Hayati BAT, Fatma Nur ALTINDAĞ, Merve Arefe YİĞİT, Şeküre Şebnem ELLİALTIOĞLU, Nuray ÇÖMLEKÇİOĞLU

Ploidy estimation in pepper and eggplant via stomata characteristics

The most reliable methods to confirm the ploidy level in plants are to count the chromosomes or to measure the DNA in the cells by flow cytometry. However, these methods are laborious, time consuming, and require special equipment. In this study, the reliability of stomatal characteristics in confirming the ploidy level was investigated in haploid and spontaneous double haploid (SDH) pepper and eggplant plants. Stomatal characteristics were measured using a digital camera and related software from light microscope images in 100 samples at each ploidy level. Stomatal density, guard cell width and length were measured in random fields of view, and chloroplasts were counted. Mean stomatal lengths were determined as 28.34 µm and 40.39 µm, and mean stomatal widths were determined as 22.52 µm and 29.50 µm respectively for haploid and SDH plants in pepper. The stomatal density was 10.71 in haploid and 27.07 in SDH. Average stomatal lengths were determined as 22.32 µm and 32.00 µm, and mean stomatal widths were determined as 17.36 µm and 22.32 µm, respectively, in haploid and SDH eggplant. The stomatal density of eggplants were found to be 29.20 in haploid plants and 12.61 in SDHs. Chloroplast numbers in guard cells of SDH plants were determined to be 2 fold more than haploids. In haploid and SDH peppers 9.93 and 18.66 chloroplasts were counted, respectively, and 6.39 and 11.19 chloroplasts were counted in eggplants, respectively. There were positive relationships between stomatal size and chloroplast number and ploidy level, and negative relationships between stomatal density and ploidy level, which can be presented as an early marker to determination ploidy levels in both species.

Keywords:

chloroplast, ploidy level, stomata, anther culture,

PDF

___

Abak. K., Comlekcioglu N., Buyukalaca S. & Sari, N. (1998). Use of stomatal characteristics to estimate ploidy level of haploid and dihaploid pepper plants. Xth EUCARPIA Meeting on Genetics and Breeding of Capsicum annuum & Eggplant. Avignon, France. p 179-182.
Açıkgöz, N., İlker, E., & Gökçöl, A. (2004). Biyolojik araştırmaların bilgisayarda değerlendirilmeleri, Ege Üniversitesi Tohum Teknolojisi Uygulama ve Araştırma Merkezi Yayınları No: 2, Ege Üniversitesi Ziraat Fakültesi ofset atölyesi (in Turkish).
Al Remi, F., Taskın, H., Sönmez, K., Büyükalaca, S., Ellialtıoglu. Ş. (2014). Effect of Genotype and Nutrient Medium on Anther Culture of Pepper (Capsicum annuum L). Turkish J. Agr. Natural Sci. 1:108–116.
Alsahlany, M. Zarka D., Coombs J. & Douches, D.S. (2019). Comparison of Methods to Distinguish Diploid and Tetraploid Potato in Applied Diploid Breeding. American Journal of Potato Research (2019) 96:244–254 https://doi.org/10.1007/s12230-018-09710-7
Anderson T.W. (1984). An introduction to multivariate statistical analysis, Second Edition. John Wiley & Sons.
Ari, E., Bedir, H., Yildirim, S., Yıldırım, T. (2016), Androgenic Responses of 64 Ornamental Pepper (Capsicum annuum L.) Genotypes to Shed-Microspore Culture in Autumn Season. Turkish Journal of Biology, 40 (3): 706-717. https://doi.org/10.3906/biy-1505-41
Beaulieu, J.M., Leitch, I.J., Patel, S., Pendharkar, A. and Knight, C.A. (2008). Genome size is a strong predictor of cell size and stomatal density in angiosperms. New Phytologist, 179: 975-986. https://doi.org/10.1111/j.1469-8137.2008.02528.x
Çömlekçioğlu N., & Ellialtıoğlu, Ş.Ş. (2018). Review on the research carried out on in vitro androgenesis of peppers (Capsicum annuum L.) in Turkey. Review Paper: Research Journal of Biotechnology. Vol. 13 (6).
Comlekcioglu, N., & Ozden M. (2019): Polyploid induction by colchicine treatment in goldenberry (Physalis peruviana), and effects of polyploidy on certain traits. The Journal of Animal & Plant Sciences, 29(5): 1336-1343. ISSN: 1018-7081.
Corral-Martinez, P. & Segui-Simarro, J.M. (2012). Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.) Euphytica (2012) 187:47–61. https://doi.org/10.1007/s10681-012-0715-z
Dumas de Vaulx, R., & Chambonnet, D. (1982). Culture in vitro d’anthe`res d’aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements a` 35°C associe´s a` de faibles teneurs en substances de croissance. Agronomie 2:983–988.
Garcia-Arias, F., Sanchez-Betancourt, E., & Nunez, V. (2018). Fertility recovery of anther-derived haploid plants in Cape gooseberry (Physalis peruviana L.). Agron Colomb 36(3):201–209
Głowacka, K., Jezowski, S. & Kaczmarek, Z. (2010). In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterisation of induced polyploids in two Miscanthus species. Ind Crops Prod 32: 88-96.
Grozeva, S.,Todorova, V. & Nankar, A.N. (2021). Creation of pepper doubled haploids and morphological characterization of androgenic plants. Euphytica 217;113. https://doi.org/10.1007/s10681-021-02840-w
Gyulai, G., Gemesne, Juhasz A., Sagi, Z., Zatyko, L., Heszky, L.,& Venczel, G. (2000). Doubled haploid development and PCRanalysis of F1 hybrid derived DH-R2 paprika (Capsicum annuum L.) lines. J Plant Physiol 156:168–174. https://doi.org/10.1016/S0176-1617(00)80302-8
Hannweg, K., Sippel, A. & Bertling, I. (2013). A simple and effective method for the micropropagation and in vitro induction of polyploidy and the effect on floral characteristics of the South African iris, Crocosmia aurea. S Afr J Bot. 88: 367-372.
Jacobs, J.P., & Yoder, J.I. (1989). Ploidy levels in transgenic tomato plants determined by chloroplast number. Plant Cell Reports 7: 662–664.
Jaskani, M.J., Kwon S.W., & Kin, D.H. (2005). Flow cytometry of dna contents of colchıcıne treated watermelon as a ploıdy screenıng method at M1 stage. Pak. J. Bot., 37(3): 685-696,
Keleş, D., Pınar, H., Ata, A., Taşkın, H., Yıldız, S., & Büyükalaca, S., (2015). Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. HortScience, 50(11), 1671-1676.
