 medic species/populations. SOV DF Leaf length Leaf width Shoot length Root length Seedling weight Shoot weight Root weight Leaf area index Water use efficiency Branch number Populations 20 30.17** 39.07** 1074.9** 966.5** 0.476** 0.712** 0.039** 3.26** 0.384** 583.7** Error 79 4.75 3.73 8.63 13.30 0.006 0.012 0.002 0.11 0.010 13.08 CV% 15.49 17.49 14.01 21.77 11.87 12.51 21.07 14.87 16.52 15.99 ** =Significant at the 1% probability level. Table 3. Means Comparison of morpho-physiological traits based on total ploidy levels in all studied alfalfa species. Ploidy levels Leaf length Mm Leaf width mm Shoot length cm Root length cm seedling Weight g/plant shoot weight g/plant Root weight g/plant Leaf area index Water use efficiency Branch number Perennial 4x (Control) 15.88 a 11.96 a 26.30 a 25.51 a 1.08 a 0.77 a 0.31 a 2.96 a 0.26 a 26.04 a Diploids 2x 13.02 b 9.90 b 18.97 b 14.46 b 0.63 b 0.47 b 0.16 c 1.89 c 0.11 b 17.64 b Induced tetraploids 14.23 b 11.70 a 20.33 ab 14.79 b 1.02 a 0.76 a 0.27 b 2.31 b 0.23 a 25.68 a Means with similar letters in each column has no significant difference at 5% probability level by Tukey test. 79Induction of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics Table 4. Means Comparison of morpho-physiological traits in perennial alfalfa cultivars. Commercial Varieties 4x Leaf length mm Leaf width mm Shoot length cm Root length cm Seedling weight g/plant Shoot weight g/plant Root weight g/plant Leaf area index Water use efficiency Branch number Baghdadi 19.60 a 13.00 a 39.18 a 27.54 b 1.32 ab 1.07 a 0.25 b 3.43 a 0.42 a 37.80 a Bami 14.10b 10.90 a 18.30 c 18.90 c 0.72 c 0.46 c 0.26 b 2.00 b 0.17 c 22.80 b Ghareyounje 14.70b 11.50 a 29.18b 32.48 a 1.27 b 0.91 b 0.35 a 3.30 a 0.29 b 18.40 bc Hamadani 15.60 ab 13.00 a 26.46b 27.68 b 1.45 a 1.07 a 0.38 a 3.25 a 0.31 b 35.00 a Renger 15.40b 11.40 a 18.38c 20.96 c 0.63 c 0.35 c 0.28 b 2.82 ab 0.13 c 16.20 c Means with similar letters in each column has no significant difference at 5% probability level by Tukey test Table 5. Mean comparison of diploid Medicago species and their induced tetraploids for Leaf length, leaf width, shoot length, root length and seedling weight. Populations Leaf length mm Leaf width mm Shoot length cm Root length cm Seedling weight g/plant 2x 4x 2x 4x 2x 4x 2x 4x 2x 4x M. sativa (Karaj1) 12.20 b 12.43 c 10.60 bc 10.57 b 14.64 b 11.30b 13.00 b 8.81b 0.59 c 0.75 cd M. sativa (Karaj2) 17.00 a 16.86 a 15.00 a 14.86 a 15.54 b 10.83b 8.60 b 5.43b 0.53 c 0.88 bc M. sativa (Tehran) 12.40 b 12.00 c 10.60 bc 10.83 b 16.78 b 12.47 b 9.50 b 5.92 b 0.60 bc 1.01 b M lupulina 12.60 b 12.86 c 13.60 ab 13.43 a 14.54 b 10.69b 11.92 b 5.77b 0.68 b 0.96 b M. radiata 13.60 ab 12.00 c 9.20 cd 7.80 c 16.78 b 11.86 b 11.28 b 7.80 b 0.61 bc 0.90 bc M. Rigidula 11.60 bc 13.40 bc 6.60 de 9.40 bc 15.10 b 11.48 b 12.10 b 8.84 b 0.64 b 0.93 b M. truncatula 15.00 ab 15.75 ab 12.80 b 13.25 a 54.52 a 50.44 a 51.58 a 40.63 a 1.32 a 1.54 a M. turbinata 15.80 ab 16.25 a 8.80 d 9.50 bc 14.86 b 12.75 b 11.08 b 9.33 b 0.43 cd 0.61 d Average 13.78 13.94 10.90 11.21 20.35A 16.48B 16.13A 11.57B 0.68B 0.95A Means with similar letters in each column has no significant difference at 5% probability level by Tukey test. Table 6. Mean comparison of diploid medicago species and their induced tetraploids for shoot weight, root weight, LAI, WUE and branch number. Population name Shoot weight g/plant Root weight g/plant Leaf area index Water use efficiency Branch number 2x 4x 2x 4x 2x 4x 2x 4x 2x 4x M. sativa (Karaj1) 0.38 cd 0.48 c 0.20 b 0.27 ab 2.10 bc 2.18 d 0.08 de 0.17 c 15.00 c 17.14 c M. sativa (Karaj2) 0.41 cd 0.63b 0.12 c 0.25 ab 2.21 bc 2.33 c 0.09 