Journal of Applied Botany and Food Quality 92, 88 - 93 (2019), DOI:10.5073/JABFQ.2019.092.012 1Department of Field Crops, Faculty of Agriculture, Eskişehir Osmangazi University, Eskişehir, Turkey 2Department of Field Crops, Faculty of Agriculture, Namık Kemal University, Tekirdağ, Turkey Autotetraploid plant production in endemic Onobrychis elata with colchicine treatments Süleyman Avci1*, Metin Tuna2, Mehmet Demir Kaya1 (Submitted: December 10, 2018; Accepted: February 22, 2019) * Corresponding author Summary This study aimed to induce autotetraploidy in endemic Onobrychis elata plants by colchicine treatment of seeds or seedlings. Colchicine was applied to O. elata directly on germinated seeds, pre-germinated seeds (root length of 3-8 mm), and apical regions (using cotton) under in vivo conditions. Out of a total of 1,210 colchicine-treated seeds that were evaluated, only 203 survived. There was an inverse re- lationship between the number of surviving plants and colchicine concentration and exposure time. The highest percentage of tetra- ploidy in surviving plants (50%) was obtained by applying 0.2% colchicine for 6 hours to pre-germinated seeds. No significant tetra- ploidy was achieved by colchicine application to seedlings. Flow cytometry observations indicated that DNA content varied between 0.99 and 1.06 pg in diploid plants (controls), while DNA content varied between 2.22 and 2.48 pg in tetraploid plants. It was con- cluded that tetraploid plants were induced successfully only in seed- lings obtained from pre-germinated seeds, with their ploidy level confirmed via flow cytometry analysis. Key words: Onobrychis, colchicine, flow cytometry, polyploidy Introduction Sainfoin (Onobrychis viciifolia Scop.) is an important forage le- gume for hay and pasture in calcareous and arid lands because of its well-developed root system and adaptability to water limitations (Jefferson et al., 1994; Borreani et al., 2003). The nutritive values of hay are similar to alfalfa, and because hay contains appreciable amounts of condensed tannins, animals grazed in pure pasture are not at risk of bloat (Wang et al., 2015; Bhattarai et al., 2016). There are approximately 170 species of the genus Onobrychis around the world, located mainly in southwestern Asia, the Mediterra- nean, and temperate regions of Europe and Asia (Cronquist, 1981; Zohary, 1987; aktoklu, 2001). Wild Onobrychis species consti- tute important genetic resources for breeding sainfoin and improving grassland (avCi et al., 2013; ÖZaslan Parlak and Parlak, 2008). In Turkey, there are 55 Onobrychis species with two subgenera (Onobrychis and Sisyrosema) and five sections (Dendobrychis, Laphobrychis, Onobrychis, Hymenobrychis, and Heliobrychis); 28 of them are endemic (aktoklu, 2001; avCi et al., 2013). Onobrychis elata Boiss. et Bal. is a valuable native species that is widely scat- tered due to its morphological similarity to cultivated O. viciifolia (avCi et al., 2013). O. elata is diploid (2n = 14) and has a basic chro- mosome number of x = 7 (Cartier, 1976). It has been considered an important potential wild genetic source for hybridization with O. viciifolia to help further forage production under biotic and abiotic stresses; therefore, it would be advantageous to double its chromo- some number. Autopolyploids originate from the combination of unreduced ga- metes in nature and can also sometimes be artificially stimulated (Chen, 2010). Chromosome doubling is used to more efficiently breed superior varieties with high yields of forage crops (aCquaah, 2007; Meru, 2012). Moreover, polyploidy plays an important role in the fertility of interspecific hybridization between cultivated species and their wild relatives (falCinelli, 1999; aversano et al., 2012). Colchicine treatment is the most effective and widely used method of producing polyploidy in forage legumes. anderson et al. (1991) obtained polyploidy in Trifolium plants and their hybrids by treating seeds, seedlings, and growing shoot apices with colchicine. Similar results were achieved by diByendu (2010) in Lathyrus sativus L., ZeinaB et al. (2012) in Trifolium alexandrinum L., and Wu et