Kramer, L.J. & Bamberg, J. (2019). Comparing Methods of Ploidy Estimation in Potato (Solanum) Species. Am. J. Potato Res. 96, 419–426. https://doi.org/10.1007/s12230-019-09729-4
Lawson, T. (2009). Guard cell photosynthesis and stomatal function. New Phytologist (2009) 181: 13–34. https://doi.org/10.1111/j.1469-8137.2008.02685.x
Mattheij, W.M., Eijlander, R., de Koning, J.R.A. & Louwes. K.M. (1992). Interspecific hybridization between the cultivated potato Solanum tuberosum subsp. tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (stone) Behrens. 1. Somatic hybrids. Theoretical and Applied Genetics 83: 459–466.
McGoey, B.V., Chau, K. & Dickinson, T.A. (2014). Stomata Size in Relation to Ploidy Level in North American Hawthorns (Crataegus, Rosaceae). Madrono 61(2), 177-193, https://doi.org/10.3120/0024-9637-61.2.177 Mir, R., Calabuig-Serna, A., & Seguí-Simarro, J.M. (2021). Doubled Haploids in Eggplant. Biology, 10, 685. https://doi.org/10.3390/biology10070685
Monakhos, S.G., Nguen, M.L., Bezbozhnaya, A.V., Monakhos, G.F. (2014). A relationship between ploidy level and the number of chloroplasts in stomatal guard cells in diploid and amphidiploid Brassica species. Agric Biol 5:44–54. https://doi.org/10.15389/agrobiology.2014.5.44eng
Monda, K., Negi, J., Iio, A., Kusumi, K., Kojima, M., Hashimoto, M., Sakakibara, H.,& Iba, K. (2011). Environmental regulation of stomatal response in the Arabidopsis Cvi-0 ecotype. Planta 234: 555–563
Omidbaigi, R., M. Mirzaee, M.E. Hassani, & S.M. Moghadam (2010). Induction and identification of polyploidy in basil (Ocimum basilicum L.) medicinal plant by colchicine treatment. Int J of Plant Prod. 4(2): 87-98.
Qin X., & Rotino, L. (1995). Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets. Plant Cell Tissue Org Cult 41(2):145–149
Rivas-Sendra, A.,Corral-Martinez, P., Camacho-Fernandez, C., & Segui-Simarro, J.M. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis Plant Cell Tiss Organ Cult. 122:759–765. https://doi.org/10.1007/s11240-015-0791-6
Robinson, D.O., Coate, J. E., Singh, A., Hong, L., Bush, M., Doyle, J.J., & Roeder, A.H.K. (2018). Ploidy and Size at Multiple Scales in the Arabidopsis Sepal [OPEN] The Plant Cell, 30: 2308–2329.
Rotino, G.L. (1996). Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 3. Kluwer, Dordrecht, pp 115–141.
Sakhanokho, H. F. & Islam-Faridi, M. N. (2014). Spontaneous Autotetraploidy and Its Impact on Morphological Traits and Pollen Viability in Solanum aethiopicum. HortScience 49(8). 997-1002. https://doi.org/10.21273/HORTSCI.49.8.997
Salas, P., Prohens, J., & Segui-Simarro, J.M. (2011). Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274. Sari, N. Abak, K.& Pitrat M. (1999). Comparison of ploidy level screening methods in watermelon: Citrullus lanatus (Thunb.) Matsum and Nakai. Scientia Horticulturae, 82; 265-277.
Şimşek, İ. , Göçmen, M. & Sarı, N. (2013). Diploid ve tetraploid karpuz bitkilerinde morfolojik ve sitolojik farklılıkların belirlenmesi. Derim, 30 (1), 1-14. http://www.derim.com.tr/tr/pub/issue/4574/62652
Soonthornkalump, S., Chuenboonngarm, N. Jenjıttıkul, T., Thammasırı, K. & Soontornchaınaksaeng, P. (2017). Morphological and Stomatal Guard Cell Characteristics of in vitro Kaempferia rotunda L. (Zingiberaceae) through Colchicine Induced Polyploidy. Walailak Journal of Science and Technology 2017; 14(3): 235-242.
Tekin M. &, Yılmaz, G. (2018). Anatomıcal and palynological studies on endemic Verbascum weidemannianum Fisch. & Mey. (Scrophulariaceae) in Turkey. International Journal of Agriculture, Forestry and Life Science. 2 (2) 6-15.
Tepe, Ş., Ellialtıoğlu, Ş., Yenice, N., & Tıpırdamaz, R. 2002. In vitro kolhisin uygulaması ile poliploid nane (Mentha longifolia L.) bitkilerinin elde edilmesi. Akdeniz Üniv. Ziraat Fakültesi Dergisi, 15(2),63-6
Widoretno, W. (2016). In vitro induction and characterization of tetraploid Patchouli (Pogostemon cablin Benth.) plant. Plant Cell Tiss Organ Cult. 125:261-267.
Xie, X., Aguero, C.B., Wang, Y. & Walker, M.A. (2015). In vitro induction of tetraploids in Vitis x Muscadinia hybrids. Plant Cell Tiss Organ Cult. 122:675-683