d 0.18 c 16.60 c 19.00 c M. sativa (Tehran) 0.48 bc 0.74 b 0.12 c 0.27 ab 1.53 c 1.65 f 0.11 cd 0.21 bc 16.40 c 18.00 c M. lupulina 0.55 b 0.71b 0.13 c 0.25 ab 3.22 a 3.41 a 0.17 b 0.23 b 35.60 a 44.00 a M. radiata 0.47 bc 0.67b 0.14 bc 0.23 ab 1.35 c 1.45 g 0.10 d 0.18 c 13.20 c 18.60 c M. Rigidula 0.46 bc 0.64b 0.18 bc 0.29 ab 1.32 c 1.42 g 0.13 cb 0.18 c 14.40 c 17.60 c M. truncatula 0.95 a 1.22 a 0.37 a 0.31 a 2.82 ab 2.96 b 0.24 a 0.37 a 28.80 b 37.08 b M. turbinata 0.29 d 0.40 c 0.14 bc 0.21 b 1.64 c 1.78 e 0.07 de 0.11 d 12.80 c 16.50 c Average 0.50B 0.69A 0.18B 0.26A 2.02B 2.15A 0.12B 0.20A 19.10B 23.49A Means with similar letters in each column has no significant difference at 5% probability level by Tukey test. 80 Elham Ansari, Mahmood Khosrowshahli, Ali Ashraf Jafari, Alireza Etminan root length, respectively. In contrast, the induced 4x had 40%, 38%, 44%, 65% and 23% higher values than 2x for seedling weight, shoot weight, root weight, WUE and branch number, respectively (Tables 5 and 6). DISCUSSION In this study, we found higher ploidy induction at the lower colchicine concentration (0.1%). Colchicine still remains the most efficient and clearly the most pre- ferred and the most used anti mitotic agents because of its widely successful mitosis inhibition ability (Touchell et al, 2020). The others anti-mitotic agents such as dini- troanilines and etc.. may also increase the ploidy levels. In addition, increasing of the ploidy levels can prob- ably be an appropriate solution for crossing of various alfalfa species and increasing their genetic diversity and transferring desirable traits. It seems polyploidy induc- tion in self-pollinated annual medic species are likely to improve the morphological and physiological character- istics in these species. The result indicated that induced 4x had 25% and 38% lower values for the shoot and root length than 2x, respectively, averaged over all of species. The reduction in root length of M. truncatula was higher than that of other species. The results of the present study were in line with results of Pickens (2004) showing shorter shoots of colchicine treated plants compared to not- treated controls. Overall, the induced 4x had 60%, 62%, 68%, 65%, 22%, 109% and 47% higher mean values than their 2x for seedling weight, shoot weight, root weight, LAI, WUE and branch number, respectively. In M. trunca- tula, the seedling weight of induced 4x was 16% higher than 2x (1.32 vs. 1.54 g/p). This result was in agreement with the result of the polyploidy induction in Catharan- thus roseus using colchicine solution that significantly increased the seedling weight of tetraploid plants com- pared to diploid plants (Hosseini et al. 2013). Shoot weight was higher in all induced species than the diploids. The higher values of shoot weight among all of population were obtained in M. truncatula. In this species, the value of induced 4x was 28% higher than its parental 2x. This result was in line with the Staji et al. (2017) that found polyploidy induction significantly increased shoot weight in Salvia leriifolia. Tavan (2014) in some endemic species of Thyme genus in Iran found that increase in ploidy level was associated with an increase in shoot weight. Similarly, Bagheri and Man- souri (2015) in cannabis (Cannabis sativa L.) found that the root and seedling weight of polyploid plants were significantly increased than diploid plants. In our study, the higher values of LAI were obtained in M. lupulina. LAI was higher in all induced species than the dip- loids. In the overall average over all species, the LAI of induced 4x was 7% higher than 2x (2.02 vs. 2.15). As the ploidy levels