al. (2015) in Stylosanthes guianensis (Aublet) Sw. However, research on polyploidy in Onobrychis species has not been found in the literature. This study aimed to produce autotetraploid plants from diploid O. elata by colchicine treatment under in vivo conditions. Materials and methods O. elata is a perennial with erect stems measuring approximately 60-120 cm (Fig. 1A). Leaves include 5-7 pairs of narrow elliptical leaflets (Fig. 1B). The raceme is many flowered with a loose structure (Fig. 1C), and the corolla of the flower is rose with deeper striations (Fig. 1D). The fruit is suborbicular and includes long and short spiny teeth on the crest and disk, respectively (Fig. 1E). The seed is roughly kidney-shaped (reniform), and seed color varies from light brown to dark brown (Fig. 1F). Seeds of O. elata were collected from Mount Erciyes (1,443 m), Kayseri, Turkey, and stored at 4 °C until used. The seed coats were very hard and impermeable; consequently, they were abraded with sandpaper as described by avCi and kaya (2013). The seeds were then surface sterilized in 96% alcohol for 2 minutes and rinsed twice with distilled water. Germination was performed in petri dishes (100 mm × 20 mm) on top of two filter papers moistened with distilled water. Each petri dish was then sealed with laboratory film, and the seeds were allowed to germinate in the dark at 20 ± 1 °C. Colchicine (Sigma catalog no.: C9754) was dissolved in water at room temperature, and the aqueous solution was stored at 4 °C. The colchicine was directly applied to germinated seeds at two concen- trations (0.05% and 0.1%) and three exposure times (24, 48, and 72 hours). Pre-germinated seeds with 3-8 mm root lengths were treated with four concentrations (0.1%, 0.2%, 0.3%, and 0.4%) and three exposure times (6, 12, and 24 hours), followed by rinsing. In addition, three concentrations of colchicine (0.05%, 0.1%, and 0.2%) at two exposure times (48 and 72 hours) were applied with cotton to the apical regions of 10-day-old seedlings. The surviving seedlings were transferred into pots containing a mixture of peat and perlite (3:1 v/v) and incubated for a 16-hour photoperiod day of 8,000- 10,000 lux, at 20 ± 1 °C and 60% humidity. Ploidy analysis was performed using flow cytometry, which is the newest, fastest, most accurate, and most economical method for this purpose. First, rice, tomato, common vetch, barley, and safflower plants were analyzed separately with diploid O. elata, and common vetch (Vicia sativa L.) and safflower (Carthamus tinctorius L.) were selected as the most suitable internal standards. Partec kits were used to isolate nuclear DNA from fresh leaf samples derived from both In vivo induction of tetraploid plants in Onobrychis elata 89 Fig. 1: Morphological characteristics of O. elata. (A) general view with erect stem; (B) leaf; (C) raceme; (D) corolla with calyx; (E) fruits; (F) seeds. 90 S. Avci, M. Tuna, M.D. Kaya diploid and colchicine-treated two-month-old plantlets, and DNA content was calculated for each sample using the standard plants. The resulting flow histogram was analyzed with the FloMax packet program, and fluorescence intensity, mean, and CV values were de- termined for each sample. The nuclear DNA content of each O. elata sample was calculated according to the following formula: DNA content = (fluorescence intensity of sample) / (fluorescence intensity of standard) × DNA content of standard. Results and discussion Out of a total of 480 germinated seeds treated with colchicine, 113 seedlings (23.5%) survived, and chromosome doubling was not observed (data not shown). In similar studies of legume species, di- rect treatment of germinated seeds with colchicine did not success- fully induce polyploidy. Patil (1992) reported that germinated seeds of Crotalaria linifolia Linn. treated with colchicine at concentrations of 0.10%, 0.15%, 0.20%, 0.25%, and 0.30% for 6, 12, and 24 hours did not produce polyploid plants. arya et al. (1988) found that colchicine treatment at a concentration of 0.15% for 10 hours was a lethal dose in germinated seeds of fenugreek, and gandhi and Patil (1997) stated that direct treatment of