Wang et al. Bot Stud (2017) 58:2 DOI 10.1186/s40529-016-0157-3 ORIGINAL ARTICLE Open Access Morphological, physiological, cytological and phytochemical studies in diploid and colchicine‑induced tetraploid plants of Fagopyrum tataricum (L.) Gaertn Lin‑Jiao Wang1,3, Mao‑Yin Sheng1,2,3*, Pei‑Cai Wen1,4 and Jia‑Ying Du3,4 Abstract Background: Tartary buckwheat are very popular as an important functional food material and its cultivation is very widespread in our whole world, but there obviously lack works in the researches of genetic breeding for agricultural and medicinal utilization. The aim of this study is to obtain good germplasm resources for agricultural and medicinal use of tartary buckwheat (Fagopyrum tataricum) by inducing the tetraploid plants. Results: Four cultivars of F. tataricum, that is, Qianwei 2#, Jinku 2#, Chuanqiao 1#, and Liuqiao 1# were selected to experiment. The tips of seedlings with two true leaves were treated by 0.25% (w/v) colchicine solution for 48, 72, and 96 h, respectively. The chromosome number of treated plants was determined by metaphase chromosome count‑ ing of root tip cells and PMCs (pollen mother cells) meiosis observation. Tetraploid induction successfully occurred in all three treatments with an efficiency ranging from 12.13 to 54.55%. The chromosome number of the diploid plants was 2n 2x 16, and that of the induced tetraploid plants was 2n 4x 32. The typical morphological and physi‑ ological= qualities= were compared between the control diploid and corresponding= = induced tetraploid plants. Results showed that the induced tetraploid plants had obviously larger leaves, flowers, and seeds. Moreover, the content of seed protein and flavonoid were also increased in the tetraploid plants. The pollen diameter and capsule size of dip‑ loid plants were significantly smaller than those of tetraploid plants. Conclusion: Fagopyrum tataricum can be effectively induced into tetraploids by colchicines. The tetraploid induction can produce valuable germplasm resources for breeding and is a practicable breeding way in F. tataricum. Keywords: Fagopyrum tataricum, Colchicine, Tetraploid, Chromosome counting, Morphological characteristics Background belongs to the big-achene group of genus Fagopyrum Buckwheat as an important, highly nutritional nonpoa- (Chen et al. 2004; Sheng et al. 2011). Tartary buckwheat ceous crop has great potential as source of food, forage, grain, as an important functional food material, contains and medicine (Lin 1994; Holasova et al. 2002; Tang and proteins with high biological value and balanced amino Wang 2010; Pan and Chen 2010). Buckwheat originates acid composition; relatively high crude fiber and vitamins from China and is widely distributed in Asia and Europe B1, B2, and B6; and more rutin than common buckwheat (Hou et al. 2016). The tartary buckwheat (Fagopyrum (F. esculentum Moench) (Pan and Chen 2010; Wang et al. tataricum (L.) Gaertn), a diploid species (2n = 2x = 16), 2015). The products of tartary buckwheat are very popu- lar and its cultivation is widespread in whole world (Lin 1994). Although lots of cultivation and utilization have *Correspondence: [email protected] 3 National Engineering Research Center for Karst Rocky Desertification been done in tartary buckwheat, there obviously lack Control, Guizhou Normal University, Baoshanbei Road 180, studies in the genetic breeding of tartary buckwheat for Guiyang 550001, P. R. China Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wang et al. Bot Stud (2017) 58:2 Page 2 of 12 agricultural and medicinal utilization (Ohnishi and Ohta water for three times and dried at a loft drier with 25 °C 1987; Chen et al. 2007). for about 24 h and finally sown into flower pots. The pots The polyploidy induction is a significant method for were covered with transparent plastic film and incubated the production of new germplasm resources applica- under greenhouse conditions. Colchicine was applied to ble for plant genetic breeding (Tang et al. 2010). Poly- the apical meristem of seedlings in the stage of two true ploidy induction has been used as a breeding method in leaves. Three treatment durations (i.e. 48, 72 and 96 h) many plants, especially, in crop, horticulture and medi- were studied to determine the optimum colchicine expo- cal botany. Induced polyploidy can enhance qualities for sure times at concentration of 0.25% (w/v) for tetraploid agriculture, medicine and horticulture utilization (Tang induction. Absorbent cotton balls sucked with a solution et al. 2010) and improve resistibility to environmental of colchicines (0.25%, w/v) were wrapped around the api- stresses and diseases (Ahloowalia 1967). Besides, poly- cal tips of seedlings when two true leaves of the emerged ploidy induction can lead to content change of second- seedlings appeared. The experiments were