increased, the average value of WUE in all studied species increased up to 65%. Con- tributing factors to WUE are related to climate factors that are essential for water use (evapotranspiration) and water supply (atmospheric rain), management factors for cultivation and operations lead to evaporation reduc- tion from the soil surface (Kafi and Damghani 2001). It seems that M. truncatula has probably been the best choice for polyploidy induction in annual species. Ploidy changes in cell size are due to an increase in the number of copies of genes and thus an increase in the amount of protein produced (Tsukaya 2013). Nowa- days, it has been found that control of morphological traits at the molecular level by ploidy level alteration via genetic (Tsukaya 2013), transcriptomic (Li et al. 2012) and epigenetic (Zeng et al. 2012, Aversano et al. 2012) modification occurs. For example, changes in the genomic dose of polyploids lead to changes in the expression of genes involved in cell cycle, photosynthe- sis, and cell metabolism (Shi et al. 2015), As a result of autotetraploidy, alterations in the expression of genes related to stress response, hormonal signaling actions, and response to phytohormones are applied Which may lead to a flexible and rapid response to external and internal stimuli (Del Pozo and Ramirez 2014). Slight but pervasive changes in gene expression are probably corre- lated with large phenotypic differences in autotetraploids (Allario et al. 2013). In some reports, autotetraploidy has caused epigenetic alteration by DNA methylation at the functional genes sites, encoding proteins and at the sequences involved in DNA replication, electron trans- port chain, and transcriptional regulation (Zeng et al. 2012). In some cases, methylation has been the result of cytosine methylation at the CG and CHG sites (Aver- sano et al. 2012). probably a combination of chromo- some duplication, along with genetic, epigenetic, and transcriptomic effects, can produce morphophysiological differences due to polyploidy induction, as in our experi- ment (Yang et al. 2011). CONCLUSION In addition to the highest survival rate and most successful induction after colchicine treatment, the induced tetraploid of M. truncatula, among all of eval- uated species, showed the highest levels of morpho- 81Induction of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics logical indices. It seems that if the goal of breeding is to increase in economical properties such as seedling weight and WUE etc. It seems that M. truncatula prob- ably is the best choice for polyploidy induction in annual species. All perennial diploids did not show any un-uni- formity by increasing ploidy level in this study, the increasing of the ploidy level of agronomic “perenni- al” tetrapleoids may be a more effective step to achieve superior new cultivars. In comparing diploid alfalfas, the annual species, showed higher mean values for morphological traits than perennial diploid alfalfas. In comparisons among the three perennial “diploid” populations, Tehran had higher mean values for shoot length, shoot weight, seed- ling weight and WUE. Therefore, Tehran population is advisable for breeding improve new variety of perennial tetraploid via induce polyploidy. As a result, increasing the ploidy level 2x to 4x is a successful way to obtain superior morphological and physiological indices in the studied species and subspecies. In overall, there were no significant differences between 2x and 4x for leaf size and LAI. The induced 4x had 25% and 38% lower values than 2x for Shoot and root length, respectively. 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