germinated seeds of Clitoria ternatea L. was not a practically efficient means of inducing tetraploid tissue. Colchicine treatment of pre-germinated seeds resulted in 75 survi- ving plantlets (12.5%) out of a total of 596 seedlings. Of those 75, 15 plants (20%) were determined to be tetraploid by flow cytome- try (Tab. 1). As the concentration and exposure time increased, the number of surviving plants decreased, and plants did not survive when treated at concentrations of 0.2%, 0.3%, and 0.4% for 24 hours. Tetraploid plants were observed for all exposure times at 0.1% con- centration, as well as for 6 hours of exposure time at 0.2% concen- tration, which was determined to produce the highest chromosome doubling percentage (50%) in surviving plantlets. Similar results were obtained from Astragalus membranaceus, with the mortality rate higher than 70% at a concentration of 0.3% colchicine over a 24- hour exposure time; the highest chromosome doubling (50%) among surviving plants was obtained from treatment with 0.2% concentra- tion for 6 hours (Chen and gao, 2007). tulay and unal (2010) and Joshi and verMa (2004) reported that when seeds of Vicia villosa Roth and Vicia faba L. were first soaked in water for 20 hours, tetra- ploid plants were attained with colchicine treatment at lower doses (0.005% concentration for 8 hours). Pathak et al. (2015) supported the finding that soaking seeds in water prior to colchicine treatment was quite efficient at promoting polyploidy. When colchicine was applied to the apical regions of 134 seedlings, only 15 (11%) of them survived, and no polyploidic plants were observed (data not shown). Increasing concentration and exposure time negatively affected seedling survival. Treatment with a con- centration of 0.2% for 48 and 72 hours led to death of the plantlets. daBkeviCiene et al. (2016) determined that colchicine treatment to Trifolium pratense L. embryos resulted in 3.3 times more polyploid plants than overhead application to cotyledon leaves of six- to eight- day-old seedlings. Wu et al. (2015) obtained 10% tetraploid induc- tion by applying colchicine to the apical region of S. guianensis at the highest concentration (0.2%) for 48 hours. BeWal et al. (2009) stated that the highest polyploidy rate obtained by colchicine treatment to the apical region of Cyamopsis tetragonoloba L. was observed at 0.2% concentration and 10 hours of exposure time for two days. In contrast to the current findings, previous studies have demon- strated that polyploid plants were obtained from applications to the apical regions of different legume species, and a reduced survival rate was observed at colchicine concentrations over 0.2%. In the fu- ture, new approaches to exposure frequency and times and applica- tion type on the apical region should be evaluated. Colchicine was applied to germinated seeds, pre-germinated seeds, and the apical region of seedlings, and out of a total of 1,210 seed- lings, 203 survived. Of the surviving plantlets, 15 of them, inclu- ding those originating from pre-germinated seeds, were determined to be tetraploid by using flow cytometry (Tab. 2). When safflower (C. tinctorius) was used as the internal standard, nuclear DNA peaks of tetraploid (2n = 28) O. elata overlapped (Fig. 2), but no overlap was observed in nuclear DNA peaks of diploid (2n = 14) plants (Fig. 3). After analysis of all samples with safflower as the internal standard, those with overlapping nuclear DNA peaks and thus considered to be tetraploid were again analyzed with the nuclear DNA of common vetch (V. sativa). As a result of these analyses, tetraploid plants were successfully identified as those without overlap (Fig. 4). The content of nuclear DNA in diploid and tetraploid plants ranged from 0.99 to 1.06 pg and 2.22 to 2.48 pg, respectively (Tab. 2). In conclusion, tetraploid O. elata plants were successfully obtained at a high percentage by treating pre-germinated seeds of diploid plants with colchicine. Increased concentrations of colchicine adversely af- fected the survival rate of the plants. For unsuccessful treatments of the apical region and germinated seeds, modifications can be