conducted ary metabolites (Majdi et al. 2010). Colchicine has been in a greenhouse and continued for the three treatment found to have a significant effect on polyploid induc- durations (i.e. 48, 72 and 96 h); the colchicines solution tion and is widely used for inducing polyploidy in plants was dropped to the absorbent cotton balls twice a day, at (Tang et al. 2010) because it can effectively arrests mitosis 8:30 and 17:30 o’clock. After the treatment durations, the at the anaphase stage (Kunitake et al. 1998; Kermani et al. absorbent cotton balls were removed from the seedling 2003). Until now, numerous researchers reported that tips, and then the seedling tips were cleaned by distilled germplasm resources have been innovated using poly- water and nursed further in the greenhouse. 50 seedlings ploidization techniques in many crop or medicinal plants were experimented for each treatment. After 2 weeks (Ahloowalia 1967; Kermani et al. 2003; Tang et al. 2010; of the durations of colchicines treatment, death rate on Ye et al. 2010; Kanoktip et al. 2014) including F. esculen- young plants was recorded. tum (Zhu and Gao 1988; Lian and Chen 2013). Limited works regarding tartary buckwheat, polyploidy induction Chromosome counting and meiosis observation works have been reported (Hou et al. 2002; Zhou et al. The ploidy levels were determined by chromosome 2012). In the present study, the tetraploid F. tataricum counting of root tip cells, and confirmed by PMCs (pol- plants were successfully induced by colchicine. The chro- len mother cells) meiosis observation. Before induction mosome number of the induced plants was confirmed of polyploidy, the chromosome number was determined by cytological identification, and the morphological fea- in root tips of germinated seeds. For the induced plants, tures of the induced tetraploid plants were recorded. The seeds were obtained by self-pollination of each treated germplasm characteristics and breeding utilization of plant and the chromosome counting was also conducted such tetraploid materials were discussed. The aim of this in root tips. When root tips grew to 1–2 cm long, root present study was to produce novel and valuable tetra- rips were cut and pretreated by the method of soak- ploid germplasm resources for the utilization of agricul- ing in a saturated solution of α-bromine naphthalin at ture and medicine in F. tataricum. room temperature for 5 h. After pretreated, root tips were washed by distilled water for three times and then Methods immersed in Carnoy’s liquid (ethanol:acetic acid = 3:1) Plant material for 12–20 h. After three rinses by distilled water, the Mature seeds of the following four F. tataricum culti- root tips were soaked in 1 N HCl at 60 °C for 8 min and vars (2n = 2x = 16): Qianwei 2#, Jinku 2#, Chuanqiao then moved to distilled water. After that, the root tips 1#, and Liuqiao 1#, were collected from Guizhou, Shanxi, were stained by improved Carbolfuchsin, and squashed Sichuan, and Yunnan province of China, respectively. for chromosome observation. For meiotic investigation, Before experiment in this study, they were stored in a PMCs in young flowers of about one week age were fixed freezer at temperature of 0–2 °C. The field testing was in Carnoy II solution and stained in 2% (w/v) aceto-car- conducted during 2012–2013 year in the experimental mine solution. Images of mitotic chromosomes in the field site of Guizhou Normal University, Guiyang, China. root tip and meiosis were photo by a DP70 digital camera The laboratory test was conducted in the State Key Labo- in a BX51 microscope (Olympus, Japan). ratory Incubation Base for Karst Mountain Ecology Envi- ronment of Guizhou Province, Guiyang, China. Morphological and physiological characteristics determinations Tetraploid induction The morphological and physiological characteristics were Before germination, seeds, firstly, were disinfected by compared between the control diploid plants and corre- 0.3% K2MnO4 for 20 min, then washed with distilled sponding induced tetraploid plants. Parameters of plant Wang et al. Bot Stud (2017) 58:2 Page 3 of 12 height, plant breadth, leaf length, leaf width, inflores- (%) and tetraploid induction efficiency (%) were obvious cence length and flower size were directly measured by differences among three different durations of colchicines a ruler. A digital caliper was used to measure the char- treatment (Table 1). All the four cultivars can be induced acteristics of leaf thickness and diameter of basal stem. tetraploids successfully, but the induction efficiency were Pollen grains were collected from flowers at anthesis, obvious different. Among the twelve treatment combi- fixed in Carnoy II solution overnight, and then stained by nations, the tetraploid induction efficiency ranged from 1% (v/v) I2-KI solution to detect pollen vitality.