made to increase the success of chromosome doubling. Thirteen tetraploid Tab. 1: Results of colchicine application to germinated seeds (3-8 mm root length) Colchicine Application time Number of planted Number of Plantlet survival rate Number of Tetraploid plant rate concentration(%) (hours) seedlings surviving plantlets (%) tetraploid plants (%) in surviving plantlets 0.1 6 30 14 47 3 21 12 50 4 8 1 25 24 60 10 17 2 20 0.2 6 50 18 36 9 50 12 50 1 2 0 0 24 41 0 0 0 0 0.3 6 45 12 27 0 0 12 50 1 2 0 0 24 70 0 0 0 0 0.4 6 30 14 47 0 0 12 50 1 2 0 0 24 70 0 0 0 0 Total — 596 75 — 15 — In vivo induction of tetraploid plants in Onobrychis elata 91 Tab. 2: Fluorescence intensity, average DNA content, and CV values in samples with and without colchicine treatment Plant Sample fluorescence Standard fluorescence Sample Standard CV1 CV2 Ploidy number intensity intensity DNA content (pg) DNA content (pg) (O. elata) (standard) level 14 (Control) 119.24 238.72 1.00 2.00* 4.75 3.00 Diploid 24 (Control) 115.42 233.24 0.99 2.00 6.15 3.68 Diploid 32 (Control) 128.94 244.13 1.06 2.00 3.95 4.99 Diploid 69 (Control) 133.78 266.69 1.00 2.00 5.24 5.99 Diploid 57 129.28 197.37 2.39 3.65** 5.32 3.43 Tetraploid 59 134.12 199.27 2.46 3.65 5.52 2.75 Tetraploid 64 119.63 186.20 2.35 3.65 3.97 2.54 Tetraploid 71 93.97 150.29 2.28 3.65 6.33 4.13 Tetraploid 77 125.74 187.67 2.45 3.65 5.98 3.02 Tetraploid 79 149.51 234.60 2.33 3.65 3.28 3.88 Tetraploid 82 123.29 196.15 2.29 3.65 6.39 4.09 Tetraploid 85 122.39 201.41 2.22 3.65 7.81 4.25 Tetraploid 89 139.64 211.13 2.41 3.65 3.63 2.47 Tetraploid 90 139.56 205.61 2.48 3.65 3.78 2.56 Tetraploid 91 118.97 182.09 2.38 3.65 3.82 2.89 Tetraploid 92 129.82 202.40 2.34 3.65 3.63 2.82 Tetraploid 94 141.21 218.06 2.36 3.65 3.27 2.74 Tetraploid 96 129.02 200.04 2.35 3.65 4.48 4.36 Tetraploid 97 143.96 218.05 2.41 3.65 3.92 3.16 Tetraploid * Safflower (C. tinctorius) was used as the standard DNA content. ** Common vetch (V. sativa) was used as the standard DNA content. Fig. 2: Overlap of G1 peaks in tetraploid O. elata and internal standard (safflower) plants were transferred to field conditions for further morpholo- gical, cytological, and palynological observation, and to hybridiza- tion facilities in the sainfoin breeding program. Acknowledgments The author thanks Eskişehir Osmangazi University for its financial support (Project number: 2016-1030). References aCquaah, g., 2007: Principles of plant genetics and breeding. UK, Oxford: Blackwell Publishing. aktoklu, e., 2001: Two new varieties and a new record in Onobrychis from Turkey. Turk. J. Bot. 25, 359-363. anderson, J.a., Mousset-déClas, C., WilliaMs, e.g., taylor, n.l., 1991: An in vitro chromosome doubling method for clovers (Trifolium spp.). Genome, 34(1), 1-5. DOI: 10.1139/g91-001 FL1 UV LED co un ts 92 S. Avci, M. Tuna, M.D. Kaya Fig. 4: Positions of G1 peaks belonging to tetraploid O. elata and standard plants (common vetch). Fig. 3: Positions of G1 peaks belonging to diploid O. elata and standard plants (safflower) arya, i.d., raMa, rao, s., raina, s.n., 1988: Cytomorphological studies of Trigonella foenum-graecum autotetraploids in three (C1, C2, C3) gene- ration. Cytologia. 53, 525-534. DOI: 10.1508/cytologia.53.525 avCi, s., kaya, M.d., 2013: Seed and germination characteristics of wild Onobrychis taxa in Turkey. Turk. J. Agric. For. 37, 555-560. DOI: 10.3906/tar-1211-29 avCi, s., sanCak, C., Can, a., aCar, a., Pinar, n.M., 2013: Pollen mor- phology of the genus Onobrychis (Fabaceae) in Turkey. Turk. J. Bot. 37, 669-681. DOI: 10.3906/bot-1207-52 aversano, r., erColano, M.r., Caruso, i., fasano, C., rosellini, d., CarPuto, d., 2012: Molecular tools for exploring polyploid genomes in plants. Int. J. Mol. Sci. 13(8), 10316-10335. DOI: 10.3390/ijms130810316 BeWal, s., Purohit, J., kuMar, a., khedasana, r., raMa, rao s., 2009: Cytogenetical investigations in colchicine-induced tetraploids of Cyamopsis tetragonoloba L. Czech J. Genet. Plant Breed. 45, 143-154. Bhattarai, s., CoulMan, B., Biligetu, B., 2016: Assessment of sainfoin (Onobrychis viciifolia Scop.) germplasm for agro-morphological traits and nutritive value. Can. J. Plant Sci. 96, 748-756. DOI: 10.1139/cjps-2015-0378 Borreani, g., Peiretti, P.g., taBaCCo e., 2003: Evolution of yield and quality of sainfoin (Onobrychis viciifolia Scop.) in the spring growth cycle. Agronomie. 23, 193-201. DOI: 10.1051/agro:2002082 Cartier, d., 1976: In IOPB chromosome number reports LIII. Taxon. 25, 492-494. FL1 UV LED co un ts G1 peak of diploid O. elata G1 peak of standard plant (safflower) FL1 UV LED co un ts G1 peak of tetraploid O. elata G1 peak of standard plant (common vetch) In vivo induction of tetraploid plants in Onobrychis elata 93 Chen, l.l., gao, s.l, 2007: In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus. Sci. Hort. 112(3), 339-344. DOI: 10.1016/j.scienta.2006.12.045 Chen, Z., 2010: Molecular mechanisms of polyploidy and hybrid vigor. Trends Plant Sci. 15 (2), 57-71. DOI: 10.1016/j.tplants.2009.12.003. Epub 2010 Jan 18. Cronquist, a., 1981: An integrated system of classification of flowering plants. USA, New York: Columbia University Press. daBkeviCiene, g., statkeviCiute, g., Mikaliuniene, J., norkeviCiene, e., keMesyte, v., 2016: Production of Trifolium pratense L. and T. hy- bridum L. tetraploid populations and assessment of their agrobiological characteristics. Zemdirbyste. 103(4), 377-384. DOI: 10.13080/z-a.2016.103.048 diByendu, t., 2010: Cytogenetic characterization of induced autotetraploids in grass pea (Lathyrus sativus L.). Caryologia, 63 (1), 62-72. DOI: 10.1080/00087114.2010.10589709. falCinelli, M., 1999: Temperate forage seed production: conventional and potential breeding strategies. J. New Seeds 1, 37-66. DOI: 10.1300/J153v01n01_04. gandhi, s., Patil, v.P., 1997: Colchicine-Induced autotetraploidy in Clitoria ternatea L. Cytologia. 62(1), 13-18. DOI: 10.1508/cytologia.62.13 Jefferson, P.g., laWrenCe, t., lrvine, r.B., kielly, g.a., 1994: Evalua- tion of sainfoin-alfalta mixtures for forage production and compatibility at a semi-arid location in southern Saskatchewan. Can. J. Plant Sci. 74(4), 785-791. DOI: 10.4141/cjps94-140 Joshi, P., verMa, r.C., 2004: High frequency production of colchicine in- duced autotetraploids in faba bean (Vicia faba L.). Cytologia. 69(2), 141- 147. DOI: 10.1508/cytologia.69.141 Meru, g.M., 2012: Polyploidy and its implications in plant breeding. In: McGregor, C., Brummer, C. (eds.), Plant Breeding in the 21st Century. UGA press. Pathak, s., Malaviya, d.r., roy, a.k., dWivedi, k., kaushal, P., 2015: Multifoliate leaf formation in induced tetraploids of Trifolium alexan- drinum L. Cytologia. 80(1), 59-66. DOI: 10.1508/cytologia.80.59 ÖZaslan Parlak, a., Parlak, M., 2008: Effect of salinity in irrigation water on some plant development parameters of sainfoin (Onobrychis viciifolia Scop.) and soil salinity. Tarım Bilim. Derg. 14, 320-325. Patil, B.C., 1992: The Induction of tetraploid in Crotalaria linifolia Linn. Cytologia. 57; 247-252. DOI: 10.1508/cytologia.57.247 tulay, e., unal, M., 2010: Production of colchicine induced tetraploids in Vicia villosa roth. Caryologia. 63 (3), 292-303. DOI: 10.1080/00087114.2010.10589739 Wang, y., MCallister, t.a., aCharya, s., 2015: Condensed tannins in sainfoin: composition, concentration, and effects on nutritive and feeding value of sainfoin forage. Crop Sci. 55, 13-22. DOI: 10.2135/cropsci2014.07.0489 Wu, f.h., yu, X.d., Zhuang, n.s., liu, g.d., liu, J.P., 2015: Induction and identification of Stylosanthes guianensis tetraploids. Genet. Mol. Res. 14 (4), 12692-12698. DOI: 10.4238/2015.October.19.13 ZeinaB, M., naBila, a.M., khalid, h.r., dina, a., 2012: Colchicine in- duction of polyploidy in Egyptian clover genotypes. J. Am. Sci. 8(10), 221-227. Zohary, M., 1987: Onobrychis. In: Zohary, M. (ed.), Flora Palaestina, 158- 164, Vol. 2. Jerusalem: Academic Science Human. ORCID Süleyman Avci https://orcid.org/0000-0002-4653-5567 Metin Tuna https://orcid.org/0000-0003-4841-8871 Mehmet Demir Kaya https://orcid.org/0000-0002-4681-2464 Address of corresponding author: Süleyman Avcı: Department of Field Crops, Faculty of Agriculture, Uni- versity of Eskişehir Osmangazi, Odunpazarı, Eskişehir, Turkey, 26160. E-mail: savci@ogu.edu.tr © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en).